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In The Name of Allah, The Most Gracious, The Most Merciful
42.Consider not that Allah is unaware of that which the Zalimum
(polytheists, wrong-doers) do, but He gives them respite up to a Day
when the eyes will stare in horror. 43. (They will be) hastening forward
with necks outstretched, their heads raised up (towards rge sky), their
gaze returning not towards them and their hearts empty (from thinking
because of extreme fear). 44. And warn (O Muhammad) mankind of the
day when the torment will come unto them; then the wrong-doers will
say: Our Lord! Respite us for a little while we will answer Your Call
and follow the Messengers! (it will be said): Had you not sworn
aforetime that you would not leave (the world for the Hereafter).
(Surah 14, Ibrahim, Part 13, The Noble Quran)
JOURNAL OF THE EGYPTIAN SOCIETY OF
PARASITOLOGY
Volume (38), Number (3), December, 2008
Contents Page
1- EPIDEMIOLOGY OF PLASMODIUM FALCIPARUM AND P. VIVAX
MALARIA ENDEMIC IN NORTHERN AFGHANISTAN By MICHAEL
K. FAULDE, RALF HOFFMANN, KHAIR M. FAZILAT AND ACHIM
HOERAUF........................................................................... ...................
2- PCR ASSAY IN MALARIA DIAGNOSIS USING FILTER PAPER
SAMPLES FROM JAZAN REGION, SAUDI ARABIA By SAEED A.
AL-HARTHI AND MANAL B. JAMJOOM
3- PCR VERSUS ELISA IN DIAGNOSIS OF HUMAN TOXOPLASMO-
SIS IN JEDDAH, SAUDI ARABIA By ABDULKADER M.D. TONKAL....
4- EFFICACY AND SAFETY OF INTRAOPERATIVE RADIOFREQU-
ENCY ABLATION IN MANAGEMENT OF HEPATOCELLULAR CAR-
679
693
707
CINOMA By HESHAM RIZK, MOHAMED MAGDY, MAHMOUD RE-
DA, TAHA ALAA, M. MOHAMED EL-SHAFIE, MAHMOUD GHANEM
AND TAREK SAED
.............................................................
5- HELMINTHES OF SYNANTHROPIC RODENTS (RODENTIA: MU-
RIDAE) FROM DAKAHLIA AND MENOUFIA, EGYPT By ATEF M. EL
SHAZLY, SOHA I. AWAD, MANAR S. AZAB, HANY M. ELSHEIKHA,
ABDEL GAWAD E. ABDEL-GAWAD, HAZEM H.M. KHALIL and TO-
SSON A. MORSY
6- HEAD LOUSE INFESTATIONS IN YEMEN: PREVALENCE AND RISK FA-
CTORS DETERMINATIONAMONG PRIMARY SCHOOL-CHILDREN, AL-MA-
HMEET GOVERNORATE, YEMENBY MOHAMED T. AL-MAKTARI ..
7- INFECTIVITY OF TRICHOMONAS VAGINALIS PSEUDOCYSTS
INOCULATED INTRA-VAGINALLY IN MICE BY EMAN M. HUSSEIN
AND MAHA M. ATWA
8- AN UPDATE REVIEW ON COMMIPHORA MOLMOL AND RELAT-
ED SPECIES By ABDULKADER M.D. TONKAL AND TOSSON A.
MORSY.
9- A SINGLE-STEP IMMUNOCHROMATOGRAPHIC LATERAL-FL-
OW ASSAY FOR DETECTION OF GIARDIA LAMBLIA AND CRYPT-
OSPORIDIUM PARVUM ANTIGENS IN HUMAN FECAL SAMPLES
By DINA M. ABDEL HAMEED, HALA S. ELWAKIL AND MONA A.
AHMED.
10- EFFECTS OF DIGENEAN LARVAL INFECTION ON METALLIC
IONS OF THE SHELLS AND SOFT PARTS OF THEIR INTERMEDI-
ATE HOST SNAIL LANISTES CARINATUS By AMAAL HASSAN MO-
HAMED HASSAN.
11- DOSE RELATED EFFECT OF SYSTEMIC ANTIBIOTICS IN PRE-
VENTION OF POSTOPERATIVE INTRA-ABDOMINAL ADHESION
FORMATION IN EXPERIMENTAL ANIMALS By MOHAMAD ABBAS,
AYMAN E. NAFEH, MAGDY ELSEBAE AND YOUSSEF FAROUK..
12- ULTRASTRUCTURE OF NORMAL AND JHA-TREATED EGGS
OF THE SOFT TICK ARGAS PERSICUS (OKEN) BY WAFAA A. RA-
DWAN, NADIA HELMY, NOHA A. GUNEIDY AND SHIMAA S. MOH-
AMMED
13- ATTACHMENT OF LEISHMANIA MAJOR AND LEISHMANIA IN-
FANTUM IN THE MIDGUT OF THEIR RESPECTIVE SAND FLY VE-
CTORS PHLEBOTOMUS PAPATASI AND PHLEBOTOMUS LANG-
ERONI (DIPTERA: PSYCHODIDAE) By BAHIRA M. EL SAWAF, SA-
ID A. DOHA, KAMAL E. KAMEL AND MOHAMED I. EMAM..
715
727
741
749
763
797
805
813
823
833
14- COMPARISON BETWEEN TRICHMONAS VAGINALIS SYMPTO-
MATIC AND ASYMPTOMATIC ISOLATES EFFECTS IN INTR-AVAG-
INALLY INFECTED RATS BY MOSHERA M. HELMY, EMAN K. EL-
GAYAR, EMAN M. HUSSEIN, AMAL M. ABDOU AND ZAKIA E. MA-
HDY. .
15- THYROXINE AS A STIMULATOR OF LIVER REGENERATION
AFTER PARTIAL HEPATECTOMY: AN EXPERIMENTAL ANIMAL
STUDY By MAHMOUD REDA AND MAHA ZICKRI..
16- Z00NOTIC HELMINTHES OF COMMENSAL RODENTS IN TAL-
KHA CENTER, DAKAHLIA GOVERNORATE By GAMAL A. EL KA-
DY, YOUSR MOSLEH GHENEAM AND IMAN M. BAHGAT...
17- IATROGENIC BILE DUCT INJURIES: MANAGEMENT OF TEN
PATIENTS By MOHAMED A. HELMY.
18- GASTROINTESTINAL STROMAL TUMORS (GISTs): CLINiCAL
PRESENTATION, DIAGNOSIS, SURGICAL TREATMENT AND ITS
OUTCOME By MOHAMED ABBAS, YOUSSEF FAROUK, MAGED M.
NASR, MAGDY M. ELSEBAE, AHMED FARAG, MAHA MAHMUD
AKL AND OLFAT HAMMAM.
19- EFFECT OF TOXOPLASMA CO-INEFECTED WITH INTESTINAL
HELMINTHES ON CELL MEDIATEDIMMUNITY TO TUBERCULOSIS
PATIENTS By MOHIEDDEN M. ABDUL-FATTAH, MONA EL-MOTA-
YAM, EMAN A. EL-SHAMI, GHADA A. SALEM, AMIRA M. SOLIMAN
AND SOHA E. KHORSHED..
20- THE UTILITY OF DIRECT AGGLUTINATION (DAT) AND FAST
AGGLUTINATION SCREENING (FAST) TESTS IN SERODIAGNOS-
IS OF EXPERIMENTAL MICROSPORIDIOSIS By IMAN F. ABOU EL
NAGA, MAHA R. GAAFAR, LOBNA A. EL-ZAWAWY, DOAA EL-SAID
and SHERIN F. MOSSALLAM..
21- EFFICACY OF FIVE CHEMICALS ON FASCIOLA GIGANTICA
ENCYSTED METACERCARIAE INFECTIVITY ByALI AWAD A. HAS-
SAN, NAHLA M. SHOUKARY, MONA EL-MOTAYAM AND AYMAN T.
A. MORSY.
22- EFFECT OF THE CHANGE IN ENERGY RESERVES ON THE
ENTOMOPATHOGENIC NEMATODE EFFICACY By FAIZA M. EL-
ASSAL, SOHEIR F. EL-LAKWAH, WAFAA S. HASHEESH AND MA-
GDA EL-MAHDI..
23- MPACT OF SEVERAL CONTROL MEASURES ON THE ENCYS-
STED METACERCARIAE OF HETEROPHYIDS By LOBNA A. EL-
ZAWAWY..
843
853
863
873
883
895
903
919
929
945
24- ULTRASTRUCTURE AND SOME PATHOLOGICAL PICTURES
OF GASTRODICUS AEGYPTIACUS (COBBOLD, 1876) IN EGYPTI-
AN HORSES By GAZAA HASSAN MORSY..
25- TWO METHODS FOR ATTENUATING TOXOPLASMA GONDII
TACHYZOITES RH STRAIN BY USING ETHANOL EXTRACT OF
CURCUMA LONGA By NAJIA A. AL-ZANBAGI AND NUHA T. ZELAI..
26- BIOCHEMICAL PARAMETERS IN CHRONIC FASCIOLIASIS By
ATEF M. EL-SHAZLY, MANAR S. AZAB, SAMAR N. EL-BESHBISHI,
TAREK I. SAKR, KHALED N. EL-FAYOUMY, AZA S. EL-GHAREEB,
ADEL O. HAFEZ AND EMAN T. EL SHERBINI.
27- GENETIC POLYMORPHISM OF GLUTATHIONE-S-TRANSFER-
ASE (GST-M1 AND GST-T1) IN SCHISTOSOMIASIS-ASSOCIATED
BLADDER CANCER IN EGYPTIAN PATIENTS By KHOLOUD A. EL-
NOUBY, AMAL H. ABD EL HAMEED, OSAMA E. NEGM, HALA E.
HAMOUDA, OSAMA M. EL GAMAL AND GHADA M. ISMAIL
28- DETERMINATION OF ALLOZYME, PROTEIN AND SCHIST-
OSOME SUSCEPTIBILITY IN BIOMPHALARIA ALEXANDRINA PR-
OGENIES PRODUCED BY SELF AND CROSS FERTILIZATION BY
HANAA M.M. EL-KHAYAT, HANAA M. ABU EL EININ AND FATHIA
A. GAWISH..
29- TREATMENT OF TOXOPLASMA GONDII BY TWO EGYPTIAN
HERBS By NENEIN M. ABDEL HADY, GEHAD T. EL SHERBIBI AND
TOSSON A. MORSY..
SOCIETY NEWS
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THE EGYPTIAN SOCIETY OF PARASITOLOGY
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 703 - 710
IMMUNOSUPPRESSANT EFFECTS OF LEISHMANIA INFECTION IN
PSAMMOMYS OBESUS TRAPPED IN SAUDI ARABIA
By
HAMDAN I. Al-MOHAMMED
Department of Medical Microbiology and Parasitology,
College of Medicine, King Faisal University, PO Box: 10590, Riyadh:
11443, Saudi Arabia. E-mail: hamdan@kfu.edu.sa
Abstract
This study was conducted to investigate the immune status of Psammomys
obesus (P. obesus) most implicated as a reservoir host of zoonotic cutaneous
leishmaniasis (ZCL) in the Al-Ahsa area, Saudi Arabia. Based on the presence
of amastigotes in the characteristic lesions and, rodents were divided into two
groups. G1was apparently healthy 10 rodents and G2 were infected 10 ones.
Reduced leukocyte count, percentage lymphocyte and lysozome activity oc-
curred in infected rodents compared to control ones. The infection significantly
reduced the macrophage phagocytic activity reflected by two fold reduction in
intravascular carbon clearance compared to control rodents. The results showed
that ZCL produced an immunosuppressant effects in P. obesus.
Key words: Leishmania, Psammomys obesus, Al-Ahsa, Saudi Arabia.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Leishmaniases are worldwide zo-
onotic diseases, particularly in the
Eastern Mediterranean Countries
(Morsy, 1995). They showed mani-
festations ranging from self-healing
to fatal infections (Desjeux, 2004).
Leishmania parasites are transmitted
by sand flies that inoculate the
metacyclic promastigotes into the
mammalian host. Parasites are
quickly ingested by host macro-
phages and differentiate inside fully
acidified phagolysosomes into
amastigotes, where they replicate
and ultimately induce disease pa-
thology. ZCL are known in many
areas in Saudi Arabia including the
Central region (Morsy and Haw-
wary, 1974; Morsy, 1975; Morsy
and Shoura, 1975; Abdel Aal et al.,
1975; Morsy and Al Seghayer,
1992; El Sebai et al., 1993; Morsy
et al., 1999), Western region (Al-
Qurashi et al., 2000; El-Badry et al.,
2008), Southwestern region (Sebai
et al., 1975; Sebai and Morsy, 1975;
Morsy et al., 1991; 1995b, 1997)
and Eastern region (Peters et al.,
1985; Al-Twafiq and AbuKhamsin,
2004)
The Eastern region includes Al-
Ahsa Oasis located approximately
175 km south of Dammam and
704
Dharan cities and 60 km west of the
Arabian Gulf Coast (Al-Twafiq and
AbuKhamsin, 2004). Sand fly-
vector (Kellick-Kendrick et al.,
1985) and the animal reservoir host,
Psammomys obesus (Elbihari et al.,
1984) were reported in Al-Ahsa
where 93% of P. obesus were in-
fected (Elbihari et al., 1987). L. ma-
jor was isolated from lesions char-
acterized by swelling, hyper pig-
mentation, ulceration, necrosis and
in advanced cases sloughing and
loss of ear tissue. P. obesus is the
incriminated animal reservoir in the
Middle East (Morsy et al., 1996).
The protective immune response
against leishmaniases group de-
pended on the established of Th1
immunity (Morsy et al., 1995a). In
ZCL the resolution or progression
depend on the outcome of the de-
velopment of CD4+ T cell subsets
Th1 & Th2 respectively (Wilson et
al., 2005). Immuno-competent man
and resistant mouse strains can con-
trol L. major by the development of
a protective Th1 response with
elaboration of certain cytokine pro-
files as IFN- & IL-12 (Reiner and
Locksley, 1995), and susceptible
BALB/ c mice exhibited Th2 im-
munity and succumbed ZCL pro-
gression (Locksley et al., 1999). In
chronic cases accumulation of addi-
tional immune cells, largely T- & B-
cells (Morsy et al., 1989) were
prominent in draining lymph nodes
(Chang et al., 2003).
This study aimed at the evaluation
of the immune of Psammomys obe-
sus implicated as the main reservoir
host of ZCL in Al-Ahsa district to
pave the way for feasible control
measure.
Materials and Methods
Psammomys obesus were trapped
in locally-made break-back spring
traps placed at entrances of burros.
Traps were placed in situ in the ear-
ly afternoon, inspected at sunset and
again early next morning. Trapped
animals were transported to labora-
tory and thoroughly inspected for
external lesions and when any were
detected; impression smears were
taken from the lesions edge, fixed
in methanol and stained with Giem-
sa (Mangoud et al., 2005). Based on
presence of amastigotes rodents
were divided into two groups. G1:
10 healthy control rodents. G2: 10
infected ones. Blood samples were
collected in heparin tubes for hae-
matological examination or in plain
tubes for separation of sera.
The total leukocytes counted in a
cello scope and differential leuko-
cytes were carried out using blood
smears stained with Giemsa and
May-Grunewald solutions. The re-
ticuloendothelial system function
was determined in rats by measuring
the intravascular clearance of car-
bon (Gnter, Wagner, Hannover)
was administered intravenously at a
dose of 0.0008 g/kg body weight
into the left Jugular vein. Blood was
then hemolyzed with 4 ml of 0.1%
sodium carbonate solution and the
carbon concentrations were plotted
as percentage of the injected dose
semi-logarithmically against time in
705
minutes and thus intravascular half
life in minutes was calculated. Se-
rum lysozome concentrations were
measured using Micrococcus lyso-
diekticus as a substrate (lysozome
reagent Kit, Worthington Biochemi-
cal, Co., NJ), according to the man-
ufactures recommendations (Al-
Ankari and Homeida, 1996). The
percentage changes in transmission
(at 510 nm) per minute were imme-
diately recorded using a spectropho-
tometer (Hitachi, Japan). Values
were compared to a standard curve
simultaneously prepared using a
known concentration of egg white
lysozome.
Statistical analysis: Data were ex-
pressed as mean SD. Analysis of
variance (ANOVA) for repeated
measures using general linear model
(GLM) procedure of statistical anal-
ysis system tested the effect of in-
fection. Comparison of means in
groups was by Duncans multiple-
range test and P<0.05 was statistic-
ally significant (Campbell and Ma-
chine, 1993).
Results
Table 1: Mean spleen weight, leukocyte count and lysozome enzyme activity in
control and infected P. obesus.
Variable G1 (normal) G2 (infected)
Animal weight ( g ) 1001.1 912.1 *
Spleen weight ( g ) 0.310.01 0.290.01
Total leukocyte ( x10
3
/ml ) 21.20.2 14.60.1 *
Lymphocyte ( % of leukocytes) 532 393 *
Neutrophil ( % of leukocytes ) 240.8 231.1
Lysozome activity ( 1u/l ) 8.30.11 5.60.11 *
*P<0.05, significantly different from group1.
Table 2: Absorbance of plasma carbon concentration versus time of rodents
treated with carbon colloid.
Time (minute) G1 ( normal ) G2 ( infected )
2 0.370.01 0.340.005
4 0.330.01 0.320.006
8 0.250.006 0.300.005
12 0.180.005 0.250.006
16 - 0.200.004
20 - 0.150.003
706
Discussion
In the present study, there was
significantly reduction of total leu-
kocyte and percentage lymphocyte
in infected rodents (G2) compared
to control ones (G1). Serum lyso-
zome activity was significantly
(P<0.05) decreased in infected ro-
dents than in controls. The clearance
rate values were 16.21.9 minutes
and 7.11.8 minutes for infected
rodents and controls, respectively.
Regarding carbon clearance by re-
ticuloendothelial system there was a
significantly (P<0.001) reduced in-
travascular carbon clearance and
delayed vascular half-life at about
two fold in infected rodents com-
pared to controls.
After internalization by the phag-
ocytes, L. major escaped the toxic
extracellular milieu and survived
intracellular in macrophages. The
dissemination of parasites was an
important factor in murine models
of L. major infection (Solbach and
Laskay, 2000). In the present study,
the significant decrease in the num-
ber of circulating leukocytes in in-
fected P. obesus was mainly due to
decrease in the number of lympho-
cytes, that was possibly affected by
reduction in the weight of lymphoid
tissue (spleen). The leucopenia was
reported in mice (Al-Mofleh, 1987),
and human (Malik, 1987; Belic et
al., 2000) infected with Leishmania.
In the present study, spleen weight
was not significantly reduced. But,
dissemination of L. major in mice
spleen was associated with a suscep-
tible phenol-type (Laskay et al.,
1995), whereas containment of the
parasite in skin and lymph nodes
was associated with a resistant phe-
notype (Nicolas et al., 2000). Dis-
seminated parasites for a long time
led to progressive increased of the
weight of spleen (Uzonna et al.,
2004) and lymph node
(Mahmoudzadeh-Niknam et al.,
2007). So, the
spleen serves as a safe harbor for
long term leishmaniasis persistence
(Wilson et al., 2005). This agreed
with the present study when the
short duration of infection did not
give prolonged immuno-suppressive
effect of L. major on rodents and
spleen was not enlarged.
In the present study, serum lyso-
zome activity significantly reduced
in infected rodents. Serum lysozome
activity, a macrophage function in-
dex (Diluzio, 1979) inhibited Leish-
mania infected mice (Al-Mofleh,
1987) and Cryptosporidium baileyi
infected chicks (Fatani et al., 1999).
In the present study, the reduced
phagocytosis efficiency of foreign
particles such as carbon colloid con-
tributed to the reduction of intravas-
cular carbon clearance and delay of
vascular half-life at about two fold
compared to controls. Moore and
Matlahewski (1994) found that the
heavily infected cells were prevent-
ed from degeneration although they
had impaired functions. Besides,
phagocytosis is a process involving
opsonisation followed by adsorption
into macrophage surface, endocyto-
sis, and eventually particle digestion
707
or phago-some-lysozome function
(Benacerraf et al., 1975). Depres-
sion of reticulo-endothelial function
occurred in Leishmania infected
mice (Al-Mofleh, 1987), exposed to
HCV (Williams and Diluzio, 1980),
and with malaria (Lo-ose et al.,
1972) and Cryptosporidium infected
chicks (Fatani et al., 1999). L. major
produced an immunosuppressant
effect in P. obseus.
In the present study, no statistical
difference was between infected and
control rats in number of neutro-
phils. Sunderkotter et al. (1993)
found that within the 1
st
1024 h af-
ter L. major infection, neutrophils
migrated to the skin. But, during the
2
nd
& 3
rd
day, macrophages recruited
and dominate in the cellular infil-
trate suggesting that L. major led to
extended cells survival in the early
phase of infection. Aga et al. (2002)
added that Leishmania affected the
survival of neutrophils via a mecha-
nism involving inhibition of caspa-
se-3 activation and that Leishmania-
mediated delay of neutrophil apop-
tosis was associated with a marked
decrease in caspase-3 activity.
Afonso et al. (2008) found that
phagocytosis of apoptotic, but not
viable, neutrophils by Leishmania-
infected macrophages led to an in-
crease in parasite burden via a
mechanism dependent on transform-
ing growth factor-beta 1 & prosta-
glandin E2. Peters et al. (2008)
found that neutrophils depletion re-
duced the ability of L. major to es-
tablished productive infection.
Conclusion
Leishmaniases are worldwide pro-
blem mainly in the Mediterranean
Countries. The fact that L. major
has immunosuppressant effect on
animal reservoir paves the way to
feasible and friendly control meas-
ure.
Acknowledgement
The author would like to thank the
Deanship of Scientific Research,
King Faisal University, Kingdom of
Saudi for financial support.
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 711- 721
DETECTION OF G1 GENOTYPE OF HUMAN CYSTIC
ECHINOCOCCOSIS IN EGYPT
By
MOHAMMAD H. ABD EL BAKI, ADEL M.G. EL MISSIRY, HEBA E.M.
ABD EL AATY, ANHAR A. MOHAMAD AND HEBA A.R. AMINOU
Department of Parasitology, Faculty of Medicine, Ain Shams University,
Cairo 11566, Egypt
Abstract
The first trial to detect G1 genotype in Egyptian human isolates of hydatid
cysts (HC) and serum samples to approach diagnosis of cystic echinococcosis
(CE) using human sera by PCR. Using strain specific primers, 27/36 confirmed
CE patients (75%) showed G1 specific band in their sera at 254 bp. Specificity
was 100% without detecting bands for either other parasitosis, or mass occupy-
ing lesions.
Using PCR, G1 genotype was detected in 83.3% of HC samples, without
significant difference between types of human isolates (pulmonary, hepatic, or
multi-organ). G1 genotype detection in human sera was in 75% of CE patients
compared to 83.3% in HC samples of the same group of patients proved satis-
factory, simple and safer than HCF sampling.
IHAT gave sensitivity of 58.3% compared to histopathological examination
of surgically removed cysts or examination of hydatid cyst fluid (HCF) for pro-
toscolices (gold standards). The specificity was 70% with false positive reac-
tions with other parasitic infections and mass occupying lesions.
PCR detection of G1 genotype in Egyptian animal hydatid cysts showed 90%
in camel isolates and 80% in sheep isolates, but pig isolates were negative. The
presence of this genotype in a high percentage in camel isolates incriminated
sheep strain as the source of CE camel infection. The results may give an ex-
planation to the contradicting results of other studies that did not relay upon
molecular aspects.
Key words: Egypt, echinococcosis, hydatid, isolates, genotype, PCR
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Cystic echinococcosis (CE) is a
major zoonosis of worldwide distri-
bution particularly in sheep raising
countries. It is an emerging disease
in various regions, e.g. Middle East,
Central Asia, and Eastern Africa
(McManus et al., 2003). Echinococ-
cus granulosus exhibits extensive

intraspecific (strain) variation. Ten


genetically distinct strains (G1-G10)
have been described with a consid-
erable diversity of morphology, de-
velopment, ,host range, infectivity
to humans, pathogenicity and anti-
genicity (Brayer et al., 2004). The
most previously characterised iso-
lates from humans could be con-
signed to G1 (common sheep strain)
and G6 (camel strain). The sheep
strain (G1) with known pathogenici-
ty to human was also identified in
sheep, goat, cattle and camels. Im-
munological tests have played an
important role in the diagnosis of
hydatid disease. The variation in
sensitivity of these tests could be
attributed to the nature and quality
of the antigen and methodological
sensitivity of the selected technolo-
gy. Moreover, subspecies and strain
differences of E. granulosus in dif-
ferent countries or even in the same
country may be a reason (Poretti et
al., 1999).
PCR does not only identify mi-
nute amounts of parasite nucleic
acids, but also characterize the bio-
logical status of parasite material.
There are no reports for molecular
detection of G1 genotype in human
CE sera in spite of good evidence
for the role of PCR technique in
detection of parasite nucleic acid in
fine needle aspirate from CE pa-
tients. However, a standardized
PCR could potentially allow the
detection of parasite DNA in other
clinical samples like patient's serum
(Siles-Lucas and Gottstain, 2001).
The present study aimed the de-
termination of G1 genotype in hyda-
tid cyst isolates extracted from
Egyptian patients compared to non
human isolates. This may serve as
an approach for strain specific de-
tection in the molecular diagnosis of
CE in Egypt using patient' sera.
Materials and Methods
The patients and control group
individuals included were unrelated
and of Egyptian ancestry. A total of
36 patients of both sexes (21 males
and 15 females) with confirmed CE,
aged from 7-70 years, were en-
rolled. CE was confirmed by histo-
pathological examination of cysts
after surgical excision or examina-
tion of aspirates for hydatid sand
(gold standard for all cases). Chest
radiography, abdominal ultrasonog-
raphy, and\or computed tomography
showed a characteristic cystic swell-
ing (all cases). The cases were se-
lected from patients referred to the
Diagnostic and Research Unit of the
Parasitology Department, Faculty of
Medicine, Ain Shams University,
General Surgery, Cardiothoracic
Surgery and Tropical Medicine De-
partments, Ain Shams University
Hospitals and from patients referred
to the Abdominal Ultrasonographic
Unit of the Tropical Medicine De-
partment, Kasr El-Aini Hospital.
Human subjects: 66 patients and
15 healthy individuals were catego-
rized into 6 groups. GrI: 15 patients
with isolated pulmonary CE, GrII:
15 patients with isolated hepatic
CE, GrIII: 6 patients with multior-

gan affection by CE, GrIV: 15 pa-


tients with other parasites, GrV:15
patients with other mass occupying
lesions and Gr:VI: 15 normal con-
trol individuals. GrIV included 3
cases hymenolepiasis nana, 4 cases
schistosomiasis mansoni, 2 cases
fascioliasis, 3 cases of ascariasis, 2
cases ancylostomiasis and one case
bancroftian filariasis. Diagnosis was
confirmed by urine and stool exam-
ination, blood film and\or (IHAT).
GrV included 5 cases bronchogenic
carcinoma, 6 cases hepatocellular
carcinoma (HCC), 3cases simple
hepatic cyst and one case liver ab-
scess, diagnosis was confirmed by
histopathological examination of a
biopsy material. Blood samples
from all the above groups were ob-
tained in vacutainers under aseptic
conditions for IHAT for hydatid
disease and DNA extraction fol-
lowed by PCR.
Protoscolices were aseptically
obtained from hydatid cysts of pa-
tients included in GrI, II & III ei-
ther by percutaneous aspiration in-
jection reaspiration (PAIR) tech-
nique (16 cases) or after surgical
excision (20 cases), and from camel,
sheep and pig isolates obtained from
Cairo slaughter-house. Protoscolices
were used for DNA extraction fol-
lowed by PCR to detect G1 geno-
type of metacestodes of E. granu-
losus. Animal isolates were pulmo-
nary cysts from 30 camels and he-
patic cysts from 10 sheep and 10
pigs, aspirated individually from
each cyst under aseptic conditions.
Six isolates were under the study,
namely human pulmonary, human
hepatic, human with multiorgan af-
fection, camel, sheep and pig iso-
lates. Processing of hydatid cyst
fluid to obtain and prepare proto-
scolices was done (Zhang et al.,
1998).
Detection of G1 genotype of CE in
different isolates and human seum
by PCR were done by a- DNA ex-
traction using QIAamp DNA Blood
and Body Fluid Mini Kit" supplied
by "QIAGEN". b- Spectrophotomet-
ric measurement of DNA concentra-
tion (Karcher, 1995). c- Amplifica-
tion of the extracted DNA by PCR
(Mullis and Faloona, 1987). Oligo-
nucleotide primers were Pri-
mer1:5'GTATTTTGTAAAGTTCA
3' (Forward-21mer) Primer2: 5'CT
AA ATCAC ATCATCTTACAAT
3' (Backward-22mer) (Dinkel et al.,
2004). d- Detection of PCR prod-
ucts on Agarose Gel electrophoresis
(Maniatis et al., 1982).
IHAT were done using commer-
cial kits (Fumouze Laboratory). Ti-
tre equal to or above 1:160 was sig-
nificant positive (Boyden, 1951).
Statistical analysis: Data were
processed to a personal computer
IBM compatible and analyzed using
SPSS (statistical package for social
science) program. The statistical
tests used were Chi-square test, di-
agnostic sensitivity, and specificity,
predictive value of positive and
negative test as well as diagnostic
accuracy of PCR technique.

Results
The results are shown in tables (1 to 6) and figures (1 & 2).
Table 1: PCR for G1 genotype on animals' hydatid cysts isolates
Hydatid cysts isolates
No. ve PCR +ve PCR at 254 bp
Camel 30 3 (10%) 27 (90%)
Sheep 10 2 (20%) 8 (80%)
Pig 10 10 (100%) 0
Total 50 15 35
Table 2: PCR for G1 genotype on CE patients' hydatid cysts isolates
Group
No. ve PCR +ve PCR at 254 bp
GrI 15 4 (26.7%) 11 (73.3%)
GrII 15 2 (13.3%) 13 (86.7%)
GrIII
6 0 6 (100%)
Total 36 6 (16.7%) 30 (83.3%)
Table 3: PCR for G1 genotype on sera of individuals in all the study groups
Group No. ve PCR +ve PCR at 254 bp X2 P
value
GrI 15 5 (33.3%) 10 (66.7%)
51.4 0.0001
GrII 15 3 (20%) 12 (80%)
GrIII 6 1 (16.7%) 5 (83.3%)
GrIV 15 15 (100%) 0
GrV 15 15 (100%) 0
GrVI 15 15 (100%) 0
Total 81 54 (66.7%) 27 (33.3%)
P<0.01 (Highly significant)
Table 4: Comparison between IHAT and PCR for G1 genotype on sera of indi-
viduals in all the study groups
Group
No. PCRs
IHAT
-ve +ve
GI
15
-ve 5 0
+ve 3 7
GII
15
-ve 3 0
+ve 2 10
GIII
6
-ve 1 0
+ve 1 4
GIV
15
-ve 9 6
+ve 0 0
GV
15
-ve 12 3
+ve 0 0
GVI
15
-ve 15 0
+ve 0 0
Total 81 51 30
IHAT titer above 1/160 was considered positive, PCR positive specific bands were at 254 bp.
P<0.01 (Highly significant)

Table 5: Comparison between PCR for G1 genotype on CE patients' hydatid


cysts isolates and sera
Group No.
PCR hydatid cysts isolates PCR serum
+ve -ve +ve -ve
GrI 15 11 (73.3%) 4 (26.6%) 10 (66.6%) 5 (33.3%)
GrII 15 13 (86.6%) 2 (13.3%) 12 (80%) 3 (20%)
GrIII 6 6 (100%) 0 5 (83.3%) 1 (16.6%)
Total 36 30 (83.3%) 6 (16.7%) 27 (75%) 9 (25%)
P > 0.01 (Non significant)
Table 6: IHAT validity in relation to hydatid cyst fluid examination (gold
standard) for the diagnosis of CE
Test Fluid examination
(+ve)
Total
GI GII GIII
IHAT -ve 8 5 2 15
+ve 7 10 4 21
Total 15 15 6 36
Table 7: IHAT for diagnosis of CE titers in all the study groups
Group
No.
IHAT Titer
1/80 1/160 - 1/320 1/640 1/1280 -1/2560
GrI 15
8 (53.3%) 4 (26.7%) 3 (20%)
GrII 15
5 (33.3%) 2 (13.3%) 6 (40%) 2 (13.3%)
GrIII 6
2 (33.3%) 1 (16.7%) 3 (50%)
GrIV 15
9 (60%) 4 (26.7%) 2 (13.3%)
GrV 15
12 (80%) 3 (20%) 0
GrVI 15
15(100%) 0 0
Total 81
51 (62.9%) 14 (17.2%) 14 (17.2%)) 2 (2.4%)
Sensitivity=58.3%, Specificity=70%, PPV=70%, NPV=70.5%

Fig. 1: PCR for G1 genotype on CE patients' and animals' hydatid cysts isolates
in 1% agarose gel electrophoresis stained with ethidium bromide
Positive isolates show typical band at 254 (Dinkel et al., 2004)
Lane 1: Marker, Lane 2: Negative control (no DNA), Lane 3: Positive human isolate (from a hepatic cyst),
Lane 4: Positive human isolate (from a pulmonary cyst), Lane 5: Positive human isolate (from a hepatic cyst
of multiorgan affection), Lane 6: Positive camel isolate (from a pulmonary cyst), Lane 7: Positive sheep
isolate (from a hepatic cyst) and Lane 8: Negative pig isolate (from a hepatic cyst)
Fig. 2: PCR for G1 genotype on CE patients' sera in 1% agarose gel electro-
phoresis stained with ethidium bromide
Positive typical band is at 254
Lane 1: Marker, Lane 2: Negative control, Lane 3: Positive human isolate (from a hepatic cyst), Lane 4:
Positive CE patient's serum (-ve by IHAT), Lane 5: Negative pulmonary CE patient's serum, Lane 6, 7 and 8:
Negative patients' sera of other parasitic infections (S. mansoni, H. nana and A. lumbricoides respectively)
and Lane 9 and 10: Negative patients' sera of other mass occupying lesions (hepatocellular and bronchogenic
carcinomas respectively).

Discussion
Studies on the stain specificities
of E. granulosus in the Middle East
revealed sheep strain (G1) present
in sheep, goats, cattle, camels and
humans (Sadjjadi, 2006). In the pre-
sent study, there was high positivity
rate of PCR for G1 (common sheep
strain) genotype detection with the
camel isolates (90%) followed by
the sheep isolates (80%) and none
of pig isolates was proved positive.
This could be simply attributed to
the fact that sheep strain was the
most common genotype of E. gran-
ulosus affecting sheep, cattle and
camels as mentioned by Harandi et
al. (2002). Abdel Rahman et al.
(1999) demonstrated a great resem-
blance between hydatid cyst fluid
proteins of sheep and human origin
which were different from hydatid
cyst fluid proteins of camel as re-
vealed by SDS-PAGE patterns.
Moreover, based on a molecular
study by Tashani et al. (2002),
when a portion of the cytochrome
C-oxidase subunit (COX1) gene
from protoscolices samples from
cattle, humans, camels and sheep
was sequenced, they were identical
to the common sheep strain of E.
granulosus.
The PCR for G1 genotype on CE
patients' hydatid cysts isolates
showed that 83.3% (30/36) of CE
patients have G1 genotype in their
hydatid cysts. This could indicate
the important role of that sheep
strain in human CE. Haridy et al.
(2000) found that sheep play the
important role in dissemination of
the disease due to the fact that their
cysts are the highly fertile ones as
compared to other animal hosts,
consisting the risk cycle in hydati-
dosis as sheep-dog-man. The in-
crimination of sheep as the main
source of human infection was
proved by El Shazly et al. (2007)
both serologically and parasitologi-
cally. However, Azab et al. (2004)
using RAPD-PCR technique sug-
gested that camels are important
hosts for the transmission of Egyp-
tian human hydatidosis. Ahmadi
and Dalimi (2006) in Iran found that
the sheep and human isolates per-
tains the same genotype while the
camel isolates pertained a different
one. The negative PCR for G1
genotype either with sheep or hu-
man hydatid cysts isolates could be
due to factors that inhibited the am-
plification reaction or occurrence of
DNA degradation (Innis et al.,
1990).
Dinkel et al., (2004) described a
sensitive and specific PCR system
for diagnosis of CE by detection of
G1 genotype in human hydatid cyst
fluid. However, although PCR is a
powerful and specific technique that
can detect minute amounts of para-
site DNA in HCF and FNAB is in-
creasingly being accepted as a com-
plementary diagnostic tool, it still
carries the danger of anaphylactic
reaction. Also, secondary echino-
coccosis may result by dissemina-
tion of HCF, containing viable
metacestode material. Thus, detec-

tion of parasite nucleic acids in clin-


ical samples other than HCF ob-
tained by FNAB has become a great
need.
In this study, detection of G1 gen-
otype, and diagnosis of CE using
human serum by PCR was highly
significant (p<0.01), when com-
pared with HCF for protoscolices
(gold standard). 27/36 (75%) of CE
patients' sera showed the specific
PCR band at 254 bp in ethidium
bromide stained agarose gel. Speci-
ficity was 100% without any false
positive PCR results with sera of
control groups' individuals. On the
other hand, there was a highly sig-
nificant difference (P<0.01) be-
tween IHAT versus PCR for diag-
nosis of CE. Sensitivities of both
tests were 58.3% & 75% and speci-
ficities were 70% & 100%, respec-
tively, which proved PCR on serum
as more sensitive and specific than
IHAT.
There was no significant differ-
ence (P>0.01) between G1 genotype
in human sera (75%) versus in HCF
(83.3%). So, using serum proved
satisfactory in sampling than HCF
obtained by FNAB with danger of
anaphylaxis and secondary CE.
In the present study, IHAT sensi-
tivity was 58.3% compared with
HCF examination for protoscolices.
Taratuto and Venturiello (1997)
explained the IHAT negative results
with known positive cases by im-
mune incompetence and formation
of immune complexes. Besides, a
wide range of evasion mechanisms
were advanced, including a barrier
for host cells due to hydatid cyst
laminated cuticle, polyclonal activa-
tion of lymphocytes by parasite sol-
uble antigens, and depression of
host cell immune responses. Kaur et
al. (1999); Ortona et al. (2000) and
Rashed et al. (2003) found a sensi-
tivity of 70.5%, 54% & 83.3% re-
spectively for CE by IHAT. This
variation of sensitivities may be at-
tributed to differences in hydatid
antigens preparation; type of anti-
gens and the cysts site (Schantz
and Gottstein, 1986). The serologi-
cal response of host was influenced
not only by Echinococcus species,
but also by strain. Extracts of proto-
scolices from different hosts gave a
source of strain-specific diagnostic
antigens for human CE (Rickard
and Lightowlers, 1986).
Besides, IHAT spcificity was
70% giving false positive reactions
with other parasites (2 with S. man-
soni, 2 with H. nana, & 1 with A.
lumbricoides) and mass occupying
lesions (2 HCC & 1 amoebic liver
abscess). Azazy and Abdel Hamid
(2000) reported IHAT cross reactiv-
ity with S. mansoni with a specifici-
ty of 97%. Gonlugur et al. (2005)
obtained IHAT specificity of 84%
in diagnosis of CE. The cross reac-
tion between hydatidosis and schis-
tosomiasis antigen may be due to
common lipoprotein antigen
(Planchart et al., 1994). However,
100% specificity without cross reac-
tions with other lesions was report-
ed (Aksoy and Inci, 2004; Rashed et
al., 2003). The patients with highest
IHAT titer had hepatic hydatid cysts

(2/15 cases). Rigano et al. (2002)


found that IHAT was related to state
of cyst rather than site, and that high
titers went with progressive more
than in cured or stable disease.
Generally speaking, echinococ-
cosis/hydatidosis is a real health
problem in Egypt. As an example,
Mazyad et al. (1998) in Cairo iden-
tified spinal cord hydatid cysts in a
primary school child. Also, Mazyad
et al. (1999) in Northern Sinai re-
ported vertebral unilocular hydati-
dosis in a shepherd and his wife. El
Shazly et al. (2007a) identified en-
demic zoonotic hydatidosis in Da-
kahlia governorate. Apart from
man, Haridy et al. (1998) reported
hydatidosis in slaughtered Egyptian
camels. El Shazly et al. (2007b,c)
identified the causative parasite of
zoonotic echinococcosis in street
dogs in urban and rural areas, Da-
kahlia Governorate.
Conclusion
This study proved the suggestion
of PCR using G1genotype specific
primers as a promising diagnostic
alternative to serological methods
for diagnosing human CE in Egypt
and in other countries with similar
epidemiological data.
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723
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 723- 729
INCIDENCE AND PREVALENCE OF EIMERIA INFECTION AMONG
THE HEDGEHOG, HEMIECHINUS AURITUS IN YEMEN
By
BADRIA M. ABDEL-WASAE AND MANAL EL GARHY
Department of Biology, Faculty of Science, University of Taiz, Yemen
and Department of Zoology, Faculty of Science, Cairo University, Egypt
Abstract
Fecal samples of ten hedgehogs, Hemiechinus auritus from Taiz Gover-
norate, Yemen were examined for coccidian parasites. Four (40%) were infect-
ed with Eimeria spp. Comparison of oocyst's criteria with other species of Ei-
meria indicated a new eimerian, E. yeminii n.sp. besides the previously de-
scribed species, E. auriti Mirza, 1970 reported in Iraq. Oocysts of E. yeminii
n.sp. are ovoidal to ellipsoidal, measuring 30x24.5m (27-36x20-28). The out-
er boundary is bilayered with a light reddish colour and perforated by a micro-
pyle. Oocyst residuum and polar granules are present. Sporocysts are spherical
in shape 10 x7.5m (11-12.2x10-11.2) with granular residuum and Stieda
body. Sporozoites are elongate, lying length-wise to the sporocyst long axis.
Sporulation takes place within 3-4 days at ~ 26C.
E. auriti Mirza, 1970 is characterized by subspherical oocysts measuring 20
x 17 (22-26x16-19.5) m, a micropyle and oocyst residuum. The oocyst wall is
bilayered, smooth and greenish in colour. Sporocysts are subspherical, measur-
ing 9.0x8.3 (8.5-1 x7.5-8) m with granular residuum and Stieda body. Fully
sporulated oocysts are observed within 48 h at ~ 26C.
Key words: Eimeria, Yemen, hedgehogs, light microscope, exogenous stages
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------
Introduction
Coccidians are obligatory intracel-
lular parasites infecting nearly all
vertebrates. Among Coccidia, the
genus Eimeria shows a wide distri-
bution in host species of wild ani-
mals. Hedgehogs are small spiny
mammals of the subfamily Erina-
ceinae and the order Insectivora.
They are found through parts of Eu-
rope, Asia, Africa and New Zealand
but there are no native hedgehogs in
the Americas or Australia. Hedge-
hogs are powerful form of pest con-
trol and can be kept as pets due to
their innocent and playful looks.
Few studies were carried out on
coccidian parasites of hedgehogs
(Brutzki et al., 1987; Laux, 1987;
Graczyk et al., 1998; Epe et al.,
1993, 2004; Enemark et al., 2002).
Eimeria of hedgehogs are limited to
four species; E. ostertagi and E.
724
perardii (Yakimoff and Gousseff,
1936) from Erinaceus europaeus in
Russia, E. bijlikuli (Svanbaev,
1962a) from Hemechinus auritus in
Kazakhstan and E. auriti Mirza,
1970 (Mirza, 1975) from H. auritus
in Iraq.
In this study, the long-eared
hedgehog, Hemiechinus auritus col-
lected from Yemen was examined
for natural Coccidia infection.
Materials and Methods
Ten hedgehogs (Hemiechinus au-
ritus) were collected during May
and July, 2008 from Osaefera, and
Warazan valley, Taiz Governorate.
Theanimals were caged separately
for 12 hr until they defecated and
were then set free in the natural hab-
itat. Fecal samples were preserved
individually in vials containing
2.5% aqueous potassium dichromate
solution. Individual samples were
con-centrated by the flotation tech-
nique (Long et al., 1976) and exam-
ined microscopically for the Coccid-
ia. Collected oocysts were aerated
for sporulation and stored at room
temperature. Measurements were
made on thirty sporulated and non-
sporulated oocysts using calibrated
ocular micrometer. All measure-
ments are in micrometers as means,
followed by ranges in parentheses.
Results
Of 10 hedgehogs examined four
(40%) showed eimerian oocysts in
their feces. Two Eimeria species
were identified; a new eimerian, E.
yemenii n. sp. and E. auriti Mirza,
1970.
E. yemenii n. sp. (Figs. 1-4): Oo-
cysts are ovoid to ellipsoid, 30 x
24.5 (27-36x20-28), L/W ratio 1.2.
A micropyle, oocyst residuum and
polar granules are present. The oo-
cyst wall is bilayered, light reddish
in colour and the outer layer is
thicker than the inner one. Sporo-
cysts are spherical, 10x7.5 (11-
12.2x10-11.2), L/W ratio 1:3 with
granular residuum and Stieda body.
Sporozoites are elongate, lying
length-wise to the sporocyst long
axis. Sporulation occurs within 3-4
days at ~26C.
Taxonomic summary: Type host:
hedgehog, Hemiechinus aurit
us. Type locality: Taiz Governorate,
Yemen. Prevalence: 4/10 (40%).
Sporulation: Exogenous, oocysts
sporulated within 3-4 days at ~
26C. Site of infection: Unkno-wn,
oocysts recovered from feces. Ety-
mology: Species name reflects the
host locality.
E. auriti Mirza, 1970 (Fig. 5-8):
Oocysts are subspherical, 20x17
(22-26x16-19.5),length/width (L/W)
ratio 1:17. Micropyle and oocyst
residuum are present. Oocyst wall is
greenish, smooth with two layers;
outer layer is significantly thicker
than inner one. Sporocysts are sub-
spherical, measuring 9.0x8.3 (8.5-
12x7.5-8), L/W ratio 1:1. Sporocyst
wall is single-layered, thin smooth
and colorless. A granular sporocyst
residuum and a small Stieda body at
pointed end are present in sporocyst.
The fully sporulated oocysts contain
725
four sporocysts each with two spo-
rozoites occurred within 2 days at ~
26C. Sporozoites are elongate and
lay length-wise to sporocyst long
axis.
Taxonomic summary: Type host:
hedgehog, Hemiechinus auritus.
Type locality: Taiz Governorate,
Yemen. Prevalence: 4 of 10 (40%).
Sporulation: Exogenous, oocysts
sporulated within 2 days at ~26C.
Infection site: Unknown, oocysts
recovered from feces. First reported
was 1970 in Iraq, near Baghdad
from H. auritus (Mirza, 1975).
Figs. 1-8: Two eimerian oocysts from hedgehog, Hemiechinus auritus.
Figs: 1-4: Eimeria yemenii n. sp., X1600: 1- 2: Unsporulated fresh oocyst with condensed zygote.
3: Sporulated oocyst with 4 sporocysts. 4: Fully sporulated oocyst with oocyst residuum.
Figs. 5-8: Eimeria auriti Mirza, 1970, X1600:5: Unsporulated fresh oocyst. 6: Unsporulated oocyst with
central sporont. 7: Condensation of sporont or zygote before sporulation. 8: Sporulating oocyst.
List of Abbreviations; IL: Inner layer, MP: Micropyle, MPC: Micropyle cap, ORB: Oocyst residuum body,
OL: Outer layer, S: Sporozoite, SPC: Sporocyst, SPS: Sporoblast (s), Z: Zygote.
726
Table 1: Comparative data of Eimeria species from hedgehogs
Variables E. ostertagi,
Yokimoff and
Gousseff, 1936
E. perardii,
Yokimoff and
Gousseff,
1936
E. bijlikuli
Svanbaev, 1962
E. auriiti
Mirza, 1970
E. auriti
(present
study)
E. yemenii
n. sp.
(present
study)
Oocyst
Shape
Subspheroid-
ellipsoid
Ovoid
Spheroid to
Subspheroid
Subspheroid
Subspheroid
Ovoid to
ellipsoid
Size (m)
33.1 x 26.5
(27-41 x 22-37)
20 x 14.9
(17-27 x 15-
16)
32.4 x 26
(26.5-39 x 23.5-
31)
20.4 x 18.6
(17-23 x 15.5-
22)
20 x 17
(22-26 x 16-
19.5)
30 x 24.5
(27-36 x
20-28)
Shape index 1.2
1.3 1.2
1.1
1.17
1.2
Residuum Absent
Absent Present
Absent
Present
Present
Sporocyst
Shape
Spheroid to
ellipsoid
Ovoid to
ellipsoid
Subspheroid Ovoid
Subspheroid
Spheroid
Size (m)
11-12.2 x 11-
12.2
Unknown
11.6 x 8.9
(10-13 x 8-10)
10 x 7.3
(8.5-13 x 6.5-
8)
9 x 8.3
(8.5-12 x 7.5-
8
10 x 7.5
(11-12.2 x
10-11.2)
Shape index 1
Unknown 1.3
1.35
1.1
1.3
Residuum Absent
Absent Absent
Present
Present
Present
Sporulation
time
Unknown
Unknown
Unknown
14 days at 20-
22
o
C
2 days at 26
o
C
3-4 days at
26
o
C
Infection site Unknown Unknown Unknown Unknown Unknown Unknown
Host
Erinaceus
europaeus
E. europaeus
Hemiechinus
auritus
H. auritus
H. auritus
H. auritus
Distribution Russia
Russia Kazakhstan
Iraq
Yemen
Yemen
Discussion
In the present study, examina-
tions of 1175 fecal samples of
hedgehogs between 1984 & 1991
revealed Isospora oocysts in 17.9%
(Epe et al., 1993), and in 5.7% of
106 hedgehogs between 1998 &
2002 (Epe et al., 2004). Oocysts of
I. rastegaievae were predominant in
wild and domestic hedgehogs (Ba-
rutzki et al., 1987). I. rastegaievae
in feces were 1.4%-12.9% in wild
ones, 44.7% in animal homes and
32.3% in home pets (Barutzki et al.,
1987). The natural infection of
hedgehogs with Eimeria spp. may
reach 40% as recorded in this study.
Isospora spp. causes liquid feces
and occasionally bloody diarrhea
(Lowenstein et al., 1991). Fatal
cryptosporidiosis was in a juvenile
captive African hedgehog (Graczyk
et al., 1998). Molecular characteri-
zation of Danish C. parvum isolates
of hedgehog, E. europaeus showed
a sub-genotype distinct from hu-
man, bovine and porcine isolates
(Enemark et al., 2002).
Four species of genus Eimeria
from hedgehogs family Erina-
ceidae; E. ostertagi and E. perardii
were identified from Eurasian
hedgehog, E. europaeus from Rus-
727
sia (Yakimoff and Gousseff, 1936).
They presented two line drawings of
E. ostertagi; one was subspheroid
with spheroid sporocysts and a se-
cond ellipsoid with elongated spo-
rocysts. Probably they dealt with
two distinct species. Glebezdin and
Kolodenko (1969) reported E. oster-
tagi and E. perardii in Turkmenia.
Glebezdin (1985) reported E. oster-
tagi (H. auritus) from southeastern
Turkmenestan. E. bijlikuli
(Svanbaev (1962) was described
from Kazakhstan having a charac-
teristic comma or pear-shaped spo-
rozoites with a residual body at its
broad end. Since then it has not
been reported.
The present E. auriti is similar to
E. auriti described by Mirza (1975).
The oocyst and sporocyst sizes are
comparable; however, the present
one has a micropyle and oocyst re-
siduum. E. yeminii n.sp, differs
from E. auriti (Mirza, 1975) in sha-
pe and size of oocysts and sporo-
cysts, in sporulation time and in
having polar granules (Mirza,
1975). By comparing Eimeria spe-
cies described here with the previ-
ous species (Tab. 1), E. yeminii
proved to be an.sp. Based on host
type and geographic distribution, E.
ostertagi, E. perardii were found in
E. europaeus in Russia, E. bijlikuli
and E. auriti in H. auritus from Ka-
zakhstan and Iraq, respectively. E.
yeminii n.sp. is recorded for the first
time from H. auritus with Yemen as
a new locality.
Generally speaking, Egyptian
hedgehogs were naturally infected
with toxoplasmosis (Rifaat et al.,
1967), zoonotic rikettsia (Loftis et
al., 2006) and Hyalomma species
(Schuster and Horak, 2008). They
proved to be useful agent in cancer
researches (Wechsler-Reya, 2003).
Conclusion
Coccidia in hedgehogs may be
zoonotic; precautions should be
considered especially in the com-
mercially offered hedgehog-pets.
Further study is ongoing to deter-
mine pathogenicity particularly in
domestic ones.
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731
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 731 - 744
SEROPREVALENCE OF HUMAN TOXOCARIASIS
(VISCERAL LARVA MIGRANS)
By
ATEF M. El-SHAZLY
1
, S.M. ABDEL BASET
2
, A. KAMAL
3
, KHAIRY A.
MOHAMMED
4
, T.I. SAKR
5
AND S.M. HAMMAD
6
Departments of Parasitology
1,3,4
, Chest Medicine
2
, Hepatology, Gastro-
enterology and Infectious Disease
5
, and Community Medicine
6
, Facul-
ties of Medicine, Mansoura University
1,2,6
, El-Minia University
3
,
Al-Azhar University
4
and Benha University
5
, Egypt
Abstract
A total of 455 patients who fulfilled the inclusion criteria were included in
the study. The enrolled patients were subjected to a questionnaire (including
sociodemographic and other risk factors) and thorough clinical examination
was done for the patients. Sera were collected from patients and tested for anti-
Toxocara IgG antibodies using ELISA. The overall anti- Toxocara sero-
positive was (7.7%). It was significantly higher than among the randomly se-
lected 30 healthy controls. There were no significant differences between the
seropositive and seronegative patients regarding age, sex, educational level and
monthly family income of the patient. However, rural residence, poor house,
pets ownership and frequent contact with soil were found to be significant.
Patients who had confirmed bronchial asthma were more than 2 times at higher
risk of developing toxocariasis (OR, 2.33; 95% CI, 1.09-4.98) than those with
other clinical diagnosis (PUO, hepato-megaly or heptosplenomegaly, lympha-
denopathy, neurological disorders, gastrointestinal troubles and dermatitis).
Patients with eosinophilia were at 149 times greater risk of being Toxocara
seropositive compared to those without eosinophilia (OR, 148.7; 95% CI: 53.5-
413.3). Multivariate regression analysis showed eosinophilia and contact with
soil were the most important predictors of toxocariasis. OD of anti-Toxocara
antibodies (ELISA) was significantly positive with eosinophilia level.
Key words: Egypt, ELISA, Human toxocariasis, eosinophilia.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Human toxocariasis is one of the
most commonly reported zoonotic
infections worldwide (Strickland,
2000) and its global importance may
be greatly underestimated (Hotez
and Wilkins, 2009). Toxocariasis
results from zoonotic transmission
of the roundworms, Toxocara canis
and T cati from dogs and cats, re-
spectively. Human infections may
732
take place by ingestion of embryo-
nated eggs, or transfer (ingestion) of
encapsulated larvae of T canis is in
the tissues of a paratenic host (Gil-
lespie and Hawkey, 1995). In cer-
tain ethnic groups, some adults tend
to eat uncooked animal tissues
which contain encapsulated infec-
tive larvae. Uncooked livers of cow
(Chang et al., 2006; Known et al.,
2006), pig (Fan et al., 2004), lamb
(Salem and Schantz, 1992), and
chicken (Nagakura et al., 1989; Mo-
rimatsu et al., 2006) have been re-
ported as sources of human infec-
tions. After swallowing, the encap-
sulated larvae are liberated after
hatching in the small intestine, pe-
netrate the intestinal wall, get into
the portal vein, and then reach the
liver and lungs. They encapsulated
and remained alive for a certain pe-
riod. The resulting host inflammato-
ry response ultimately overwhelms
and either kills the migrating larvae
or forces them into a state of ar-
rested development, but not before
they cause both mechanical and tis-
sues immunopathological damage
(Fitzgerald and Mansfield, 1970).
Toxocara larvae secrete at least 50
distinct macro-molecules as excreto-
ry-secretory (E/S) antigens that ac-
counts for immune evasion and pa-
thogenesis (Nunes et al., 1999), and
are diagnosed by ELISA-E/S anti-
gens specific Igs (Dubinsky et al.,
2000) or western blot procedure
(Sarimehmetoglu et al., 2001). The
prevalence estimates for toxocaria-
sis are based on serological surveys
with banked sera that detect Tox-
ocara specific antibodies (Jones et
al., 2008; Won et al., 2008). ELISA
have high sensitivity and specificity
with very low levels of cross-
reactivity when the excretory and
secretory antigens of the second-
stage T. canis larvae were used
(Magnaval et al., 1991; 1992) VLM
sym-ptoms include hepatomegaly,
myocarditis, nephritis, epilepsy,
fever, urticaria, central nervous sys-
tem disorders, asthma, severe
breathing difficulty, wheezing
cough and abdominal pain. Labora-
tory findings include marked eosi-
nophilia, leukocytosis, hypergam-
maglobulinemia and liver function
disorders (Gillespie, et al., 1993;
Magnaval et al., 1993). There are
few studies on the association be-
tween asthma and Toxocara infec-
tion among adults. Feldman and
Parker (1992) have reported high
anti-Toxocara IgG levels in a 48-
year-old patient with hypereosino-
philic syndrome and asthma attacks.
Minvielle et al. (1999) found ele-
vated levels of anti-Toxocara IgG in
asthmatic patients.
Globally, high rates of toxocaria-
sis have been noted in middle-
income countries, with prevalence
up to 40% or higher in Indonesia
and Brazil (Noordin et al., 2005; De
Andrade et al., 2005). In Egypt tox-
ocariasis showed seroprevalence
rate of 18.1% in patients with tox-
oplasmosis (Azab et al., 1990),
10.7% of children with renal
troubles in Sharkia (Nada et al.,
1996), 6% of children with hepato-
megaly in Zagazig (Hassan et al.,
733
1996), and 6.2% in children with
respiratory symptoms or PUO and
18% in adults with PUO in Tanta
(Antonios et al., 2008).
This study was conducted to de-
termine seroprevalence of human
toxocariasis in patients attending
Mansoura and Benha Universities
hospitals and to iden-tify factors
associated with infection.
Subjects, Materials and Methods
This study was conducted from
May to December 2008, and com-
prised 455 patients aged 1-67 years
presented to Pediatric, Internal Med-
icine, Derma-tology, Chest, Tropi-
cal Medicine and Neurology de-
partments of the two Universities
Hospitals. Patients were enrolled
after obtaining verbal informed con-
sent from them or from their parents
or guardians in case of diseased
children. Exclusion criteria were
history of allergy, hepatitis B or C,
maligna-ncy, autoimmune diseases,
and systemic steroid therapy or im-
munotherapy.
The patients or their parents or
guardians in case of diseased child-
ren were directly interviewed using
a questionnaire to assess the risk
factors associated with Toxocara
infection. Pets ownership, frequent
contact with soil and the socio-
economic status of the family were
considered as the risk factors for
acquiring toxocariasis. The family
socio-demographic status was as-
sessed by patients education, fami-
ly income and house type. Houses
were classified as good or poor de-
pending on the materials used for
roof, floor and walls.
All were subjected to thorough
clinical examination. Bronchial
asthmatic patients were subjected to
pulmonary function tests including
peak expiratory flow arte and spi-
rometry reversibility test to confirm
asthma (GINA, 2006).
Blood samples were collected
from all patients and submitted to:
number of eosinophils, was meas-
ured by a chamber technique and
expressed as 10
9
/L. Values from
0.4-0.9x 10
9
/L were classified as
low eosinophilia, between 1.0-3.0x
10
9
/L as high eosinophilia and over
3.0x 10
9
/L as very high eosinophi-
lia. Blood samples were centrifuged
and sera stored at -20
o
C until tested.
AntiToxocara antibody was de-
tected by a commercial ELISA kit
(Bordier Affinity Products SA,
Crissier, Switzerland). The test is
based on E/S antigens of Toxocara
larvae. The optical density (OD)
was read at 490 nm using ELISA
reader. Each plate had a positive
control pooled sera from patients
diagnosed as visceral larva migrans
(VLM) syndrome and a high anti-
body level, and a known negative
serum sample. The mean OD of the
negative control was 0.234 0.159.
The cut off value (0.711) was calcu-
lated as the mean negative control
3 SD. Some cross reactions may
occur in other human helminthiasis,
such as trichinosis, fascioliasis, and
strongyloidiasis, titers in the hel-
minthiasis were lower than in posi-
734
tive control of toxocariasis patients
(Jacquier et al., 1991).
Cases were also subjected to urine
and stool examination, IHA for
Fasciola, Schistosoma and Toxop-
lasma, blood film, hepatitis viral
markers and auto-antibody tests for
autoimmune diseases to exclude
cases with other parasitic diseases,
viral hepatitis and autoimmune dis-
eases.
Statistical analysis: Data were
analyzed with statistical package
SPSS version 15.0. Association be-
tween anti-Toxocara antibodies in
patients and characters (epidemio-
logical, clinical, and eosinophilia)
was studied by univariate analysis
with unadjusted odds ratios (ORs).
Variables significant upon univa-
riate were analyzed by multivariate
logistic regression using maximum
likelihood ratio test. Based on step-
wise forward method, considering p
=0.05 for model entry and p = 0.10
for removal. Significance was con-
sidered at P < 0.05.
At the time of the study there
was no Ethical Review Board in the
Faculty of Medicine, Mansoura or
Benha University, Egypt, so Ethical
clearance was obtained from the
different departments sharing in the
study.
Results
The overall seroprevalence of
human anti-Toxocara IgG antibo-
dies among studied patients was
7.7% (Tab. 1). It was significantly
higher than among the 30 randomly
selected healthy controls (P<
0.0001). There were no significant
differences between the seropositive
and seronegative patients in respect
of age, sex, educational level and
monthly family income of the pa-
tient. However, rural residence, poor
house, pets ownership and frequent
contact with soil were found to be
significantly correlated with positive
serology for anti-Toxocara IgG an-
tibodies. The seroprevalence of hu-
man anti-Toxocara IgG antibodies
among patients in relation to clinical
presentation and eosinophilia was
presented (Tab. 2). Patients with
confirmed bronchial asthma were
more than 2 times at higher risk of
developing toxocariasis (OR, 2.33;
95% CI, 1.09-4.98) than those with
other manifestations (PUO, hepato-
megaly or hepato-splenomegaly,
lymphadenopathy, neurological dis-
orders, gastrointestinal trou-bles or
dermatitis). Patients with eosinophi-
lia were at 149 times greater risk of
Toxocara seropositive compared to
patients without eosinphilia (OR,
148.7; 95% CI: 53.5-413.3).
The significant variables that cor-
related with positive serology for
anti-Toxocara antibodies (rural resi-
dence, poor housing, pets owner-
ship, frequent contact with soil),
together with confirmed asthma and
eosinophilia, were submitted to
multivariate analysis. In the final
model, only the frequent contact
with soil and eosinophilia remained
significantly correlated with Tox-
ocara infection, indicating a higher
risk of infection for individuals with
735
eosinophilia (odds ratio = 154.45,
95% CI = 53.3 to 447.8) and fre-
quent contact with soil (odds ratio =
3.0, 95% CI = 1.02 to 8.82) (Tab.
3). The association between OD of
ELISA-IgG and eosinphilia showed
significant positive correlation be-
tween them (r= 0.77; P <0.001)
(Tab. 4).
Table 1: Anti-Toxocara IgG related to sociodemography and characters of pa-
tients and ownership to pets and frequent contact with soil:
Patient characteristic Total No (%) Positive No (%) P value OR (95% CI)
Age (years):
1-<15
15+
99 (21.8)
356 (78.2)
10 (10.1)
25 (7.0)
0.309 1.44 (0.71-2.89)
1
a
Male
Female
280 (61.5)
175 (38.5)
24 (8.6)
11 (6.3)
0.79 1.36 (0.69-2.71)
1
a
Rural
urban
276 (60.7)
179 (39.3)
27 (9.8)
8 (4.5)
0.038* 2.19 (1,02-4,71)
1
a
Illiterate/read and write
primary through preparatory
Secondary+
147 (32.3)
180 (39.6)
128 (28.1)
15 (10.2)
12 (6.7)
8 (6.3)
0.237
0.884
-
.63 (0.72-3.72)
1.07(0.45-2.53)
1
a
Housing poor
Housing good
160 (35.2)
295 (64.8)
18 (11.3)
17 (5.8)
0.036* 1.95 (1.03-3.68)
1
a
Income not satisfactory
income satisfactory
300 (65.9)
155 (34.1)
28 (9.3)
7 (4.5)
0.068 2.06 (0.92-4.62)
1
a
Pet ownership: Yes
No
65 (14.3)
390 (85.7)
9 (13.8)
26 (6.7)
0.044
*
2.25 (1.0-5.05)
1
a
Frequent contact with soil:
Yes
No
137 (30.7)
318 (69.9)
18 (13.1)
17 (5.3)
0.004
*
2.68 (1.34-5.37)
1
a
1
*
= reference category, * significant, CI= confidence interval
Table 2: Anti-Toxocara IgG related to patients clinical picture & eosinophilia:
Patient characteristic Total No (%) Positive No (%) P value OR (95% CI)
Pyrexia of unknown origin: No
Yes
400 (87.9)
55 (12.1)
31 (7.8)
4 (7.3)
0.901 1
a
0.99 (0.92-1.08)
Hepato or hepatosplenomegaly: No
Yes
375 (82.4)
80 (17.6)
28 (7.5)
7 (8.8) 0.696
1
a
1.19 (0.5-2.83)
Lymphadenopathy: No
Yes
385 (84.6)
70 (15.4)
32 (8.3)
3 (4.3)
0.245 1
a
0.49 (0.15-1.66)
Neurological disorders: No
Yes
415 (91.2)
40 (8.8)
31 (7.5)
4 (8.8)
0.566 1
a
1.38 (0.46-4.12)
Bronchial asthma: No
Yes
375 (82.4)
80 (17.6)
24 (6.4)
11 (13.8)
0.025
*
1
a
2.33 (1.09-4.98)
Gastrointestinal troubles: No
Yes
395 (86.8)
60 (13.2)
32 (8.1)
3 (5.0)
0.401 1
a
0.59 (0.17-2.01)
Dermatitis: No
Yes
385 (84.6)
70 (15.4)
32 (8.3)
3 (4.3)
0.245 1
a
0.49 (0.15-1.66)
Eosinophilia: No
Yes
416 (91.4)
39(8.6)
7 (1.7)
28 (71.8)
0.0001
*
1
a
148.7(53.5-413.29)
736
Table 3: Multivariate logistic regression analysis of significant predictors of
anti-Toxocara positivity
Patient characteristic P value OR (95% CI)
Frequent contact with soil 0.045
*
3 (1.02-8.82)
Eosinophilia 0.001
*
154.45 (53.26-447.8)
Constant
Model x
2
Percent correctly classified
0.001
*
133.45
96.0
Table 4: Correlation between of ELISA-IgG and eosinophilia.
Eosinophilia No (%)
N=455
ELISA
Less than 0.7
No (%)
0.7-0.9
No (%)
>0.9-1.1
No (%)
>1.1
No (%)
No
Low
High
Very high
417 (91.6)
21 (4.6)
13 (2.9)
4 (0.9)
410 (98.3)
10 (47.6)
0 (0.0)
0 (0.0)
6 (1.4)
3 (14.3)
0 (0.0)
0 (0.0)
1 (0.2)
8 (38.1)
5 (38.5)
1 (25.0)
0 (0.0)
0 (0.0)
8 (61.5)
3 (75.0)
Spearman's rank correlation 0.769
P value 0.001
*
Discussion
Human toxocariasis is an impor-
tant and common zoonotic disease
worldwide. Demographic and so-
cioeconomical factors may lead to
increase in Toxocara seropreval-
ance. Infections were generally
asymptomatic, and sero-prevalence
varied from 3% to 86% in different
countries (Alderete et al., 2003).
Total seroprevalence rate of Tox-
ocara antibodies was 7.7% or lower
than 29.8% in Nigeria (Ajayi et al.,
2000), 38% So Paulo, Brazil (Al-
derete et al., 2003), 19%in Lebanon
(Kanafani et al., 2006) and 12.95%
in North West-ern Turkey (Nihal et
al., 2007). The results controversy
can be due to several factors; study
designs, heterogeneity of individu-
als (blood donors, hospitalized pa-
tients or toxocariasis high risk
group); non-standardized lab pro-
cedures and different cutoff points
in sero-diagnosis.
The present study did not find any
relationship between the seroposi-
tivity rate and sex difference and
age groups. Ajayi et al. (2000) and
Nihal et al. (2007) reported that nei-
ther age nor gender seemed to be
important factors related to a posi-
tive serology. However, literature
reported a predominance of T. canis
in malesthat may be due to differ-
ences in social behaviors of boys,
resulted in increasing exposure to
infection (Overgaauw, 1997; Ka-
nafani et al., 2006; Antonios et al.,
2008). Also, a predominance of se-
ropositivity in children was due to
the higher frequency of geophagia
in ages (Chieffi et al., 1990). Theo-
doridis et al. (2001) reported that
females were significantly more
infected than males and Genchi et
737
al. (1990) reported significant in-
crease of seropositivity with age.
In the present study on univariate
analysis we found higher Toxocara
seroprevalence in rural than urban
patients (P<0.005). This data agreed
with Nihal et al. (2007) and Anto-
nios et al. (2008). But, Alavi and
Sefidgaran (2008) in Iran found
similar Toxocara sero-positive be-
tween rural and urban.
In the present study, significant
association between Toxocara posi-
tivity and socioeconomic indicators
such as level of school and head
family income probably due to the
fact that the differences in educa-
tional level and family income be-
tween selected patients were small.
But, Alderete et al. (2003) and Ka-
nafani et al. (2006) found signifi-
cant association due to bad hygienic
conditions, and other factors asso-
ciated to low income, facilitate
transmission of Toxocara as well as
concomitant diseases.
Controversy exists regarding the
importance of pets ownership as a
risk factor for toxocariasis, a higher
frequency of infection in persons in
contact with dogs (Schantz et al.,
1980; Nihal et al., 2007; Antonios et
al., 2008). Others did not find any
association between ownership of
dogs and Toxocara infection (Gen-
chi, et al., 1990; Ajayi et al., 2000).
In the present study, there were sig-
nificantly higher Toxocara seroposi-
tivity, among pet owners than non-
owners of pets (13.8% & 6.7% re-
spectively) but this statistical sig-
nificant difference disappeared on
multivariate regression analysis.
The present study revealed that
frequent contact with soil is signifi-
cantly associated with a higher risk
of Toxocara infection; a finding in
line with that reported by others
(Genchi et al., 1990; Mizgajska,
1997). This showed that the main
source of toxocariasis was environ-
mental contamination by eggs.
In humans, three clinical forms of
toxocariasis were recognized, vis-
ceral larva migrans, ocular larva
migrans and covert toxocariasis
(Pawlowski, 2001). Visceral toxoca-
riasis can present clinically as bron-
chial asthma, neurological manife-
stations, hepatomegaly, dermatitis,
and pyrexia of unknown origin (Ho-
tez, 1993; Humbert et al., 2000;
Marx et al., 2007). Pulmonary
symptoms are common in toxoca-
riasis (Taylor et al., 1988; Buijs et
al., 1994, 1997; Chan et al., 2001;
Kuk et al., 2006) and repeated ex-
posure to Toxocara can trigger al-
lergic manifestations, including
bronchospasm, in response to the
migrating larvae or their E/S prod-
ucts.
In the present study, the highest
sero-positive was among bronchial
asthma patients (13.8%) followed
by hepatomegaly (8.8%). Glickman
et al. (1987) studied the frequency
of different signs and symptoms for
37 patients with serologically prov-
en VLM; they found that 19
(51.4%) patients had allergic ma-
nifestations including pruritis, rash
or chronic urticaria. Specific treat-
738
ment with fluoromebendazole
showed clinical improvement in ten
patients. Buijs et al. (1994) investi-
gated the relation between Toxocara
positivity and allergic asthma in
Dutch school children aged 4-6
years. They found that recurrent
bronchitis/asthma occurred signifi-
cantly more often in Toxocara sero-
positive than in the negative group;
yet the relation with eczema was
marginally significant. Bouchard et
al. (1994) reported a case of acute
sever asthma which with the exis-
tence of positive T. canis serology
as well as a contamination risk of
the patient in the domestic environ-
ment led them to integrate the clini-
cal picture into larva migrans syn-
drome. It has also been reported that
asthma patients showed a seroposi-
tive rate of 9.3% to Toxocara ES
antigen (Choi et al., 2003). Hassan
et al. (1996) tested 300 children suf-
fering from hepatomegaly in both
Sharkia and El-Minia Govemorates
by ELISA against Toxocara anti-
gen. They found an incidence of 6%
among the tested group. Antonios et
al. (2008) examined the sero-
prevalence of T. canis in 150 Egyp-
tian patients with presumptive clini-
cal syndromes. One hundred twenty
eight were children suffering from
respiratory symptoms or PUO and
22 were adults with PUO. Anti-
Toxocara-IgG were detected in sera
by ELISA. Sero-positivity was 6.2%
in children (4% &13.3% with respi-
ratory symptoms and PUQ respec-
tively). In adults, 18% were anti-
Toxocara IgG positive that may be
attributed to more outdoor activity.
In Korea, of 97 healthy people with
over 10% eosinophilia, 60% was
positive to Toxocara larvae excreto-
ry-secretory (TES) antigen by both
immunoblot and ELISA (Park et al.,
2002). In Europe, the seropositive
rate is 2 to 5 % in apparently
healthy urban adults, while the rate
is 14.2% to 37% in rural areas
(Magnaval et al., 1994). In India, of
94 people of the general population
and 30 patients with clinically sus-
pected toxocariasis, 6 (6.4%) and 7
(23.3%), respectively, were seropo-
sitive to Toxocara (E/S) antigen
(Malla et al., 2002). In Egypt, Is-
mail and Khalafallah (2005) stated
that toxocariasis is a common para-
site worldwide with high sero-
prevalence rates even among
asymptomatic individuals and that
diagnosis revealed a wide scope of
clinical syndromes, including der-
matological diseases and chronic
urticaria specially among those ex-
posed to an increased risk of envi-
ronmental toxocariasis.
The causes of eosinophilia are
known to be mainly allergic diseas-
es, parasitic diseases, cancer, and
rheumatic diseases. Among the pa-
rasitic diseases, toxocariasis may be
the most common cause of eosino-
philia.
Consistent with previous reports
(Aguiar-Santos et al., 2004; Nash,
2005), our study revealed that 28/39
(71.8%) patients with eosinophilia
were seropositive. Also, eosinphilic
patients were at 149 times greater
risk of being Toxocara seropositive
739
compared to patients who had no
eosinphilia (OR, 148.7; 95% CI:
53.5-413.3) and eosinophilia was
the most important predictor of
Toxocara infection on multivariate
regression analysis. Gueglio et al.
(1994) tested 1836 eosinophilic pa-
tients by ELISA E/S Toxocara anti-
gen and found positive results in
29% of cases. In Korea, out of 127
sera from patients with eosinophilia
(more than 500 /ul or more than or
equal to 10% WBC) who visited the
hospital, 70 sera (68%) were posi-
tive to ES antigen and patients with
lesions in the liver showed higher
serum eosinophil cationic protein
(ECP) values (Kwon et al., 2005).
Toxocara larvae induced an eosi-
nophilic response followed by the
presence of specific IgG anti-
Toxocara in sera within 2-3 weeks.
During the process of destruction of
larvae in tissue, which is a slow one
up to years, the circulating eosino-
phils decrease but anti-Toxocara
remains for a longer time? Another
cause for the lack of eosinophils in
sero-positive individuals may be the
low intensity of infection. Only in
fully developed VLM syndrome,
diagnosed clinically, can intensity of
infection assumed to be high. The
intensity of infection might be as-
sessed indirectly and imprecisely
only by measuring the sero-
prevalence rates and the values of
titers or O.D. of the serological tests
(Luzna-Lyskov, 2000). However,
Kim et al. (2008) did not find sero-
positive toxocariasis in population
with eosinophilia (more than or
equal to 10% WBC)
To detect a current active infec-
tion of toxocariasis, IgE needs to be
tested by ELISA and an assessment
of eosinophil cationic protein (ECP)
is required. People that test positive
may have covert toxocariasis, so
that a screening for toxocariasis
among those with eosinophila
should be done. This would prevent
misdiagnosis of toxocariasis as idi-
opathic hypereosinophilic syn-
drome, which may result in inap-
propriate clinical interventions.
In Egypt, many authors dealt with
zoonotic toxocariasis as Oteifa and
Mous-tafa, 1997), El Shazly et al.
(2002a, b; 2009). Elsheikha et al.
(2008) studied the kinetics of eosi-
nophilia and IgE production in ex-
perimental murine toxocariasis. Ha-
ridy et al. (2009) reported toxoca-
riasis in street and in pet dogs.
Conclusion
Toxocara seroprevalence was
much higher in studied patients es-
pecially those with bronchial asthma
(13.8%) than in the general popula-
tion (0.0%). These results suggest
that bronchial asthma presents a
high risk for Toxocara infection
specially if associated with eosino-
philia. A large population-based
survey is needed to assess the mag-
nitude of the problem and identify
more details about different risk
factors.
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745
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009

J. Egypt. Soc. Parasitol., 39 (3), 2009: 745- 756

STUDIES ON MOSQUITO BORNE DIEASES
IN EGYPT AND QATAR

By
MICHEAL.W. MIKHAIL
1
, KHALIFA M. AL-BURSHEED
2
,
AZZA S. ABD EL-HALIM
1
, AND TOSSON A. MORSY
3

Research Institute of Medical Entomology, Ministry of Health, Dokki,
Giza, Egypt
1
, Pest control Division, Health affairs, Qatar
2
and Depart-
ment of Parasitology, Faculty of Medicine, Ain Shams University Cairo
11566
3
, Egypt

Abstract
Mosquitoes identification, distribution and densities in representative Egyp-
tian Governorates and five Qatarain Municipalities (Al Rayyan, Doha, Al
Daayan, Al Khor & Al Zahakira and Al Shamal) were studied. In Qatar the
larvae were Culex pipiens complex, C. univettatus, C. pusillus, Aedes caspies,
Anopheles multicolor and A. stephensi. C. pipiens and C. univettatus were
mainly in Al Rayyan, Doha, and Al Khor & Al Zahakira. C. pusillus was in
Doha and Al Daayan while A. caspies was in Al Daayan and Al Shamal.
Anopheles multicolor and A. stephensi were mainly in Al Shamal with low
density in Al Rayyan (only in Ain- Khalid locution).
The Egyptian mosquitoes were C. pipiens, C. antennatus, C. thelerei, C.
univittatus, C. perexiguus, C. poicilipes, C. pusillus, Aedes caspius, Ae. detri-
tus, A. sergentii, A. pharoensis, A. multicolor, A. detali, A. algeriensis, A. tene-
brosus, A. gambiae (formerly), A. superpictus, A. tarkhadi, A. hispaniola, A.
rhodesiensis, A. stephensi, A. coustani and Culiseta longiareolata.
As an example in Sharkia Governorate, larvae were C. pipiens (68.77%), Ae.
caspius (15.75%), Culiseta sp. (=Theobaldia) and C. pusillus. In Greater Cairo,
parts of Qualyoubia G., C. pipiens was the most dominant and the least was C.
perexiguus. In parts of Giza G., C. pipiens was the most dominant and least
was Cs. longiareolata. In Cairo G., C. pipiens was the most dominant and least
was Ae. caspius. The overall in Greater Cairo was C. pipiens (61.74%),
Cs. longiareolata (15.56%), Ae. caspius (15.3%), C. pusillus (4.0%) and
C. perexiguus (3.16%).
Key words: Egypt, Qatara, Culex species, Anopheles species, Aedes species.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------



746
Introduction

Members of order Diptera are by
far the most important order of the
Class Insecta as regards Tropical
Medicine and Infectious Diseases
(Wilson, 1991). At least 3500 spe-
cies of mosquitoes are known allo-
ver the world and being found in
almost every country (Stone, 1977).
At least 3500 mosquitoes species
are known allover the world and
found in almost every country
(Robert, 2001). They have a world
distribution throughout the tropical
and temperate regions and extend
their range northwards into the Arc-
tic Circle. They are found at eleva-
tions of 5500 m and down at depths
of 1250 m below sea level (Service,
1996). They are the most important
vectors of human diseases and the
most serious bloodsucking arthro-
pods that feed on amphibians, rep-
tiles, birds, and mammals. Some
species exhibit considerable host
specificity, while others have more
catholic tastes (Lane and Crosskey,
1993). Surprising through it seems,
the world faces almost as great a
threat today from arthropod-borne
diseases as it did in the heady days
of the 1950s when diseases eradica-
tion by controlling vectors with syn-
thetic insecticides seemed to be a
real possibility (Lane and Crosskey,
1993).
Family Culicidae includes the
flies, which are called mosquitoes, a
word that is derived from the Span-
ish meaning little flies. This family
includes 2 sub-families Culicinae
(Linnaeus 1790) and Anophelinae
(Meigen 1818). Culicinae contains
33 genera. The mosquitoes of med-
ical and economic importance are
Culex, Aedes, Haemagogus, Psoro-
phora, Mansonia, Sabethes and
Anopheles.
C. pipiens plays the main role in
transmission of filariasis bancrofti
as in Saudi Arabia (Sebai et al.,
1974) and Egypt (Harb et al., 1993)
and Rift Valley Fever (El Gebaly,
1978), West Nile virus (Wilson,
1991), and Sindbis fever as in Saudi
Arabia (Wills et al., 1985) and
Egypt (Monath, 1991), and causes
biting nuisances (Morsy et al.,
2003). Hassan et al. (2002; 2003)
reported that Egyptian C. pipiens
complex from homes of HCV pa-
tients or among the same organs of
symbiotic and aposymbiotic mos-
quitoes fed on HCV positive blood
by an artificial membrane feeder,
HCV-RNA was detected in heads of
the indoor symbiotic mosquitoes at
3 & 6 hrs post-feeding. HCV was
detected at 3
rd
day & 8
th
day in the
gut. HCV transmission by mosquito
did not yet prove, but paves the way
to study the gut-bacteria of C.
pipiens for anti-HCV agent.
Approximately 300 millions of the
world population is infected with
malaria and up to 1.5 million pa-
tients die by every year (WHO,
1998).


747
C. pipiens complex was highly
distributed mosquito not only in
Egypt and Qatar but also world-
wide, as it breeds in pools, sub-
canals, swamps, water containers
and even in any water collection.
The work aimed at studying the
mosquito fauna in Egypt and Qatar,
species, distribution, densities and
vector-borne diseases.

Materials and Methods

The collection of Egyptian larvae
was in Greater Cairo, Sharkia,
Menoufia, Suez, El Faiyoum and
Assiut, and in Qatar was in Doha
(the capital), El Rayan, Al Daayen,
Al Khour, Al Zakira and Al Shamal.
Larval surveys were done on geo-
graphical/topographical sampling
bases for fauna, distribution, breed-
ing sites and environmental factors.
Larvae were collected from seep-
age, drains, wells, cesspits, and the
likes by dipping and netting (Morsy
et al., 2003). At least two month
visits was randomly paid to each
site. Identification was carried out
using the standard keys (Kirkpat-
rick, 1925, Gad, 1963; Habach,
1985; Lane and Crosskey, 1993;
Morsy et al., 1990; 1995; 2004).

Results
The results are shown in tables (1 and 2).
Table 1: Mosquitoes in Egypt by selected publications

Authors species genus
Kirkpatrick, 1925
Gad, 1963
Kenawy, 1988,1990
Morsy et al., 1995a,b
algeriensis Anopheles
detali
gambiae (formerly)
hispanioal
multicolor
pharoensis
rhodesiensis
sergentii
stephensi
superpictus
tarkhadi
tenebrosus (= coustani)
Kirkpatrick, 1925
Gad, 1963
Harbach et al.,1988
Morsy et al., 1990, 2003, 2004
Mostafa, 2002
El-Bashier et al., 2006
antennatus Culex
perexiguus
pipiens
poicilipes,
pusillus
quinquefasciatus
thelerei
univittatus
Gad, 1963
Mostafa et al., 2002
Morsy et al., 2003, 2004
aegypti (formerly) Aedes
caspius
detritus
Mostafa et al., 2002
Morsy et al., 2003, 2004
longiareolata Culiseta (=Theobaldia)

Table 2: 3
rd
to 4
th
larval instars of mosquitoes collected from Qatar (2006).


748
Munici
pality
Locality Species
Sites
No.
3
rd
&
4
th
larva
Pupa
No.
Total
No.
larval &
pupa/ dip
PH
value
Temp.

o
C.
Al-
Rayyan
Al Sailiyah
C. univettatus
C. pipiens
40 1288 249 1537 38.4 9.1 29.4
Ain Khalid
A. multicoler
A. stephensi
8 66 22 88 11 6.7 26.7
Abu Nakilah
C. univettatus
C. pipiens
95 5890 881 6771 71.3 9.2 27.6
Shaniya
C. univettatus
C. pipiens
8 568 82 650 81.3 8.9 28.4
Messeaid C. univettatus 28 62 14 76 2.7 8.1 29.


Doha
Misaimeer
C. pipiens
C. univettatus
C. pusillus
75 2475 392 2867 38.2 8.8 28.
Dahal
C. pipiens
C. univettatus
6 148 29 177 29.5 9.3 30.
Neagaa C. pipiens 12 1092 284 1376 114.7 9.3 30.4
Al
Daayen
Wadi Al
Wosail
C. pipiens
C. pusillus
Ae. caspics
20 224 41 265 13.3 7.8 28.5
Umm Qarn C. pipiens 4 27 4 31 7.8 9.2 31.2
Al Khor
& Al
Zhakira
Ras laffan
C. pipiens
C. univettatus
9 112 21 133 14.8 8.6 28.4
Al Zhakira
C.pipiens
C. univettatus
17 232 29 261 15.4 9.3 30.5
Al Khor
C. pipiens
C. univettatus
70 432 105 537 7.7 9.1 29.8
Al-
Shamal
Al Areesh
C. pipiens
Ae. caspius
25 119 21 140 5.6 6.8 29.9
Al. Mam-
lahah
A. multicolor 15 88 16 104 6.9 8.4 27.7
Al. Kharee-
jah
A. multicolor 30 92 9 101 3.4 8.3 27.5
Al-
Jassasiyah
A. multicolor 20 31 4 35 1.7 8.4 27.9

Discussion

In the present study, Qatarian
mosquito's fauna comprised six in-
digenous species representing 5 zo-
ogeographical Municipalities fau-
nas: C. pipiens, C. univettatus. C.
pusillus, Ae. caspies, A. multicolor
and A. stephensi. Mosquito surveys
showed that C. pipiens and C. uni-
vettatus were mainly present inAl
Rayyan, Doha and Al Khor & Al
Zakhira. C. pipiens had the highest
density in all Municipalities. C. pu-
sillus was found in Daha and Al
Daayan. Ae. caspies was found in
Al Daayan and Al Shamal. The pre-
sent results also showed that A. mul-
ticolor and A. stephensi were mainly
present in Al Shamal and had low
density in Al Rayyan (Ain Khalid
location). Micheal et al. (2007)
found that C. pipiens complex in
Qatar is the most important insect


749
high a incidence and prevalence due
to availability of pools sewage,
swamps for breeding water.
In Egypt, Kenaway (1988) studied
anopheline mosquitoes in malaria
trans-mission in Egypt. Anopheline
species were A. algeriensis, A. tene-
brosus, A. pharoensis, A. sergentii,
A. gambiae, A. detali, A. multicolor,
A. superpictus, A. tarkhadi, A. his-
paniola, A. rhodesiensis and A. ste-
phensi. Kenaway (1990) reported
that A. pharoensis and A. sergentii
are proven vectors. A. multicolor
and A. superpictus are suspected
vector. Others; A. stephensi, A. de-
tali are vector abroad but their role
in Egypt was not yet determined at
that time.
Morsy et al. (1988; 1990) studied
Culex species in Suez Canal Gover-
norates. Morsy et al. (1995) in El
Faiyoum G. concentrated only on
genus Anopheles where malaria was
endemic, identified A.pharoensis, A.
sergenti, A. multicolor and A. tene-
brosus (= coustani).
Mostafa et al. (2002) studied the
abundance and distribution of mos-
quito species monitored by three
phases. The first was in 1999 in five
governorates, Qalyobia, Menoufia,
Behaira, El Fayium and Assuit. The
second was in 2000 in Kafr El
Sheikh, Giza, Sharkia, Menia and
Aswan. The third was in 2001 in
Kena, El Wady El Gadeed, Dakahl-
ia and South Sinai. Culex species
were the commonest mainly C.
pipiens, C. antennatus and C. univit-
tatus. C. thelerei was found only in
El Kharga Oasis. Culiseta sp. was
found in Qalyoubia, Menou-fia,
Behaira, El Fayium, El Wady El
Gadeed, Dakahlia and South Sinai
and as larvae in Kafr El Sheikh, Gi-
za, and El Menia. Aedes detritus
was found in Assiut, El Fayium,
Giza, Aswan, El Wady El Gadeed
and South Sinai. Ae. caspius was
found in Assiut and Aswan and as
larvae in Kena and El Wady El
Gadeed. A. pharoensis was found in
Behaira and El Fayium, while A.
algeriensis in Aswan. A. multicolor
and A. sergentii were found in El
Fayium, Aswan and El Wady El
Gadeed; but in Kena A. sergentii
was found as larvae and A. multi-
color as adults.
Morsy et al. (2003, 2004) in Qal-
youbia Governorate (G.) reported
four mosquito larvae in a fixed site
during August 2002. These were C.
pipiens (52.08%), Cs. longiareolata
(27.08%), C. perexiguus (12.5%)
and Ae. caspius (8.33%). In Decem-
ber 2002, the collected larvae from
same site were only two species; C.
pipiens (64.7%) and Ae. caspius
(35.29%). This indicated that C.
pipiens was the most common and
most predominant species followed
by Ae. caspius. Besides, C. perex-
iguus and Cs. longiareolata were
found only in August. C. pipiens
and Ae. caspius have a bimodal life
cycle, while C. perexiguus and Cs.
longiareolata have a unimodal life
cycle. In Giza G. four species of
mosquito larvae were encountered


750
during August 2002. In a descend-
ing order were C. pipiens (64.6%),
C. pusillus (15.92%), Ae. caspius
(11.5%) and the least was Cs. longi-
areolata (7.96%). During December
2002, from the same site only two
species were recovered; C. pipiens
(69.69%) and Ae. caspius (30.3%),
but neither C. pusillus nor Cs.
longiareolata was detected. The
overall recovered larvae showed
that C. pipiens was the most domi-
nant one (65.75%), then Ae. caspius
(15.75%), C. pusillus (12.32%) and
lastly Cs. longiareolata (6.16%).
In Cairo G., only two species
were detected during August 2002;
C. pipiens (61.9%) and, Cs. longi-
areolata (38.09%). During Decem-
ber of the same year, C. pipiens
were (69.56%) and Ae. caspius were
(30.43%). The overall number of
larvae was C. pipiens (63.95%), Cs.
longiareolata (27.9%) and Ae. cas-
pius (8.13%). This proved that C.
pipiens has a bimodal life cycle,
while Cs. longiareolata has a uni-
modal life cycle.
In Greater Cairo during August
2002, five species were recovered.
They were C. pipiens (59.5%), Cs.
longiareolata (21.68%), Ae. caspius
(8.36%), C. pusillus (6.61%) and C.
perexiguus (4.41%). During De-
cember of the same year, only two
species were collected C. pipiens
(67.28%) and Ae. caspius (32.71%).
In Greater Cairo five larvae were
detected C. pipiens (61.74%), Cs.
longiareolata (15.56%), Ae. caspius
(15.3%), C. pusillus (4%) and C.
perexiguus (3.16%).
Apart from Egypt and Qatar,
mosquitos species in Arabian Gulf
Countries was reported. In Saudi
Arabia, Sebai et al. (1974) reported
bancroftian filariasis and C. pipiens
in southern region. Rodhain et al.
(1976) reported human filariasis in
part of Asia, but otherwise filariasis
only east of Pakistan. He added that
the largest focus for intensity of in-
fection and for size of population
was in India. Wills et al. (1985) iso-
lated sindbis virus from C. univitta-
tus, C. tritaeniorhynchus and C.
pipiens complex. Harbach (1988)
recognized twenty species of Culex
of subgenus Culex in southwestern
Asia and Egypt. They were C.
pipiens, C. quinquefasciatus, C. ve-
gans, C. torrentium, C. decens, C.
antennatus, C. univittatus, C. perex-
iguus, C. theileri, C. laticinctus, C.
mattinglyi, C. simpsoni, C. sinati-
cus, C. duttoni, C. sitiens, C. poi-
cilipes, C. mimeticus, C. bi-
taeniorhynchus, C. tritaeniorhyn-
chus and C. pseudovishani. Abdul-
lah and Merdan (1995) reported
mosquitoes in the south-western
district. They were A. arabiensis, A.
sergentii, A. multicolor, A. tene-
brosus, C. pipiens, C. quinquefasci-
atus, C. theileri, Ae. caspius and
Culiseta subochrea. Abdoon and Al
Shahrani (2003) sampled 180 sites
in the malaria endemic areas of Asir
Province for Anopheles. Seven spe-
cies were identified, A. dethali, A.


751
rupicolus, A. sergentii, A. ara-
biensis, A. multicolor, A. turkhudi
and A. pretoriensis. A. arabiensis
and A. sergentii were known malar-
ia vectors. Goddsey et al. (2003)
reported that A. (stegomyia) unilin-
eatus was the first species record
from Asir, Jizan, and Makkah, and
known malaria-vector in Africa,
Pakistan and India. Abdoon (2004)
identified three species as new
country record; C. duttoni, C. de-
cens and C. bitaeniorhynchus.
In Libya, Vermeil (1953) identi-
fied A. broussessi in Ferran. Amer
and Mehl-horn (2006) studied the
effect of plant extracts against lar-
vae of Ae. aegypti, A. stephensi and
C. quinquefasciatus.
In Yemen, Kuznetsov (1971) dis-
covered A. coustani and A. squamo-
sus. Kravchenko (1979) showed that
malaria was recorded in the inhabit-
ants of the six landscape zones. A.
gambia, A. dethali, A. sergenti, A.
rupicolus and A. gambiae were the
main vector. Muqbil and Subhasini
(1999) identified A. arabiensis and
A. culicifacies as vectors of P. falci-
parum in endemic malaria areas. Al-
Maktari and Bassiouny (1999)
found three anopheline species in
Al- Hodeidah Governorate. A. ara-
biensis was the most prevalent spe-
cies (84.2%), followed by A. culic-
ifacies adenensis (14.9%), then A.
rhodesiensis (0.9 %).
In Oman, Beidas and Gillies
(1980) described the hitherto un-
known egg of A. culicifacie, A. ad-
enensis christophers. Chadee and
Bennett (1988) identified eggs of C.
sitiens. Irving et al. (1991) reported
that in Al- khod Oman, when gran-
ite rock pools were artificially
flooded during the long (4-5
months) dry season, Ae. vittatus lar-
vae appeared. The source of these,
whether from gravid females or
from eggs surviving in desiccated
state? Roberts (1996) showed that in
Barka vicinity 4 species were abun-
dant in concrete reservoir tanks con-
taining brackish water that ranged
from 16 to 39% sea water. They
were C. sinaiticus, C. quinquefasci-
atus, C. sitiens, and A. stephensi.
Roberts and Irving-Bell (1997)
identified 16 mosquito species in
southern Oman. The commonest
ones were C. sitiens (35%), C. tri-
taeniorhynchus (32%) and C.
sinaticus (11%). Aedeomyia fur-
furea, A. sergentii, Ae. aegypti, Ae.
albopictus and Ae. vittats were
found only in fresh water. A. ste-
phensi was in brackish water and C.
sitiens in salt water. All other spe-
cies were found in salinity water up
to 34%. The majority (75%) of the
larvae were found in microhabitats
separate from the main water body.
The 3 Aedes species and C. laticinc-
tus were restricted to rain-filled rock
hollows, C. sitiens to mud pools.
Footprints were the main habitat of
A. dethali, A. sergenti, C. quinque-
fasciatus and C. sinaiticus. A.
coustani and Aedeomyia furfurea
were found in the main water body


752
among the emergent vegetation.
Shidrawi and Gillies (1987) de-
scribed A. paltrinierii from Oman
and the United Arab Emirates.
In Iran, Manouchehri et al. (1976)
found that A. stephensi mysoremsis
was an important malaria vector in
Southern region.
Again, in Egypt, C. pipiens is the
main vector of filariasis which has
natural and artificial breeding sites
in endemic and non-endemic villag-
es (Harb et al., 1993). The relative
importance of the indoor-vector has
a significant risk factor in the
transmission of W. bancrofti (Gad et
al., 1994). Many factors may be
responsible for the increase of C.
pipiens, in spite of its control
measures of which, poor sanitation,
the continuous floating of liquid
waste and sewage every where the
presence of water in roofs of many
new buildings which create good
breeding places (Mahdi et al., 1963;
Farid et al., 1997). The larval stages
may become more resistant to the
usual insecticides used. Other spe-
cies of mosquitoes were recovered,
C. antennatus (Gad et al., 1987), C.
univittatus, Theobaldia longiareola-
ta and Ae. caspius (Gad, 1963); but
C. pipiens were predominant 99.5%
(Mohamed et al., 1981). Under la-
boratory conditions, Rifaat et al.
(1971) found that C. antennatus
may transmit filaria. Turell et al.
(1996) evaluated the ability of Ae.
caspius, C. pipiens, C. antennatus,
C. perexiguus, C. poicilipes, and A.
pharoensis collected in Aswan and
C. pipiens to transmit RVF virus
reintroduction into Egypt in 1993.
All mosquito species were suscepti-
ble to RVF virus infection, with A.
pharoensis and Ae. caspius being
the most sensitive to infection. But,
none of 12 A. pharoensis, including
10 with a disseminated infection,
transmitted RVF virus by bite. In
contrast, nearly all C. pipiens (87%,
n=15) and C. perexiguus (90%,
n=10) with a disseminated infection
transmitted virus. Overall trans-
mission rates for mosquitoes ex-
posed to hamsters with a viremia >
or=10(7) plaque-forming units/ml
were Ae. caspius, 20% (n=5); C.
pipiens, 7% (n=102); C. antennatus,
7% (n=30); C. perexiguus, 11%
(n=9); and A. pharoensis, 0% (n
=7). Based on abundance, suscepti-
bility to infection, ability to transmit
virus, and feeding behavior, Ae.
caspius was the most efficient vec-
tor, while less susceptible than Ae.
caspius, C. pipiens, C. antennatus,
and C. perexiguus were also poten-
tial vectors during this RVF out-
break in Egypt.
Apart from filariasis, Culicini,
mainly C. pipiens transmit Rift Val-
ley fever (El Gebaly, 1978), Sindbis
virus (Wilson, 1991) & C. pipiens
complex was incriminated as vector
of HCV (Hassan et al., 2002, 2003).
Jamjoom et al. (2006) in Saudi Ara-
bia documented the endemicity of
malaria particularly malignant type.
El-Bahnasawy and Morsy (2008)


753
stated that human babesiosis is not
in mind at least in the Middle East-
ern Countries where many parasitic,
bacterial and viral diseases are en-
countered. Erroneous interpretation
of the blood film was confused with
malaria, mainly Plasmodium falci-
parum due to the abundant small
rings within the RBC.

Conclusion

No doubt, in the Arab countries
there is a marked civilization and
modern establishment of housing,
roads' network, agricultural and in-
dustrial projects particularly in new-
ly reclaimed areas. Nevertheless,
Culicinae and Anophelines mosqui-
toes are still encountered world-
wide, playing an important role in
the mosquito-borne diseases. The
Public Health and Veterinary au-
thorities must find out feasible con-
trol measures against the adult and
the immature stages of mosquitoes.

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757
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 757 - 768
ELISA-BASED COPROANTIGEN IN HUMAN STRONGYLOIDIAISIS:
A DIAGNOSTIC METHOD CORRELATING WITH WORM BURDEN
By
AYMAN A. EL-BADRY
Department of Parasitology, Faculty of Medicine, Cairo University,
Egypt, Mobile: 0020108300075, email: aymanbb@hotmail.com
Abstract
In order to overcome the false negative diagnosis of strongyloidiasis in the
absence of rhabditiform larvae in stools, an ELISA-based Strongyloides ster-
coralis-specific coproantigen detection assay in stools of infected patients was
evaluated. In a sandwich ELISA, a rabbit hyperimmune serum against S. ster-
coralis ES (excretory/secretory) adult antigen succeeded in capturing S. ster-
coralis coproantigen from infected patients and did not react with copro-
antigens prepared from the stool samples of patients infected with Schistosoma
mansoni, Fasciola gigantica and Capillaria philippenensis. Coproantigen was
able to detect anti-S. stercoralis IgG antibodies in sera of infected patients at
the same OD level as produced with S. stercoralis E/S worm antigen using an
indirect ELISA did not cross-react with sera from patients with S. mansoni, F.
gigantica and C. philippenensis. S. stercoralis coproantigen detection proved a
sensitive, simple, reliable and inexpensive ELISA-based, and an alternative to
coproscopical methods in copropositive (with larvae in stool) and copro-
negative (without larvae in stool) stool samples. Fecal ELISA showed a posi-
tive relationship between copro-Ag and worm burdens, and considered a start-
ing point for the development of species-specific copro-immunological diag-
nostic assays using monoclonal antibodies and dipstick technology.
Key Words: Stongyloides stercoralis, Fecal ELISA, Coproantigen.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Strongyloidiosis is a chronic intes-
tinal helminthiasis caused by S.
stercoralis; a soil-transmitted intes-
tinal nematode that infects humans
worldwide with higher incidences in
tropical and subtropical areas
(Grove, 1989). Prevalence esti-
mates range from 0.5- 4% among
population in the Southern Unit-
ed States to 40-80% in tropical
Africa and South America
(Grove, 1990).
S. stercoralis is unique in its abil-
ity to reinvade the same host by au-
to-infection and retrofection. Alt-
hough most strongyloidiosis cases
are asymptomatic, they occasionally
758
become severe and complicated,
involving different organs and de-
veloping into hyperinfection if
chronically infected persons become
immuno-suppressed. Studies have
linked hyperinfection with various
immuno-suppressive conditions
such as malignant tumours, severe
malnutrition (Neva, 1986), steroid
therapy (Cruz et al., 1966; Genta,
1992), organ transplantation
(Phelps, 1993), and concomitant
HTLV-1 (Gotuzzo et al., 1999;
Newton et al., 1992). Prisoners of
the 2
nd
World War living in non-
endemic areas revealed that human
infection persisted for > 40 years
(Grove, 1980). The chronic cases
were difficult to diagnose as they
were usually asymptomatic with
low num-bers or no larvae in feces.
In Egypt, strongyloidiosis has been
reported by many authors (Wilson,
1991; Massoud et al., 2006).
Diagnosis of strongyloidiosis is
based on microscopic examination
of stool specimens for the presence
of larvae; however, negative results
do not rule out infection. Owing to
the intrinsic low intensity of infec-
tion; excretion of larvae is sporadic
and scanty, and local transmission
factors such as environment and
socio-economic status make the
probability of finding larvae low
(Grove, 1980; Dreyer et al., 1996;
Neva, 1986; Genta, 1989; Conway
et al., 1995; Grove, 1996; Appleton
et al., 1999; Uparanukraw et al.
1999). A single stool examination
failed to detect larvae in up to 70%
cases (Siddiqui and Berk, 2001).
Dreyer et al. (1996) recommended
that fecal examinations should be
done at least 4 times. Concentration
techniques such as the Baermann
method, Harada Mori culture, and
the Entero-test was established. De-
layed diagnosis, tedious follow-up
of patients, and handling by labora-
tory personnel are the main disad-
vantages. However, the develop-
ment of agar culture plate technique
(ACPT) (Arakaki et al., 1990; Koga
et al., 1991) improved detection of
S. stercoralis proved superior to
other stool examinations (de Ka-
minsky, 1993; Sato et al., 1995;
Jongwutiwes et al., 1999).
Several immunological methods
were developed and antigenic com-
position of different larval Strongy-
loides sp. using different techniques
(Grove and Blair, 1981; Sato et al.,
1985, Genta, 1988; 1920; Sato et
al., 1990; Conway et al., 1993; Lin-
do et al., 1994; Neva et al., 2001;
Rodrigues et al., 2001). The widely
used one is indirect ELISA that de-
tected S. stercoralis-specific-IgG
(Neva et al., 1981; Sato et al., 1985;
Gam et al., 1987; Conway et al.,
1993).
The current study evaluated the
ELISA-based S. stercoralis-specific
copro-antigen in the stool, as a non-
invasive, simple immunodiagnostic
method for human strongyloidiasis
in absence of larvae in stool.
Materials and Methods
All cases were chosen from the
Cairo University hospital and pri-
759
mary health care centers in Giza.
Forty-eight subjects were included
eighteen with strongyloidiaisisof
whom ten had larvae in stool and
eight were seropositive by ELISA
based-S. stercoralis L3 Ag but
without larvae in stool, fifteen cases
for detection of cross-reactivity
(five with each of Schistosoma
mansoni, Fasciola gigantica and
Capillaria philippenensis) and fif-
teen parasite free individual as a
healthy control group. Informed
consents were taken from them.
Three stool samples were collect-
ed on consecutive days and stored in
plastic receptacles without preserva-
tives at 4
o
C. Copro-parasitological
diagnoses were carried out within 3-
6 hours of collection. A part of each
fresh stool sample was kept in a
small tube at -70
o
C for coproantigen
preparation. Venous blood (5 ml)
was drawn and allowed to clot at
room temperature for an hour. Then
serum was separated and put into
small tubes in a separate icebox at -
20
o
C until used.
Stool analysis was carried out
within 3-6 hours using the direct
smear test (DST), the modified for-
malin-ethyl acetate technique
(MFEAT) according to Elikins et al.
(1990) and modified Baermann`s
method (MBM) proposed by Her-
nndez-Chavarra and Avendao
(2001) to detect S. stercoralis larvae
(El-Badry, 2004) in which the fun-
nel with the hose and Mohr clamp
was replaced by a 16x100 mm test
tube with a rubber stopper that is
perforated to allow introduction of a
plastic pipette tip. 2 g feces was put
in the tube containing 8 ml of saline
solution and missed well. The tube
was sealed with the tipped rubber
stopper, inverted on top of another
tube containing 6 ml of saline solu-
tion at 37C and incubated in a
37C water bath for at least 2 hours.
The second tube was centrifuged
and the sediment was examined by
light micro-scope. The worm bur-
dens, larvae per gram of stool were
calculated for copropositive stools
of patients infected with S. ster-
coralis using MBM.
S. stercoralis ES (excretory/secr-
etory) adult worm antigen (Ag)
preparation:
S. stercoralis adult worms were
harvested from collected rhab-
ditifrom larvae (recovered from fae-
ces of infected patients) by the mod-
ified Harada Mori technique on fil-
ter-paper (Sato et al., 1995) and
they were used to prepare ES adult
worm Ag (Richard et al. 2005);
briefly adults were washed several
times with sterile 0.05 M phosphate-
buffered saline (PBS) at pH 7.2 and
used to prepare ES products by in-
cubation in sterile PBS (10
worms/ml) for six hours at 37
0
C.
The worms were removed and the
raw ES products were centrifuged at
3300xg for 15 minutes to remove
particulates. The supernatant was
aspirated, protein content was de-
termined (Lowry et al., 1951), di-
vided into aliquots and stored at -
70
o
C until used.
Copro-antigen was prepared from
stools of S. stercoralis (Coproposi-
760
tive) infected cases (El-Bahi et al.,
1992). The stools were mixed with
an equal amount of PBS (PH.7.4),
sieved through a double layer of
gauze, homogenized for 3 minutes
in a homogenizer, sonicated for 5
minutes in an ice bath and centri-
fuged at 10,000 rpm for ten minutes.
The supernatant was aspirated and
its protein content was estimated.
The antigen was aliquoted and
stored at -70
o
C until used.
Coproantigens for cross-reactivity
with patients sera were prepared
from stools of cases with S. man-
soni, F. gigantica and C.
philippenensis.
Rabbit hyperimmune sera were
prepared against the adult worm ES
Ag and copro-Ag of S. stercoralis
(Langley and Hillyer, 1989) with
modification. Copro-Ag prepera-
tions was sterilized by filteration
through vacuum-operated Steriflip
filter with Durapore membrane
(Millipore Corporation, Billerica,
USA). A primary dose of Ag was
adjusted to contain 400g protein
and mixed in an equal volume of
Freund's incomplete adjuvant and
injected deeply intramuscular into a
rabbit followed by three consecutive
booster doses of the Ag, each con-
tains 100g protein and mixed in an
equal volume of Freund's complete
adjuvant, at weekly intervals. The
immunized animals blood was col-
lected 10-14 days after the last in-
jection and serum was separated and
stored at -70C.
An indirect ELISA of coproanti-
gens and adult worm ES S. ster-
coralis Ag (Uparanukraw et al.,
1999) was used to test sera from all
cases against S. stercoralis copro-
Ag. Copro-Ags prepared from the
stools of cases infected with S. man-
soni, F. gigantica, C. philippinensis
and parasite free individuals (as
positive and negative controls) were
tested. Standardization and assay
development were done in polysty-
rene microplates. The wells were
sensitized with the Ag preparation
solution at 4C overnight at a pro-
tein conc. of 2.0 g/ mL in car-
bonate buffer 0.05 M, pH 9.6. Ex-
cess antigen was washed 3 times
with 0.05% Tween 20 in PBS, at pH
7.4. Excess binding sites were
blocked with 200l of bovine serum
albumin (2% PBS/0.1%Tween-20)
and incubated for 30 min at 37C.
The wells were washed 3 times,
100l of the sera were added to each
well (dilution 1:1500 PBST in du-
plicate) and incubated at 37C for 1
hr. After 3washings, 100l of
1:5000 dilution of horseradish pe-
roxidase conjugate goat anti-human
IgG (Zymed, San Francisco, CA,
USA) in PBST were added and in-
cubated at 37C for 1 h. Wells were
washed 3 times and 100l of the
substrate solution (0.4 mg/ml of o-
phenylenediamine, 0.001% H
2
0
2
in
citrate buffer, pH 5.0) was added.
After 15 min at 37C, reaction was
stopped by adding 100l of 4 N
H
2
SO
4
. OD was measured by mi-
croplate reader at 490 nm wave
length. Cut-off OD for a positive
reading was equal to mean value
plus 3 SDs required with negative
761
control sera. OD> 0.482 was con-
sidered positive.
S. stercoralis copro-Ag and copro-
Ags was prepared from stools of
cases infected with S. mansoni, F.
gigantica, C. philippinensis and
parasite free individuals were tested
against two different laboratory pre-
pared anti-S. stercoralis hyperim-
mune sera of rabbits (Rose et al.,
2002). The plates were coated with
100ul/well of tested rabbit anti-S.
stercoralis hyperimmune serum in
carbonate/bicarbonate buffer (pH-
7.4) overnight (required concentra-
tion of IgG was obtained after
checkerboard titration). After wash-
ing, 100l/well from each Ag (di-
luted 1: 1 in PBS-Tween 20) were
added in 2 separate rows and incu-
bated at room temperature for 2
hours. After another wash, horse-
raddish peroxidase conjugated goat
anti-rabbit IgG (Sigma) diluted 1:
100 in PBS was added in 100l/well
and the plates were incubated again
for 1 hour then washed before the
addition of 100l of the phenylene-
diamine substrate to each wel1. The
reaction was allowed to proceed for
15 minutes at room temperature in
dark and stopped by addition of 1 M
sulphuric acid in 100l /well. Posi-
tive results were evaluated as be-
fore.
Statistical analysis: Data was ana-
lyzed by a computer using SPSS
(statistical package for social sci-
ences; SPSS XI II program version
XIII, Inc., Chicago, Illinois) for data
entry and statistical analysis.
Results
Coproantigen obtained from stool
samples of copropositive S. ster-
coralis cases reacted with sera of
patients infected with S. stercoralis
in direct ELISA with a mean O.D of
0.786-0.683 (copropositive &
copronegative respectively). Read-
ings were lower than in S. ster-
coralis ES Ag and same serum
samples 0.832 & 0.711 (coproposi-
tive & copronegative respectively)
without signi-ficant difference.
Coproantigen proved specific with-
out cross reactions with sera of oth-
er parasites and control (tab. 1).
Copro-Ag from stool samples of
copropositive S. stercoralis cases
reacted with anti-S. stercoralis
copro-Ag hyperimmune rabbit sera
in fecal ELISA with a mean value
0.869. The readings were lower than
in reaction of S. stercoralis copro-
Ag with anti-S. stercoralis ES Ag
hyperimmune rabbit sera in fecal
ELISA with mean value 0.856 but
without significant. No reaction oc-
curred between C. philippinensis
copro-Ag, F. gigantica copro-Ag, S.
mansoni copro-Ag and copro-Ag
from control (-ve) versus both anti-
S. stercoralis copro-Ag (fig. 1) and
ES Ag hyper-immune sera (fig. 2)
by ELISA. To assess quantita-tive
relationship between S. stercoralis
copro-Ag and intestinal S. ster-
coralis burden, individual fecal
samples recovered from ten S. ster-
coralis patients, differing in rhab-
ditifrom larval number recovered
from stool by MBM, were analyzed
762
by fecal ELISA. There was a posi-
tive correlation between number of
excreted larvae in stool and O.D
values of faecal ELISA (fig. 3).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20
Cases
O
.
D

v
a
l
u
e
Cut off point
S. stercoralis CA
C. philippinensis CA
F. gigantica CA
S. mansoni CA
CA from control (-ve)
Linear (Cut off point)
Fig. 1: Coprantigens (CA) reactivity Vs rabbit hyperimmune sera against S. stercoralis CA using
faecal ELISA.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20
Cases
O
.
D

v
a
l
u
e
Cut off point
S. stercoralis CA
C. philippinensis CA
F. gigantica CA
S. mansoni CA
CA from control (-ve)
Linear (Cut off point)
Fig. 2: Coprantigens (CA) reactivity Vs rabbit hyperimmune sera against S. stercoralis ES Ag
using faecal ELISA.
0
1
2
3
4
5
O.D of Copro+ve
No. of larvae/gram stool
Fig. 3: O.D value of faecal ELISA to detect S. stercoralis coprantigen/number of larvae in stool
of infected patients.
763
Table 1: Reactivity of sera of cases assayed with S. stercoralis ES Ag, S. ster-
coralis Copro-Ag and Copro-Ag from control by ELISA.
Antigen
S. stercoralis C. philip-
pinensis
F. gigan-
tica
S. man-
soni Control Copro+ve Copro-ve
S
.
s
t
e
r
c
o
r
a
l
i
s
E
S

A
g
Mean O.D 0.832 0.711* 0.285 ** 0.238 0.239 0.199
No. of +ve 10/10 7/8* 1/5** 0/5 0/5 0/15
SD 0.341 0.275 0.031 0.012 0.017 0.006
DF 2 2 2 2 2 2
P value 0.090 0.077 0.005 0.001 0.002 <0.001
S
.
s
t
e
r
c
o
r
a
l
i
s
C
o
p
r
o
-
A
g
Mean O.D 0.786 0.683 0.233 0.252 0.235 0.207
No. of +ve 10/10 8/8 0/5 0/5 0/5 0/15
SD 0.197 0.159 0.018 0.007 0.010 0.003
DF 2 2 2 2 2 2
P value 0.290 0.011 0.033 0.043 0.011 <0.001
C
o
p
r
o
-
A
g
f
r
o
m

c
o
n
-
t
r
o
l
Mean O.D 0.220 0.221 0.229 0.228 0.208 0.196
No. of +ve 0/10 0/8 0/5 0/5 0/5 0/15
SD 0.006 0.003 0.001 0.003 0.004 0.000
DF 2 2 2 2 2 2
P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
* one negative case. **a case showed cross reactivity, An OD sample greater than 0.482 = positive
DF: Degree of freedom, SD: Standard deviation, P value: significant at 0.01 level (2-tailed).
Table 2: Reactivity of different in coproantigens versus anti-S. stercoralis
Copro-Ag and ES Ag hyperimmune sera by fecal ELISA.
Antigen
S.stercoralis
Copro-Ag
C. philippinensis
Copro-Ag
F. gigantica
Copro-Ag
S. mansoni
Copro-Ag
Copro-Ag
control (-ve)
F
e
c
a
l

E
L
I
S
A

u
s
i
n
g

h
y
-
p
e
r
i
m
m
u
n
e

s
e
r
a

a
g
a
i
n
s
t
S
.
s
t
e
r
c
o
r
a
l
i
s
C
o
p
r
o
-
A
g
Mean O.D 0.869 0.206 0.241 0.260 0.214
No. of +ve 10/10 0/5 0/5 0/5 0/15
SD 0.193 0.095 0.059 0.105 0.045
DF 9 4 4 4 11
P value <0.001 0.008 0.001 0.005 <0.001
S
.
s
t
e
r
c
o
r
a
l
i
s
E
S

A
g
Mean O.D 0.856 0.224 0.232 0.237 0.186
No. of +ve 10/10 0/5 0/5 0/5 0/15
SD 0.231 0.086 0.084 0.064 0.030
DF 9 4 4 4 14
P value <0.001 0.004 0.003 0.001 <0.001
DF: Degree of freedom, SD: Standard deviation, P value: significant at 0.01 level (2-tailed)
Discussion
Strongyloidiasis is a chronic hu-
man intestinal nematode infection
with an ability to reinvade the same
host (autoinfection), a unique char-
acteristic of S. stercoralis and C.
philippinensis among nematodes,
with a tendency to persist for dec-
ades, usually as asymptomatic cas-
es. Strongyloidiasis remains a major
global health challenge for which
novel diagnostic methods would be
expected to improve epidemiologi-
cal studies and control efforts. Be-
cause of the difficulty of obtaining
sufficient amounts of S. stercoralis
filariform larvae, heterologous anti-
gens from S. stercoralis were used
as a source of antigens. It has be-
come necessary to standardize the
source of antigen which can be used
764
as reliable source of antigens for
immunodiagnostic in human stron-
gyloidiasis, thus replacing heterolo-
gous S. stercoralis antigen. In this
study, a simplified and inexpensive
protocol was developed to detect of
S. stercoralis by copro-antigen fecal
ELISA that represented an attractive
diagnostic alternative to fecal larva
assays. Copro-antigen ELISAs have
previously been reported for nema-
todes such as Haemonchus contor-
tus (Ellis et al., 1993) and Telador-
sagia circumcincta (Johnson et al.,
2004) in sheep, Ostertagia ostertagi
in cattle (Agneessens et al., 2001),
Heligmosomoides bakeri (Johnson
et al., 1996) and Trichinella spiralis
(Boulos et al., 2001) in mice, S. rat-
ti in rats (Nageswaran et al., 1994),
hookworms in hamster (Richard et
al., 2005) and C. philippinensis in
human (El-Dib et al., 2004). Copro-
antigen obtained from stool samples
of copropositive S. stercoralis cases
reacted with the sera of all patients
infected with S. stercoralis (copro-
positive and copronegative) in a
direct ELISA. ODs were lower than
in the reaction between S. ster-
coralis ES Ag and same serum
without significant difference. The
coproantigen was more specific as
no cross reactions occurred neither
with sera of other parasites nor with
sera of control group. El-Bahi et al.
(1992) reported that the specificity
of coproantigens may be due to the
nature of the antigens which lack
one or more of the cross reacting
epitopes, which usually lead to high
ELISA OD values and occurrence
of cross reactions with other related
antibodies. Copro-Ag obtained from
stool samples of copropositive S.
stercoralis cases reacted with anti-S.
stercoralis copro-Ag rabbit hy-
perimmune sera in fecal ELISA
without cross reaction with C. phil-
ippinensis copro-Ag, F. gigantica
copro-Ag, S. mansoni copro-Ag and
copro-Ag from control (-ve) versus
both anti-S. stercoralis copro-Ag &
ES Ag hyperimmune sera by fecal
(sandwich) ELISA.
The current results showed that
fecal ELISA offered a means for
diagnosis of low intensity S. ster-
coralis not detected coproscopical-
ly, allowing for accurate estimation
of S. stercoralis prevalence. The
results also showed a positive rela-
tionship between fecal ELISA ODs
and S. stercoralis burden, and the
assay gave a positive diagnosis
when the reactivity of S. stercoralis
copro-Ag was assayed against sera
of S. stercoralis copropositive and
copronegative cases. The data
agreed H. contortus (Ellis et al.,
1993), T. circumcincta (Johnson et
al., 2004), and hookworms (Richard
et al., 2005) suggesting that estima-
tion of S. stercoralis worm burdens
in humans proved possible by fecal
ELISA and represent an attractive
alternative to methods that rely on
extrapolation from larval counts or
recovery of expelled worms after
treatment (Anderson and Schad,
1985). Also, fecal ELISA enabled
detection of single coproantigens
and made it possible to develop spe-
cies-specific diagnostic assays using
765
monoclonal antibodies and dipstick
technology (Allan et al., 1993).
Measure-ment of coproantigens by
fecal ELISA gave new insights in
the molecular pathogenesis of
strongyloidiasis by examining the
excretion kinetics of individual
coproantigen, and detection of pre-
patent infection.
Conclusion
A simple and inexpensive fecal
ELISA-based method for detection
of S. stercoralis copro-Ag repre-
sents an attractive diagnostic alter-
native to faecal larva assays, and
would improve epidemiologic stud-
ies and control efforts. The fecal
ELISA demonstrated a positive rela-
tionship between copro-Ag and
worm burdens. In addition, to serv-
ing as a starting point for the devel-
opment of novel future immunodi-
agnostic methods, the adaptation of
the fecal ELISA to study single
copro-Ags provided new insight in
molecular pathogenesis of infection
and to develop species-specific di-
agnostic assays by mono-clonal an-
tibodies and dipstick technology.
Acknowledgement
The author would like to thank the
staff and technical members of Cai-
ro University hospital and Giza
health care units for kindly supply-
ing the cases and Faculty of Veteri-
nary Medicine, Cairo University, for
kindly supplying all laboratory fa-
cilities. To Dr. Nadia A. El-Dib,
Professor of Parasitology, De-
partment of Medical Parasitology,
Faculty of Medicine, Cairo Univer-
sity for her valuable help, support
and kindly supplying the C.
philippenensis cases. Dr. Khaled
Heissam, Lecturer of Family Medi-
cine, Primary Care Division, Not-
tingham University, UK kindly re-
vised the statistical analysis.
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769
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 769- 787
IDENTIFICATION, CLONING AND EXPRESSION OF CORE
PROTEIN GENE OF HEPATITIS C GENOTYPE 4A
By
SALWA F. SABET
1
, MAGED M. AL-SHERBINY
2
AND PAUL HAGAN
3
Department of Zoology, Faculty of Science, Cairo University
1, 2
Division of Infection and Immunity, Joseph Black Building (B4-09d), In-
stitute of Biomedical and Life Sciences, University of Glasgow, Glasgow
G12 8QQ, UK
3
Abstract
Although Egypt has very high rates of HCV, not much is known about geno-
type 4a which is the most predominant genotype in Egypt. In the present study,
core (C_ED43) gene of the Egyptian strain ED43 of HCV genotype 4a was
first analyzed using PC/GENE program. Computer analysis of Core region of
the isolate ED43 revealed that the Egyptian genotype 4a is different from those
isolated from Europe and Central Africa and that it is closely related to geno-
type 1b. The DNA region coding for the Core was amplified from
HCV_ED43/PUC19 plasmid. The PCR product was then cloned and expressed
in E. coli M15 using pQE-30 vector. The expression and antigenicity of the
core (Core_4a) protein in E. coli was confirmed by SDS-PAGE and western
blotting, which will make it useful for developing assay systems for detecting
anti-HCV antibodies and HCV antigen, respectively and which might help in
the design of a vaccine against the Egyptian genotype 4a.
Keywords: HCV, Genotype 4a, Egyptian strain ED43, E. coli (M15), Core_4,
C_ED43, Cloning, Expression
-----------------------------------------------------------------------------------------------
Introduction
Hepatitis C virus (HCV) infection
is a major public health problem in
Egypt (Hibbs et al., 1993; Abdel-
Wahab et al., 1994). Prevalence of
antibodies to HCV (anti-HCV) is
approximately 10-fold greater in
Egypt than in the United States and
Europe (Strickland et al., 2002).
There is no vaccine against HCV so
far, recent advances in biotechnolo-
gy have improved the understanding
of HCV immunology and brought to
light several new challenges. An
effective HCV vaccine must be able
to prevent infection or the progres-
sion of infection, protect against the
predominant genotypes within the
geographic region of use, and be
safe and effective for widespread
administration (Strader and Seeff,
2001). Although Egypt has a very
770
high prevalence of HCV and a high
morbidity and mortality from chron-
ic liver disease, cirrhosis, and hepa-
to-cellular carcinoma, not much is
known about genotype 4a which is
the most predominant genotype in
Egypt (Ray et al., 1994; El-Sadawy
et al., 2004).
Core protein is considered as a
major target in HCV vaccine re-
search as the core protein-encoding
sequence is among the most con-
served genes in HCV genome
(Bukh et al., 1994; Liu et al., 2002).
Also, core protein has several func-
tions other than its role in nucle-
ocapsid formation. In addition to the
viral translational regulation of the
core protein (Shimoike et al., 1999),
it plays a role in the regulation of
cellular transcription, the modula-
tion of cellular signal transduction
pathways, cellular transformation,
apoptosis and immuno-suppression
(Lai and Ware, 2000; Jin et al.,
2000). The nucleotide sequence
(573 bp) of the core protein-
encoding region is well conserved
(homology of more than 90%) be-
tween virus strains, but about 10%
variation at the nucleotide level is
seen between different genotypes
(Bukh et al., 1995) The HCV core
protein (21-23 kDa) is the most N-
terminal protein of the viral poly-
protein, whose cleavage from the
nascent polyprotein is mediated by
host-signal peptidase (Yasui et al.,
1998).
In the present work, the DNA se-
quence of core region was analyzed
using PC/GGENE program and will
then be amplified from
HCV_ED43/PUC19 plasmid. The
PCR product will then be cloned
and expressed in M15 strain of E.
coli using pQE-30 vector. The spe-
cific HCV antigenicity of the
Core_4a fusion protein will be iden-
tified by western blotting.
Materials and Methods
Sequence analysis of the core
gene in HCV genotype 4a genome:
Isolate ED43 of HCV genotype 4a
DNA genome (HCV_ED43) insert-
ed in PUC19 vector was kindly pro-
vided by Dr. Richard M. Elliott lab
(Institute of Virology, University of
Glasgow, Church Street, Glasgow).
The complete nucleotide sequence
of HCV_ED43 which is 9355 nu-
cleotide long is deposited in the
EMBL database under accession
number Y11604.
PC/GENE software programs (In-
telligentics, Inc.) were employed for
the analysis of the nucleic acid se-
quence data and the deduction of the
amino acid sequences of the core
(C_ED43) gene of HCV_ED43. The
PC/GENE analysis programs have
been used to determine the open
reading frame, restriction enzymes
cleavage sites as well as the prima-
ry, secondary and tertiary structure
analysis of the deduced amino acid
sequences. The nucleotide of the
C_ED43 was subjected to similarity
search using standard nucleotide-
nucleotide BLASTN option of the
internet (http://www.ncbi.nlm .gov/
BLAST) (Altschul et al., 1997).
Multiple DNA and protein align-
771
ments of C_ED43 with some of the
isolates which showed a high degree
of homology were done using
PC/GENE program.
Amplification and cloning of
C_ED43 gene: The original DNA
concentration of the HCV_ED43
genome was 0.98g/l and was di-
luted to a final concentration of
0.1g/l by adding 10 l of DNA to
90 l sterile distilled water (dH
2
O).
Amplification and cloning strategies
of the C_ED43 and gene was done
(Sambrook et al., 1989) and as men-
tioned in the QIAexpressionist kit
instruction manual. DNA sequences
coding for whole core protein was
amplified from PUC19/HCV_
ED43. The sequence of the forward
and reverse primers was used:
C_ED43F primer: 5'-
CGCGGATCCAGCACGAATCCT
A AACCTCAA-3'.C_ E D43R pri-
mer: 5'CGCAAGC
TTGGCCGAAGCGGGGA
CAGTCAG-3'. The amplified re-
gion was cloned into pQE30 cloning
and expression vector between the
BamHI and HindIII Sites.
Expression and purification of
core (Core_4a) fusion protein: Re-
combinant M15 cells were grown
overnight at 37C in 20 ml of Lu-
riaBertani (LB) broth (1% tryp-
tone, 0.5% yeast extract, 1% NaCl)
containing 100g/ml ampicillin, 25
g/ml kanamycin (LB
kan,amp
). The
20 ml culture was inoculated into 1
litre of LB
kan,amp
media and cultured
at 25 C with vigorous shaking for 1
h. Expression of recombinant pro-
tein was induced by adding isopro-
pyl b-D-thiogalactoside (IPTG) to a
final concentration of 1 mM. After 4
h of induction, cells were harvested
by centrifugation 4000 rpm for 20
min and frozen overnight at 20 C
until used for purification.
Preparation of cleared lysates was
done under denaturing conditions on
Ni2+-nitrilotriacetate (NTA)aga-
rose (Qiagen) according to manu-
facturers instructions. The cell pel-
let was thawed for 15 min on ice
and resuspended in buffer B (100
mM NaH
2
PO
4
, 10 mM TrisCl, 8 M
urea, pH to 8.0) at 5 ml/g wet
weight (25 ml buffer was added to 5
g cells). The cells were gently vor-
texed for 20 min at RT until the so-
lution became translucent. The ly-
sate was centrifuged at 10,000 rpm
for 30 min at RT to pellet the cellu-
lar debris. The supernatant (cleared
lysate) was saved and the cell debris
was discarded. Five ml of the 50%
Ni-NTA slurry was added to 25 ml
lysate and mixed gently by shaking
(200 rpm on a rotary shaker) for 60
min at RT. The lysateresin mixture
was carefully loaded into an empty
column with the bottom cap still
attached. The bottom cap was re-
moved and the flow-through was
collected. The column was washed
twice with 4 ml buffer C (100 mM
NaH
2
PO
4
, 10 mM TrisCl, 8 M urea,
pH to 6.3). The recombinant protein
was eluted by adding 2.5 ml buffer
D (100 mM NaH
2
PO
4
, 10 mM
TrisCl, 8 M urea, pH to 5.9) 4 times
and followed with 2.5 ml buffer E
(100 mM NaH
2
PO
4
, 10 mM TrisCl,
8 M urea, pH to 4.5) 4 times. A 10l
772
5 X SDS-PAGE sample buffers was
added to 40 l of each elution and
stored at 20C for SDS-PAGE
analysis. The samples were analysed
in 12% SDS-PAGE. The protein
content of purified samples was
measured using BioRad protein as-
say reagent (BioRad)
For western-blot analysis, proteins
were electrotransferred onto nitro-
cellulose membrane (Wattman). The
membrane was blocked with 1%
bovine serum albumin in Tris-
buffered saline (TBS, 20 mM Tris-
Cl, pH 7.5, 500 mM NaCl) for 2 h at
RT with shaking. The membrane
was then incubated with anti-HCV
pooled human sera (from Egyptian
blood donors infected with HCV),
washed, then incubated with second
antibody [horseradish peroxidase
(HRP)-labelled Protein A (Sig-
ma)]for 1 h at 37C. After washing,
the membrane was developed using
the enhanced chemiluminescent
(ECL2) kit from Amersham. The
same steps were repeated using sera
from healthy Egyptian donors to
ensure the proteins recognition
specify.
Results
The complete nucleotide sequence
of isolate ED43 HCV genotype 4a
(HCV_ED43) which is 9355 nu-
cleotide long is deposited in the
EMBL database under accession
number Y11604. GenBank search
revealed that HCV_ED43 has a sin-
gle open reading frame that starts at
base 280 (the first ATG start codon)
and ends at base 9306 encoding a
polyprotein of 3008 AA. The core
(C_ED43) gene of HCV_ED43 lies
between nucleotide bases 280 and
852 of the genome. Amino acid
translation of C_ED43 gene using
PC/GENE program revealed a pro-
tein that consists of 191 AA and
have a molecular weight of 20.699
kDa (Fig. 1).
PC/GENE program was analyzed
the main protein deduced from the
C_ED43 DNA sequence translation.
The following analysis was per-
formed. Amino acid composition
analysis of the C_ED43 protein re-
vealed the presence of a high per-
centage of glycine (13.6%), arginine
(12%), proline (10.9%) and leucine
(9.4%). The isoelectric point (pI) of
the C_ED43 deduced protein was
found to be 11.88. Antigenic deter-
minants of the C_ED43 protein
were predicted by analyzing the
amino acid sequence in order to find
the point of greatest hydrophilicity
(Hopp and Woods, 1981). The three
highest points of hydrophilicity
have been determined. The first
point was located from amino acid
69 to 74 with an average hydro-
philicity (Ah) of 2, the second point
was located from amino acid 50 to
55 with an Ah of 1.98 and the third
was from amino acid 8 to 14 with an
Ah 1.97 (Fig. 2). The estimated
half-life is considered to be 30 hours
in mammalian reticulocytes, in
vitro, more than 20 hours in yeast in
vivo and more than 10 hours in E.
coli, in vivo. Sites annotated analy-
sis of the C_ED43 protein revealed
the presence of 2 cAMP- and
773
cGMP-dependent protein kinase
phosphorylation sites, 5 protein ki-
nase C phosphorylation sites, 9 N-
myristoylation sites and one ami-
dation site.
The BlASTN (for DNA) program
was used to search the nucleotide
sequence similarity between
C_ED43 DNA sequence of HCV-
ED43 and other DNA sequences,
using non rebundant Gen-
Bank+EMBL+DDBJ+PDB se-
quences database. The DNA of nine
isolates from those resulted from
BlASTN search were chosen for
multiple alignments with the
C_ED43 DNA. BLASTN homolo-
gy search revealed DNA similarities
of 95% with isolate 25 of geno-type
4a (Is. 25), 93% with isolate Z7 of
genotype 4c (Is. Z7), 92% with
DK13 of genotype 4d (Is. DK13),
92% with isolate 24 of genotype 4d
(Is. 24), 91% with clone FR12 of
genotype 4f (Is. FR12), 90% with
clone CAM 736 of genotype 4e (Is.
CAM 736), 90% with isolate Z4 of
genotype 4a (Is. Z4), 87% with iso-
late Z1 of genotype 4b (Is. Z1) and
87% with isolate 21 of genotype 1b
(Is. 21). Amino acid translation of
the nucleotide sequences of the nine
isolates that were aligned with
C_ED43 gene was performed and
multiple protein alignments of the
C_ED43 protein with the deduced
proteins were done using PC/GENE
program. Alignments revealed an
identity of 85% between the
C_ED43 protein and the nine pro-
teins (Fig. 3).
A restriction map of the C_ED43
DNA was done by using PC/GENE
software programs (Intelligentics,
Inc.). The restriction map revealed
that BamHI and HindIII which are
present in the MCS of the pQE-30
vector do not have internal re-
striction sites in the C_ED43 region.
The primers for PCR were designed
by adding BamHI site to the forward
primer and HindIII site to the re-
verse primer. Corresponding
C_ED43 DNA coding sequence was
amplified by PCR reaction. After
amplification, electrophoresis re-
vealed a band of the expected size
(0.6 kb) of the PCR product (Fig. 4).
The PCR product of the amplified
C_ED43 DNA was purified from
gel using High Pure PCR Purifica-
tion Kit (Roche), which was then
double digested with the restriction
enzymes BamHI and HindIII result-
ing in sticky ends C_ED43 DNA
product. After digestion, PCR prod-
uct was purified from solution using
High Pure PCR Purification Kit.
The concentration of the purified
double digested DNA was estimated
to be 30 ng/l for each band \(Fig.
5). The PCR product was ligated to
pQE30, transformed into M15 cells,
miniprep took place and electropho-
resis of the double digested DNA
with BamHI and HindIII showed the
presence of a band around 0.6 kb
which is the size of the Core_4a
insert (Fig. 6). 5 DNA sequence
analysis of the Core_4a region of
the recombinant pQE-30/Core_4a
plasmid revealed that Core_4a insert
was ligated in frame with the 6xHis-
774
tagged DNA of the pQE-30 vector
(Fig. 7) and alignment search with
the original HCV_ED43 using
BLAST program showed that the
sequence of the insert is similar to
that of the original C_ED43 (Fig. 8).
Expression and purification of
Core_4a protein: M15 bacterial cells
contain-ing recombinant plasmids
pQE 30/Core_4a was induced with
1mM IPTG. Four hours after induc-
tion, the cells were harvested, sam-
ples of whole-cell lysates were pre-
pared and analysed by SDS-PAGE.
A band of approximately 22 kDa
was observed, in good agreement
with predicted molecular mass (Fig.
9). Nearly all of the expressed fu-
sion protein was found in the insol-
uble fraction after sonication (Fig.
10). Core_4a protein was solubil-
ized with 8 M urea and was purified
on Ni2+-NTAagarose under dena-
turing conditions. After washing,
bound proteins were eluted at low
pH, and fusion protein of high puri-
ty was obtained (Fig. 11). Final
yield was about 2.703 mg/2litre cul-
ture.
Western blot analysis of purified
Core_4a protein was done to check
protein antigenicity. Expressed
Core_4a protein was recognized by
anti-HCV anti-bodies in pooled hu-
man sera of Egyptian HCV patients
and a band appeared approximately
at 22 corresponding to Core_4a
(Fig. 12). Specificity was confirmed
by sera from healthy donors without
specific recognition of Core_4a pro-
tein.
Discussion
HCV leads chronic liver disease
that infects approximately 3% of the
worlds population. Worldwide,
more than one million new cases of
infection are reported annually, and
HCV is believed to be more preva-
lent than hepatitis B virus infection
(HBV) (Cooreman and Schoonder-
mark-Van de Ven, 1996; Barth et
al., 2006).
Advances in biotechnology have
improved the understanding of hep-
atitis C virus immunology and
brought to light several new chal-
lenges. An effective HCV vaccine
must be able to prevent infection or
the progression of infection, protect
against the predominant genotypes
within the geographic region of use,
and be safe and effective for wide-
spread administration (Strader and
Seeff, 2001).
Genotype 4a is the most widely
distributed of type 4 sequences, be-
ing the principal genotype in the
Middle East and Egypt, and ac-
counts for a major proportion of
cirrhosis and hepatocellular carci-
noma in these populations (Dush-
eiko et al., 1994; Ray et al., 2000;
Elkady et al., 2009). Despite the
success after introduction of combi-
nation therapy with IFN- and Rib-
avirin which is the current treatment
of chronic HCV (Pawlotsky et al.,
1995), about 60% of patients with
HCV genotype 4 fail to respond (El-
Zayadi et al., 1996; Al-Traif et al.,
2004; Zekri et al., 2007).
775
Fig. 2: Hydrophilicity profile of C_ED43 protein sequence, computed by an average group
length of 6 amino acids.
776
777
Fig. 4 Fig. 5
Fig. 6
Fig. 4: Agarose gel electrophoresis of PCR product after amplification of C_ED43 gene from HCV_ED43
DNA.Lane M: 1kb ladder marker. Lane 1: control sample. Lane 2: PCR product of C_ED43 gene.
Fig. 5: Agarose gel electorophoresis of C_ED43 PCR product digested with BamHI and HindIII and purified
from solution. Lane M: 1 kb ladder marker. Lane 1: sample 1 of pure digested C_ED43 PCR product. Lane
2: sample 2 of pure digested C_ED43 PCR product.
Fig. 6: Double digestion of miniprep samples of recombinant pQE-30/Core_4a plasmids with Bam-
HI/HindIII. Lane M: 1 kb Ladder marker. Lane V: pQE-30 digested with BamHI/HindIII. Lane 1-5: DNA
from minipreps digested with BamHI/HindIII. V: vector.
778
Fig. 7: DNA sequence analysis of Core_4a insert inside pQE-30 (C1) using ABI PRISM model 310 DNA automated se-
quencer. Bases 1-36=pQE-30 vector sequence, bases from 37 ~ 590 =Core_4a sequence. Sequence of 6xHis. BamHI site
and beginning of Core_4a DNA sequence without first ATG
779
Fig. 9
Fig. 11
Fig. 10 Fig. 12
Fig. 9: SDS-PAGE (12% gel) of bacterial cultures from small expression of Core_4a protein in M15 bacteria.
Lane M: prestained wide range MW marker. Lane 1: uninduced culture. Lane 2: induced culture (arrow
points to expressed core protein). Lane 3: Flow-through. Lane 4: Wash. Fig. 10: SDS- PAGE (12% gel) of
bacterial cultures from expression of Core_4a protein in M15 bacteria to determine solubility. Lane M:
prestained wide range MW marker. Lane 1: uninduced culture of Core_4a protein. Lane 2: soluble fraction of
induced culture of Core_4a protein. Lane 3: insoluble fraction of induced culture of Core_4a protein.
Fig. 11: SDS- PAGE (12% gel) of Core_4a protein after purification from large-scale culture. Lane M: pres-
tained wide range MW marker. Lane 1: elution 1 of Core_4a protein. Lane 2: elution 2 of Core_4a protein
Fig. 12: Western blot of purified Core_4a protein recognized by pooled sera of Egyptian HCV patients.
780
In the present study, DNA seque-
nce of the core region was analyzed
using PC/GGENE program.
C_ED43 gene of HCV-ED43 lies
between nucleotide bases 280 and
852 of the genome. The sequence
analysis revealed that C_ED43 has a
relatively higher proportion of GC
bases than AT bases with a base
percentage of 29.5 % G, 30.3 % C,
18.8% A and 21.3% T.
The predicted translation of
C_ED43 gene resulted in a deduced
protein of 191 aa long, having a mo-
lecular weight of 20.699 kDa. The
deduced protein has shown to be
rich in glycine (13.6%), arginine
(12%), proline (10.9%) and leucine
(9.4%) with an isolelectric point at
pH 11.88. These findings agree with
those of the other core proteins (Hi-
jikata et al., 1991; Santolini et al.,
1994). The 10 arginine and lysine
residues within aa 39-62 found in
the C_ED43 protein are invariant
among all HCV isolates, suggesting
that this domain may represent an
important RNA-binding site. Also,
additional methionine at position 20
specific for the genotype 4 groups
(Bukh et al., 1994) was found in the
C_ED43 protein. C-terminal se-
quence of the C_ED43 protein con-
sisted of Pro-Ala-Ser-Ala, which is
also seen in all the genotypes except
genotype 3 (Mori et al., 1992),
which suggests that the C/El cleav-
age site is conserved in C_ED43
protein. The N-terminal of the
C_ED43 protein sequence is M
(Met).The estimated half-life is con-
sidered to be 30 hours in mammali-
an reticulocytes, in vitro, more than
20 hours in yeast, in vivo, and more
than 10 hours in E. coli, in vivo.
The ability to predict antigenic
sites on proteins is of major im-
portance for the production of syn-
thetic peptide vaccines and synthetic
peptide probes of antibody structure.
Many predictive methods, based on
various assumptions about the na-
ture of the antigenic response have
been proposed and tested (Arnon
and Van Regenmorte, 1992). Since
the hydrophilic regions are consid-
ered the most Polar Regions, they
may provide the greatest interac-
tions for the bonding between the
antigen and the antibody (Hopp,
1993). Hydrophilicity is associated
with antigenic determinants because
hydrophilic regions are on the out-
side of a protein. Antigenic deter-
minants of the protein were predict-
ed by analyzing the amino acid se-
quence in order to find the point of
greatest hydrophilicity (Hopp and
Woods, 1981). The three highest
points of hydrophilicity have been
determined in C_ED43. The first
point was located from amino acid
69 to 74 with an average hydro-
philicity (Ah) of 2, the second point
was located from amino acid 50 to
55 with an Ah of 1.98, and the third
was from amino acid 8 to 14 with an
Ah 1.97. Several studies have indi-
cated that these three major hydro-
philic regions of the core protein
contain linear immunogenic
epitopes (Sllberg et al., 1992; Jo-
livet et al., 1997). In one of the
studies, antibodies against synthetic
781
peptides from aa 1-18, 51-68, and
101-118 were detected in infected
patients (Sllberg et al., 1992).
However, while these immunogenic
regions are highly conserved, geno-
type-specific differences were ob-
served at several amino acid posi-
tions that may influence the speci-
ficity and sensitivity of the serologi-
cal tests (Bukh et al., 1994). A sin-
gle substitution at aa 110 affected
seroreactivity (Sllberg et al., 1992).
Despite the high degree of conserva-
tion in the immunodominant regions
of the core protein among the dif-
ferent genotypes, it is possible that
genetic heterogeneity of the core
protein could lead to false-negative
results in serological tests (Bukh et
al., 1994).
Sites annotated analysis of the
predicted protein encoded by the
C_ED43 gene revealed the presence
of 2 cAMP- and cGMP-dependent
protein kinase phosphorylation sites
at positions 16 and 53 which are
known to catalyze phosphrylation of
a number of different enzymes and
non enzymic proteins and have an
important role in regulation of met-
abolic activity. Five protein kinase
C phosphorylation sites were also
defined along the C_ED43 protein
at positions 11, 15, 49, 53 and 99.
Protein kinase C plays a key role in
cell growth and gene expression
(Post and Brown, 1996). Also, pro-
tein kinase appears to play a role of
crucial importance in transmitting
various extracellular information
signals across the cell membrane
(Kishimito et al., 1985). Thus, it is
likely that protein kinase C trans-
duces distinctly different pieces of
information into the cell through
their own specific protein phos-
phorylation. In vivo, protein kinase
C exhibits a preference for the
phosphorylation of serine or the-
ronine residues close to a C-terminal
basic residue (Kishimito et al.,
1985). Other investigators found
that protein kinase-catalyzed phos-
phorylation is an important mecha-
nism by which the activity of the
ion channels, including K
ATP
chan-
nel can be controlled (Light et al.,
2000).
The C_ED43 protein contained 9
N-myristoylation sites at positions
28, 45, 87, 90, 102, 142, 161, 167 &
171. They are post-transitional mod-
ification of the protein sequence.
Myristoylation (transfer of myristate
from myristoyl-co-enzyme A in am-
ide linkage to the amino-terminal
glycine residue of the proteins) is
essential for the biological function
of most proteins. Attachment of
myristoyl residue to glycine resi-
dues provides hydrophobicity and
promotes the protein-protein inter-
actions (Johnson et al., 1994). The
presence of N-myristoylation sites
in along the C_ED43 protein se-
quence may be responsible for the
hydrophobic nature of the protein.
The hydrophobic domains are likely
to be involved in protein-protein
and/or protein-RNA interactions
during assembly of the nucleocapsid
as well as in interaction with the
lipoprotein envelope were suggested
for flaviviruses (Rice et al., 1986).
782
In general, the hydrophobicity of
residues located within the hydro-
phobic protein core is highly con-
served because it is needed for
proper protein folding and stability
(Penin et al., 2004). One amidation
site was found at position 59 in the
C_ED43 sequence. A high propor-
tion of peptide transmitters and pep-
tide hormones terminate their pep-
tide chain in a C-terminal amide
group which is essential for their
biological activity (Bradbury and
Smyth, 1987). So, it was obvious
that the predicted C_ED43 protein
shared the main features of the core
proteins of the different HCV geno-
types.
Homology analysis of the
C_ED43 DNA with the other clones
from the GenBank was carried on.
Nine isolates from those which
showed significant homology with
C_ED43 DNA were chosen for mul-
tiple alignments with C_ED43.
DNA sequence homology search of
the C_ED43 gene with the 9 chosen
isolates revealed similarities of 95%
with isolate 25 of genotype 4a (Is.
25), 93% with isolate Z7 of geno-
type 4c (Is. Z7), 92% with a DK13
of geno-type 4d (Is. DK13), 92%
with isolate 24 of genotype 4d (Is.
24), 91% with clone FR12 of geno-
type 4f (Is. FR12), 90% with clone
CAM 736 of genotype 4e (Is. CAM
736), 90% with isolate Z4 of geno-
type 4a (Is. Z4), 87% with isolate
Z1 of genotype 4b (Is. Z1) & 87%
with isolate 21 of genotype 1b (Is.
21).
Amino acid translation of the nu-
cleotide sequences of the nine iso-
lates was done and multiple protein
alignments of the C_ED43 protein
with the deduced proteins were done
using PC/GENE program. The ami-
no acid alignments revealed an iden-
tity of 85% among the HCV core
isolates. Nucleotide and amino acid
sequence homologies showed that
the C_ED43 region of the HCV
genotype 4a and other HCV isolates
genotype 4 were more closely relat-
ed to genotype 1 sequences which
agree with previous studies (Bukh et
al., 1994; Franco et al., 2007). From
these homology results, it was no-
ticed that the C_ED43 region of
HCV genotype 4a which is isolated
from an Egyptian patient is slightly
different from the core protein of
the European HCV4a (Is. 25) (Fran-
co et al., 2007) with a similarity of
95% and from Central African
HCV4a (Is. Z4) with a 90% similar-
ity (Bukh et al., 1994). The results
agreed with the fact that type 4a
from Egypt is a different subtype
from the type 4a of Central Africa
(Bukh et al., 1995). Although the
core region is conserved among the
different HCV genotypes, there are
slight differences between C_ED43
region and the core region of the
other HCV genotypes.
Following computer analysis of
C_ED43, it was cloned and ex-
pressed in pQE-30/M15 E.coli. The
Gram-negative bacterium E. coli
remains one of the most attractive
expression systems because it is
able to grow rapidly and at high
783
density on inexpensive substrates, it
is also well-characterized genetical-
ly and has availability of large num-
ber of cloning vectors and mutant
host strains (Baneyx, 1999). Alt-
hough bacterial expression systems
lack most post-translational pro-
cessing mechanisms found in eu-
karyotic cells, their expression level
is usually higher than eukaryotic
expression systems, especially
mammalian cell systems. This con-
stitutes a big advantage for purifica-
tion and cost reduction (Liu et al.,
2001). Fragments of core (Hand-
schuh and Caselma-nn, 1995; Song-
sivilai et al., 1996; Zhang et al.,
2003) were successfully cloned and
expressed by E. coli.
The cloning strategy for C_ED43
gene was done (Sambrook et al.,
1989) where it started by amplifica-
tion of C_ED43 DNA using
PUC19/HCV_ED43 plasmid as a
template. To get oriented ligation of
core insert into pQE30 vector, t
BamHI and HindIII restriction sites
were introduced to the forward and
reverse primers respectively. pQE-
30 vector having the PCR product
of the core region (designated as
Core_4a) was used to transform
M15[pREP4] bacteria supplied with
the kit. Successful transformation
was confirmed by plasmid miniprep
and gel electrophoresis that revealed
the presence of DNA bands of the
expected size (0.6 kb).
Optimized conditions for expres-
sion of Core_4a protein was done
according to those stated in the
manufacturers manual of QIAex-
pressionist kit with the only excep-
tion that before induction, the bacte-
rial cultures of Core_4a was grown
at 25C instead of 37C because
core protein is known to have a hy-
drophobic region which often has a
toxic effect on host cells, most like-
ly due to the association of the pro-
tein with or incorporation into vital
membrane systems. It is possible to
express proteins containing signal
peptides that target the protein mol-
ecules into the periplasmic space,
albeit at a lower rate by lowering the
growth temperature to 25C before
induction. SDS-PAGE of the whole
induced cultures before purification
showed the presence of bands of
Core_4a fusion protein of 22 kDa.
In the present study, the Core_4a
protein appeared at the expected
MW which contradicts with a previ-
ous study (Handschuh and Casel-
mann, 1995) who found that the full
length core protein inserted in pQE
vector and expressed in E.coli mi-
grated faster than expected. They
concluded that intracellular proteo-
lytic processing, which gave rise to
a truncated protein or to mobility in
SDS-PAGE gels caused by the hy-
drophobic nature of the full-length
protein. In the present study, growth
of Core_4a bacterial culture at 25C
before induction might have de-
creased hydrophobic effect of the
protein. The solubility of expressed
Core_4a protein was checked before
preparing the proteins on large
scales, and it was found that the
Core_4a protein was expressed in
the insoluble form.That may have
784
been due to the fact that hydropho-
bic proteins or proteins synthesized
in bacteria in high quantities often
accumulate in intracytoplasmatic
inclusion bodies (Songsivilai et al.,
1996). Thus, large scale prepara-
tions of the Core_4a protein was
done under denaturing conditions
and Core_4a protein content was
estimated to be around 1.35mg/liter.
Reactivity of Core_4a against hu-
man sera in western blot was ana-
lysed. The expressed Core_4a pro-
tein was recognized by the anti-
HCV present in the pooled human
sera of Egyptian patients infected
with hepatitis C and a band ap-
peared approximately at 22 corre-
sponding to the Core_4a. Specificity
of this recognition was confirmed
by using sera from healthy donors
which showed no specific recogni-
tion of the two proteins. This
showed that this E. coli-derived
Core_4a protein displayed specific
antigenicity.
Conclusion
Analysis of Core region of the
isolate ED43 revealed that the
Egyptian genotype 4a is different
from those isolated from Europe and
Central Africa but closely related to
genotype 1b. Construction of pQE-
30/Core_4a recom-binant plasmid
and expression of Core_4a gene and
E. coli (M15) was success-ful with
immunogenicity in Western blot
useful for developing assay systems
to detect anti-HCV antibodies, HCV
antigen and in a vaccine design
against the Egyptian genotype 4a.
Further studies are recommended in
experimental animals to specify the-
se proteins antigenicity. Characteri-
zation of more HCV isolates from
Egyptian patients is crucial for the
improvement of diagnostic, epide-
miological, clinical treatment and
development of a candidate vaccine
against HCV genotype 4a.
Acknowledgments
The authors would like to express
their deep gratitude to Dr. Paul Ha-
gan, Professor of Parasitology, Insti-
tute of Virology, Glasgow Universi-
ty, for his instructive guidance,
helpful support, suggestions and
facilities provided through this
work.
Thanks are also due to the authori-
ty of Institute of Virology, Glasgow
University, who has always shown
the spirit of a team, especially Prof.
Dr. Richard Elliot for his assistance
and advices.
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789
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 789 - 802
EARLY DEVELOPMENT OF OSTEOPOROSIS IN MALE SMOKERS
WITH HYPOANDROGENISM DUE TO FASCIOLIASIS WITH OR
WITHOUT SCHISTOSOMIASIS ADDED BY LIFE STYLE
By
YASSER FOUAD KILANY
1
, SAHAR A. ABOU HOLW
2
, MOHAMED
FATHY ABOUEL-NOUR
3
, and AYMAN T.A. MORSY
4
Departments of Tropical Medicine
1
, Parasitology
2
, and Zoology
3
, Facul-
ties of Medicine
1
, and Science
3
, Al Azhar University
1
, Cairo, and
Mansoura University
3
, Mansoura, Medical Research Institute, Alexan-
dria University
2
, Alexandria, and Tropical Medicine Unit, The Ministry of
Interior Hospitals
4
, Egypt
Abstract
The multifactor outcome of hypoandrogenemia with the impact of oxidative stress
induced by glucose intolerance, fascioliasis with or without schistosomiasis and cumu-
lative smoking influence on bone remodeling and the early development of osteoporot-
ic manifestations were studied.
The effect on vascular endothelium immune mediated mechanisms and antioxidant
capacity were monitored in cases of youth aged selected male smokers involving 20
with hypoandrogenemia who were either subjected to sedentary life style, glucose in-
tolerance fascioliasis hepatic fibrosis (FHF) (G1) or without (G2) and GI after follow-
ing 6 months therapy (G3). Monitoring of clinical picture and biochemical assessments
of osteoporotic indices (osteocolcin, bone alkaline phosphatase, parathyroid hormone,
urinary cyclic AMP), hypoandrogenism (dehydroepiandrosterane sulphate or DHEAS
& testosterone) glycemic determinant (insulin) immuno-infla-mmatory response (inter-
leukein-6, tumor necrosis factor , E-selectin, ceruloplasmin) smoking index (serum
cotinine), total antioxidant capacity (AOC) and lipid peroxidation (malonedialdehyde)
was done before and after 6 months therapeutic program involving supplement of
DHEAS, mirazid, chromium picolinate, and megavit zinc alongside smoking cessation
and physical exercise daily for at least 30 minutes. Treatment with Mirazid supplied as
10 mg/kg for 6 successive days resulted in 100% cure of fascioliasis whether single or
combined with schistosomiasis.
Key words: Egypt, fascioliasis, osteoporosis, hypoandrogenemia, schistosomiasis,
Male, smoking.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
In Osteoporosis an overall mass
of skeleton is reduced without
change in the ratio of mineral to
matrix (Raef et al., 1984). It is
characterized by low bone mass
790
density and micro architectural dete-
rioration of bone tissue, and en-
hanced bone fragility and increased
fracture risk (Vanasse et al., 2005).
It is an inevitable health problem of
aging in both sexes, its modulation
and delaying of its occurrence espe-
cially at a younger age is greatly
related to life style, and sex steroid
hormones influence bone growth
and metabolism. At time of male
puberty, epiphyseal growth is accel-
erated by testosterone while in older
age; testosterone interferes with
bone resorption (Khosla et al.,
2005).
The Egyptians showed that seden-
tary life style and smoking repre-
sented the highest risk factors lead-
ing to early development of osteo-
porosis (Ward and Klesges, 2001).
Sedentary life enhances bone re-
porption and smoking reduces intes-
tinal absorption of calcium which
antagonizes the attainment of peak
bone mass (Lobbardi and Ross,
2001). Cigarette smoke extracts
contain a great amount of oxygen
anion or hydrogen peroxide. This
can initiate or propagate the process
of lipid peroxidation represented by
malondialdehyde production
(MDA) and facilitate the develop-
ment of atherosclerotic plaque and
tissue damaging effects (Barnoya
and Glantz, 2005). The adverse ef-
fect of cigarette smoking on hypo-
thalamic pituitary axis has been re-
ported. Evidently, nicotine and its
major metabolite cotinine were
measured in various biological flu-
ids and related to the magnitude of
smoking. The cigarette smoking
suppresses the immune mechanisms
and induces vascular endothelial
damage as sequalae of decreasing
antioxidant capacity. This was po-
tentiated by Schistosoma mansoni
and imparting schistosomal hepatic
fibrosis (Mangoud et al., 2004). A
positive correlation between male
hypoandrogenism and some endem-
ic liver parasites was reported (Joz-
kowicz et al., 1999). Also, a posi-
tive relationship between the insulin
resistance, increased free radicals
and antioxidants decreased levels,
association with impaired glucose
homeostasis and the increased vas-
cular endothelial injury was report-
ed (Paolisso et al., 2006).
In Egypt, fascioliasis (Farag,
1998) and schistosomiasis (Abdel
Wahab et al., 1996) are endemic in
many foci. As an example, Abou-
Basha et al. (2000) studied fascio-
liasis and/or schistosomiasis in Abis
1 village (Alexandria area) found
fascioliasis, alone or combined with
schistosomiasis was more prevalent
among children aged between 5 and
15 years than in adults. Serum pro-
collagen III peptide levels as an in-
dicator of active. Liver histopathol-
ogy and ultra-sonography were used
as indicators of established fibrosis.
The levels were significantly higher
in children than in adults and in
mixed infections than in fascioliasis
alone. In adults, fibrosis around
granulomata detected by histo-
pathology and grade 3 periportal
fibrosis detected by sonography was
791
encoun-tered more frequently in
dual than in single infections.
The present study identified the
influence of multidisciplinary risk
factors concerned the life style on
early development of osteoporotic
manifestations in the hypoandro-
genemic youth, as sedentary life
style, smoking, fascioliasis with or
without schistosomiasis and glucose
intolerance.
Subjects, Materials and Methods
Twenty smoking men (35-40
years) with hypoandrogenism were
subjected to history taking and full
clinical examination. The exclusion
criteria was those with other parasit-
ic diseases (apart from fascioliasis
with or without schistoso-miasis
mansoni), or other chronic diseases,
or treated with drugs affecting he-
patic metabolism in last 6 months,
or with evidence of liver failure.
The patients were classified into
two groups. G1: 10 smokers with
glucose intolerance, fascioliasis (3
concomitant with schistosomiasis)
and sedentary life style. G2: 10 non-
smokers, non-glucose intolerant free
from fascioliasis and/or schistoso-
miasis and with active life style. G1
was treated by Mirazid

(Pharco) at
a dose of 10mg/kg (2 capsules) on
empty stomach an hour before
breakfast for 6-8 days for both
schistosomiasis and fascioliasis (El
Baz et al., 2003; Abou-Madyan et
al., 2004a,b). Treatment strategy
was a six month program to change
the life style, comprising cessation
of smoking, participation in sportive
exercise twice weekly for at least an
hour and nutritive diet enriched with
minerals and antioxidants (vegeta-
bles, fruits, beans, nuts, honey,
datesetc). Treatment was Dehy-
droepiandrosterone sulphate (100
mg/day), Chromium preslinate 200
mcg daily capsules (Mepaco), and
Zinc intensified multivitamins, min-
eral supplement as a daily capsule
of megavit zinc (Amriya Pharma-
ceuticals). Cured cases of G1 were
referred to as G3.
Fascioliasis was diagnosis by ab-
dominal ultrasonography (Haseeb et
al., 2003b), Kato thick smear (Katz
et al., 1972) and modified formol
ether method. They were also sub-
jected to sigmoidoscopy rectal snip
biopsy for schistosomiasis (Harries
and Speare, 1988) and ELISA-
Fasciola (Fhes) antigen for fascio-
liasis (Haseeb et al., 2003a). Cure of
fascioliasis with or without schisto-
somiasis was confirmed by clinical
improvement, ultrasonography &
SOPT for schistosomiasis and drop
in eosinophilia and ELISA-Fasciola
(Fhes) antibody.
The impaired glucose tolerance
depended on 2 hours blood glucose
levels in the range of 7.8-11 mol/L
which were elevated but still of
lower diagnostic levels for diabetes,
after an overnight fasting. Blood
was taken to separate serum for
complete liver function tests, kidney
function tests. ALT & AST were
determined colorimetrically (Reitm-
an and Frankel, 1957). Total serum
bilirubin (Bartels and Bohmer,
1970), serum albumin and creatiine
792
were colorimetrically estimated
(Dumas et al., 1997). Serum dehy-
droepiandrosterone sulphate
(DHEA-S), insulin, testosterone
were assessed, lipid peroxidation
was estimated by malonedialdehyde
(Hennery, 1974). Smoking index
(cotinine) (Satoh, 1978), total anti-
oxidant capacity (Bonnectont et al.,
1989), immunoinflammatory index
IL-6 (Hirano, 1989), TNF- (Ledur
et al., 1995), E-selectin (Blann et
al., 1995), ceruloplasmin & PTH
(Buffone et al., 1979) electro-
chemilu-minescence immunoassay
(ECLIA) were measured by Elec-
cys-2010 kits (Diagnostic Co.,
USA).
Statistical analysis were done us-
ing SPSS version 9.0 Software. Stu-
dent t-test was used to compare
group with control before and after
therapy using paired t-test. Student
t-test was between groups of G1, G2
& G3 (Campbell and Machine,
1993).
Results
All fascioliasis patients with or with-
out schistosomiasis were cured (100%).
They were verified free of current infec-
tion by eosinophilia (about normal level
3 months after therapy and drop of anti-
Fasciola antibody). Maintenance of
liver and renal function, absence of liv-
ing S. mansoni eggs in rectal snips and
non detectable S. mansoni ova in stools
was confirmed.
The patient hepatic fibrosis (HF) was
graded by Ultrasound (Mangoud et
al., 2004) as Grade 1 with periportal
thickening of 3-5 mm, Grade II from
5-7 mm, and Grade III > 7 mm. Data
identified cases with FHR as Childs
grade. A. GI had HOMA-R from 0.61-
10.2 with a mean of 2-90.41 while
subjects of GII had a mean 1.70.36
and differed from treated patients with
mean1.620.62. G1-G3 expressed sig-
nificant variation in indices of osteopo-
rosis, hypoandrogenemia and glycemic
determinant, immuno-inflammatory
response, antioxidant capacity and
smoking index. G3 gave changes to
near normal parameters, insignificant
increase in bilirubin ALT, AST and
creatinine versus decrements albumin
were recorded. Details are in tables (1,
2 & 3).
Discussion
In the present study, G1 & G2
early developed osteoporosis by
reduction of bone mass due to mul-
tiple factors where the rate of bone
resorption exceeds that of bone for-
mation. The findings agreed the fact
that men more common than women
develop osteoprosis secondary to an
underlying disease or metabolic de-
rangement whereby at least 50% are
ascribed to life style choices. The
equilibrium between bone formation
and bone resorption are balance of
functions of three bone cells types;
osteoblasts, osteocytes and osteo-
clasts. So, the lower osteocalcin
level in the present cases reflected
the cumulative impact of several
risk factors associated with life
style. The National Osteoporosis
Foundation reported that about 25
million people have osteoporosis
and that 1.5 million osteoporotic
fractures annually caused morbidity
and mortality (Olszynki et al.,
793
2004). Osteoporosis is a preventable
and treatable disease characterized
by low bone mass and micro-
architectural deterioration of bone
tissue leading to enhanced bone fra-
gility and a consequent increase in
fracture incidence (Raisz, 2005).
Modifiable risk factors of human
osteoporosis were the smoking, sed-
entary lifestyle; low calcium and
Vitamin D level or lack to sun ex-
posure (Meknight et al., 1995).
Table 1: Clinical and ultrasonography pictures of smokers (G1), non-smokers (G2) and
treated smokers patients (G3).
Features G 1 G 2 G 3
Average liver size - - -
Hepatomegaly 10 - 10
Shrunken Liver - - -
Average size 6 0 10
Splenomegaly 4 0 0
Ascites 0 0 0
- Normal Abd U/S 0 10 0
- Echogenic liver 0 0 0
- Hepatomegaly 10 0 10
- Shrunken liver 0 0 0
- Normal spleen 6 10 6
- Splenomegaly 4 0 4
Histological active index 5.11.3 - 5.11.3
Periportal fibrosis
Grade I 4 - 4
Grade II 4 - 4
Grade III 2 - 2
Table 2: Age, liver and kidney function tests in subjects:
G 1 G 2 G 3
Age (year) 35-47,38.13.2 37-46,40.13.1 35-47,38.13.2
AST 70.418.2 70.116.8 66.3217.2
t1 (p1) 3.28 3.51 2.79
t2 (p2) 0.04 3.28 3.12
ALT 64.118.3 55.317.2 38.39.9
t1 (p1) 4.78 3.69 1.36
t2 (p2) 1.11 3.92 4.78
Total bilirubin 0.810.19 0.930.45 0.810.31
t1 (p1) 0.78 1.2 1.34
t2 (p2) 0.77 1.55 1.05
Albumin 3.30.96 3.890.41 4.71.14
t1 (p1) 2.00 1.07 0.51
t2 (p2) 1.79 2.97 2.0
Creatinine 1.060.17 0.930.14 0.830.12
t1 (p1) 3.28 5.78 2.45
t2 (p2) 5.42 1.79 4.78
794
Table 3: Osteoporosis and different index markers in subjects:
Specific index G 1 G 2 G 3
Osteocalcin 6.3 1.5 5.1 1.1 4.8 1.06
t1 (p1) 4.70** 3.11** 2.50*
t2 (p2) 2.04* 2.58**
Alkaline phosphatase 136 24 119 23 106 21
t1 (p1) 5.36** 3.74** 2.51*
t2 (p2) 1.62 (NS) 2.97**
Parathyroid hormone 93.2 19.1 81.7 17.4 73.113.2
t1 (p1) 4.42** 3.00** 3.80**
t2 (p2) 1.41 (NS) 2.74**
cAMP 6.2 1.2 5.1 1.04 4.3 0.9
t1 (p1) 6.64** 4.69** 3.05**
t2 (p2) 2.19* 4.01**
DHEAS 146 58 189 60 314 117
t1 (p1) 5.44** 4.50** 1.54 (NS)
t2 (p2) 1.63 (NS) 4.07**
Total testosterone 15.9 3.1 17.8 3.8 21.6 4.1
t1 (p1) 3.82** 2.58** 0.60 (NS)
t2 (p2) 1.23 (NS) 3.51**
Fast insulin 17.9 5.3 15.8 4.7 13.1 2.9
t1 (p1) 5.47** 4.77** 4.49**
t2 (p2) 0.94 (NS) 2.51*
IL-6 89.3 17.9 70.8 14.1 34.9 7.1
t1 (p1) 9.14** 7.44** 0.73 (NS)
t2 (p2) 2.57** 8.93**
TNF- 17.6 4.1 12.9 2.7 8.3 2.1
t1 (p1) 8.48** 7.15** 2.99**
t2 (p2) 3.03** 6.38**
E-selectin 123 29.1 93.2 24.6 67.1 11.3
t1 (p1) 7.50** 5.15** 3.58**
t2 (p2) 2.47* 5.66**
Ceruloplasmin) 18.7 4.8 15.7 3.4 13.6 2.9
t1 (p1) 4.29** 3.26** 1.93**
t2 (p2) 1.61 (NS) 2.88**
Serum cotinine 139.7 40 ND ND
AOC% 67.1 11.1 72.4 13.2 83.7 14.8
t1 (p1) 5.22** 4.10** 2.27*
t2 (p2) 0.97 (NS) 2.84**
Malondialdehyde 9.1 0.9 7.8 0.6 6.3 0.6
t1 (p1) 10.11** 8.10** 3.71**
t2 (p2) 3.80** 8.19**
* Significant< 0.05, **significant< 0.01, t1 (p1): t-test and its significance between control and others,
t2 (p2): t-test and significance between G I and others.
In the present study, lower oste-
ocalcin (OC) in G1 > G2 was poten-
tiated by smoking, FHF, glucose
intolerance and hypoandrogenemia.
The laboratory markers of bone
turnover predicted the rate of bone
loss by reflecting the rate of bone
formation or degradation assessed
by measuring either an enzymatic
activity of the osteoblastic or osteo-
clastic cells (alkaline phosphates) or
the components of bone matrix re-
leased into the circulation during
formation or resoprtion of bone
795
(Gumdnerg et al., 2002). This ef-
fect the role of osteocalcin (a small
protein 49 amino acid residues) ex-
isted in bone and played a role in
bone calcium regulation (Lombardi
and Ross, 2001). G1a protein, a Vit-
amin K dependent protein binds
strongly to hydroxyapatite crystals
of bone and synthesized by osteo-
blasts and fraction of newly synthe-
sized protein released directly into
the circulation to the rate of bone
formation (Brown et al., 1984). As
osteocalcin is the only marker of
bone metabolism exclusively found
in mineralized tissue being capable
of predicting remolding rate and is a
specific marker of bone formation
whereby high osteocalcin level is an
index of low skeleton mass, and
evaluation of other osteoporotic in-
dices as alkaline phos-phates
(Takeda, 2005), osteocalcin (bone
GIa protein), parathyroid hormone
(PTH), and urinary adenosine
monophosphate (cAMP) (Broadus
et al., 1997).
The premature osteoporosis in
G1>G2 versus treated patients,
greater evaluated for bone GIa pro-
tein and urinary cAMP were parallel
to hyper parathyroidism aligned
with additive effect of cigarette
smoke inhalation (CSI) on PTH in-
crease. Link between androgen de-
ficiency (potentiated by CSI) and
PTH induced increase in cAMP ac-
tivity alongside PTH-induced inhi-
bition of phosphodiestrase. In-
creased secretion of PTH and de-
creased in glomerular filtration with
aging are the main causes patho-
genesis. Higher PTH in G1> G2
was influenced by hypoandro-
genemia in FHF smokers caused
elevation of urinary cAMP with de-
crease of renal function (Rapuri et
al., 2000). The results coordinated
with sedentary life and implicate
CSI influence on bone resorption as
well as formation. Biosynthesis of
bone was at least mediated by oste-
oblast involved the intracellular syn-
thesis of the precursor of bone ma-
trix components including osteocol-
cin and ALP, but osteoclasts control
the reportion process initiation and
the coupling was altered by CSI
(Gamero and Delmas, 1998).
Several factors may contribute to
the pathogenesis of osteoporosis
including various cytokines, certain
associated immuno-inflammatory
indices, hormonal milieu and ele-
ments (McGowan et al., 2001). So,
secondary hyperparathyroidism,
highly prevalent in the elder with
vitamin D deficiency probably,
worsens the increase in bone turno-
ver related to estrogen deficiency
and hypoandrogenemia as evident in
the present study. Tobacco was
mildly antiestrogenic, and women
who smoke have menopause one to
two years earlier than non smoker
ones, and the regular exercise re-
duced the performance of hy-
perparathyroidism (Friel, 2004). It
aligns with the role of PTH as the
principal regulator of bone remodel-
ing in adult skeleton (Bilezikian,
1999).
On the other hand, the assessed
increments in levels of bone alkaline
796
phosphatase measured the rate of
bone formation by evaluating the
enzymatic activity of the osteo-
blastic cells. So, an index of osteo-
porosis was by defining increased
osteoblastic activity (Kobayashi et
al., 2000). Parathyroid hormone
increased bone resorption by stimu-
lation of osteoclastic activity by ad-
enylate cycles stimulation and in-
crease of intracellular cAMP may
serve as a mediator for hormone
action. Renal tubular cells contained
PTH receptors, cAMP levels in-
creased in response to hormone by
cAMP formed in renal tubular cells
and released in urine. The urinary
cAMP levels markedly increased in
the hyperparathyroidism. This
agreed with Broadus et al. (1997).
Serum dehydroepiandrosterone
sulphate (DHEA-S) is an indicator
of adrenal androgen function and
considered a precursor of androgen,
secreted primarily by adrenal cortex
and well antioxidant potency. It
was used as a replacement therapy
to decelerate manifestation of early
development of osteoporosis and in
coronary heart diseases (Fukui et
al., 2005). DHEA-S supplements
increased testosterone production as
verified by a lower level of DHEA-
S and testosterone. The testosterone
affects bone metabolism by interfer-
ing with bone resoprtion. Changes
of insulin physiological levels an-
tagonized bone resorption due to
PTH by suppression of protein ki-
nase C activity in osteocytes (Beutel
et al., 2005). Increased insulin lev-
els increased free radical damage to
vascular endothelium leading to ox-
idative stress. The relative fasting
insulin level confirmed the impact
of oxidative stress by glycemic sta-
tus and delineates how metabolic
determinants could affect the devel-
opment of premature osteoporosis
which is influenced by life style in
hypoandrogenic smokers with FHF
with or without schistosomiasis and
GIT.
The present results showed how
that the smokers patients most likely
to develop osteoporosis had the
lowest initial bone mass and
strength and reflected the impact of
environmental factors on bone loss
which is most rapid in area of the
skeleton containing the greatest
proportion of trabecular bone such
as the spine, hip, distal radius, pel-
vis and ribs as noted elsewhere.
The present laboratory markers of
osteoporosis were more pronounc-
edly altered reflecting the impact of
oxidative stress induced by lower
antioxidant capacity. This was
proved by lower AOC% versus
higher MDA as a marker of lipid
peroxidation potentiated by SH,
smoking, GIT and hypoandro-
genemia. The imunoinflammatory
response reflected influence of SHF
together with sedentary life and
smoking due to increased free radi-
cal mechanisms affected by im-
paired glucose metabolism. The
end product of nicotine was exclu-
sively detected in G1 (Rubin et al.,
2002). The overall events was mon-
itored by reduced total AOC which
ultimately influenced osteoporosis
797
associated with increased lipid pe-
roxidation by increased MDA level.
TheIL-6, TNF, cerulo-plasmin
(CP) as immuno-inflammatory and
MDA were parallel to total anti-
oxidant capacity. This may be al-
leged association of hypoandro-
genemia and life style as well as the
CSI role on the influx and activation
of inflammatory cells verified by the
assessed increments of cytokines
(TNF & IL-6) and E-selection
acute phase reactant protein (CP &
MT). They showed an early re-
sponse of vascular endothelial cell
damage. TNF & IL-6 involved in
upreglating the acute phase reactant
gene and caused an increased hepat-
ic synthesis in liver metallothionien
(MT) and ceruloplasmin (Leone,
2005). This agreed with the present
data. Zinc uptake was essential co-
factor and substrate requirements
associated with inflammation, im-
mune reactivity, and tissue injury or
repaid (Tudor et al., 2005). The
lowest zinc versus higher copper
was associated with osteoporotic
manifestations in coordination with
increased levels of cytokines TNF
& IL-6 agreed with Faker et al.
(2000). The role of ceruloplasmin as
lipid peroxidation inhibitor repre-
sented a compensatory defense
strategy to reduce the vascular dam-
age extent from lipid peroxides in-
duced by ROS in smokers.
The present study confirmed the
implication of CSI as a risk factor
for bone mineral density. Osteopo-
rosis gave a decrease in bone miner-
al density (BMI) and emergence of
focal skeletal changes (Vonda and
Wright, 2006). This could have de-
veloped alongside the cumulative
impact of smoking that influences
osteoporosis by oxidative stress
known to be associated changes in
trace elements (El-Dardiry and El-
Marzouki, 2001) and colinked with
GIT & SHF posed by factors repre-
sented a higher magnitude of oxida-
tive stress in GI > GII the gas phase
of cigarette smoke inhaled as well
as from generation of ROS via cel-
lular inflammatory response to CSI
(Johnson et al., 1990)
In the present study, TNF, IL-6
showed response to fascioliasis with
or without schistosomiasis in chron-
ic immunoinflammatory lesions
produced by eggs and adult worms
and ended to hepatic fibrosis. The
present study also showed increased
levels of adhesion molecule E-
selectin in parallel to the magnitude
of vascular injury. This activated
the release of acute phase proteins
including ceruloplasmin. In agree-
ment with reports on fascioliasis to
be associated with cytokine affec-
tion, decreased antioxidant capacity
and alteration of serum trace ele-
ments and correction of these al-
tered parameters and immune as-
pects changes observed after treat-
ment with fasciolicidal drugs
(Rehim et al., 2003; Massoud et al.,
2004). This fact was supported by
the present results where TNF, IL-
6 & ceruloplasmin decrements to-
gether with increments in AOC by
supplementation and treatment of
798
fascioliasis with or without schisto-
somiasis. Efficacy of Mirazid in
treating fascioliasis and/or schisto-
somiasis must be added to many
previous reports (El Baz et al.,
2003; Hegab and Hassan, 2003;
Abo-Madyan et al., 2004a,b; Mas-
soud, et al., 2007; Tonkol and
Morsy, 2008).
Generally speaking, both fascio-
liasis and schistosomiasis are en-
countered in Egypt. Zoonotic fascio-
liasis is increasing in Egypt (Soli-
man, 1998; Haseeb et al., 2002) and
worldwide (Mas-Coma and
Bargues, 1997). Besides, Marcos et
al. (2009) reported that fascioliasis
is an example of an emerging tropi-
cal infe-ction which can be present
in chronic liver diseases, requiring
greater clinician awareness especial-
ly in endemic rural areas.
On the other hand, osteoporosis
can develop either secondary to
fascioliasis or as idiopathic (50%)
cause of life style. Early osteoporo-
sis development was influenced by
androgenic profile, smoking and
free radical mechanisms by fascio-
liasis and/or schistosomiasis and
sedentary life. Vascular endotheli-
um damage and dual in-fluence of
smoking and hepatic efficiency by
toxic metabolites deposits as co-
tinine led to imbalance between
hormonal milieu, increased lipid
peroxidation and immuno-
inflammatory indices affected &
increased bone resorption. Treat-
ment control androgenic imbalance
by hormonal milieu, affecting oste-
oporosis and antioxidant causing
immuno-inflammatory response
influencing bone formation.
Conclusion
The adjustment of hormonal status
and antioxidant potential is a must
by treatment of parasitosis and
smoking cessation seriously to
maintain physical fitness and to stop
the early onset of osteoporosis. The
impact of life style on bone, risk
factors as smoking, hypoandro-
genemia and/or glucose intolerance,
metabolic and immunoinflammatory
derangement added by fascioliasis
and/or with schistosomiasis fibrosis
induced oxidative stress and early
development of osteoporosis in
middle aged smokers. The cessa-
tion of smoking and adopting active
life style and proper treatment read-
justed osteoporotic manifestations.
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 803 - 8
PREVALENCE OF DIPTEROUS FLIES ASSOCIATED WITH HUMAN
AND ANIMAL DISEASES IN MATRUH AND SOUTH SINAI
GOVERNORATES, EGYPT
By
AZZA S. ABD EL-HALIM; M.I. SOLIMAN and M.W. MIKHAIL
Research Institute of Medical Entomology, the General Organization
for Institutes and Teaching Hospitals, The Ministry of Health, Dokki,
Giza, Egypt.
Abstract
The present study identified the dipterous flies associated with human and
animal diseases in Matruh and South Sinai Governorates. The results indicated
that 49817 belonging to 13 families, 24 genera and 33 species were trapped
from Matruh Governorate and 3708 flies belonging to 9 families, 13 genera and
16 species were trapped from South Sinai Governorate from January to De-
cember 2009. M. domestica was the most abundant in both Governorates. Sta-
tistical analysis showed that species of all families were significantly higher in
Matruh Governorate than South Sinai Governorate due to spread of garbage,
fermented fruits and human & animal excreta.
Key words: Egypt, Dipterous flies, Matruh and South Sinai Governorates.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Dipterous flies are among the
most important insect that affect the
health of human and animals (Wil-
son, 1991). They act as vectors
pathogenic organisms either me-
chanically or biologically (Smith,
1973). The majority of species
breed in Carrion, decaying vegeta-
ble matter from which they may
carry pathogens to human food or
drink or directly to the human body
(Smart, 1965). Others attack the
human body producing different
types of myiasis.
This work aimed to survey and
identifies the dipterous flies of med-
ical and veterinary important in
Matruh and South Sinai Gover-
norates.
Materials and Methods
This work covered the whole year
2009 Dipterous flies was collected
from Matruh and South Sinai Gov-
ernorates during the period from
January to December 2009. The col-
lections were done by using a stand-
ard insect net around garbage accu-
mulation, garbage boxes (El-Bashier
et al., 2006) or sticky traps held
harm-pests. Also infested fruits,
vegetables, fishes excreta were ob-
tained and kept in small cages at

laboratory, till emergency of adults.


Identifications were carried out us-
ing keys given by Zumpt (1965);
Steyskal and El- Baily (1967); So-
liman and Morsy (1976); Shaumer
et al. (1977, 1985, 1989); Shaumer
and Kamal (1982, 1983); Mohamed
and Shoukry (1991); Morsy et al.
(1991) and Amin et al. (1997a,b).
Results
The results are shown in tables (1 to 4).
Table 1: Flies collected from Matruh and South Sinai Governorates.
Matruh XSEM
(Min. Max.)
South Sinai XSEM
(Min Max.)
Calliphora vicina R.O. 0.08 0.08 (0- 1) 0.8 0.2 (0 -2)
Chrysomyia albiceps (Wied.) 24.8 9.3 (0 -76) 7.4 2.4 (0 -24)
Lucilia sericata (Meig.) 18.2 5.4 (1- 54) 0.0 0.0 (0 -0)
Musca domestic a L. 1644.8 + 436.5 (201-4163) 289.5 30.3 (131-440)
Musca Sorbans wied. 12.8 5.4 (0-51) 3.0 1.2 (0 -12)
Muscina stabulance (Fallen) 1.8 1.1 (0 -13) 0.3 0.1 (0 -1)
Fannia canicularis (L.) 0.4 0.2 (0 -2) 0.0 0.0 (0 -0)
Stomoxys calcitrans (L.) 3.9 1.9 (0 -25) 0.0 0.0 (0 -0)
Hydrotaea meteorica (L.) 48.7 31.6 (0 -365) 0.0 0.0 (0 -0)
Synthesiomyia nudiseta (Van.) 0.2 0.1 (0 -1) 0.0 0.0 (0 -0)
Limnophora variegata (stein) 0.6 0.3 (0 -3) 0.3 0.1 (0 -1)
Limmophora multipunctata (s) 0.4 0.3 (0 -3) 0.3 0.1 (0 -1)
Adia cinerella (Fallen) 0.2 0.1 (0 -1) 0.0 0.0 (0 -0)
Physiphora demandata (Fabr.) 0.5 0.2 (0 -2) 0.0 0.0 (0 -0)
Piophila casie (L.) 22.9 6.1 (0 -64) 0.8 0.3 (0 -2)
Parasarcophaga hirtipes wied. 2.2 0.9 (0 -11) 0.0 0.0 (0 -0)
Coproica vagans (Haliday) 2147.7 529.4 (31 -5796) 1.6 0.4 (0 -4)
Cop. Ferruginata (Stenh.) 28.4 11.7 (2 -111) 0.0 0.0 (0 -0)
Cop. Digitata (Duda) 99.1 45.01 (0 -407) 0.0 0.0 (0 -0)
Ceroptera algira (Vill.) 5.4 2.4 (0 -23) 0.3 0.1 (0 -1)
Copromyza costalis Zetter. 0.5 0.5 (0 -6) 0.0 0.0 (0 -0)
Limosina brivicostata Duda 0.4 0.2 (0 -2) 0.0 0.0 (0 -0)
Limosina bifrons Stenh. 0.08 0.08 (0 -1) 0.0 0.0 (0 -0)
Meoneura vagans (Falln) 15.3 3.2 (0 -32) 1.5 0.8 (0 -9)
Hippelate pusio Low 30.5 11.2 (0 -134) 2.2 0.9 (0 -10)
Drosophila melanogaster Meig. 5.3 2.1 (0 -24) 0.6 0.2 (0 -2)
Drosophila histrioides O&K 5.08 2.05 (0 -19) 0.3 0.2 (0 -2)
Sepsis thoracica (Rob.- Des.) 27.3 15.04 (0 -187) 0.0 0.0 (0 -0)
Sepsis lateralis Wied. 0.8 0.4 (0 -3) 0.2 0.2 (0 -2)
Sepsis fissa Becker 1.6 0.9 (0 -12) 0.0 0.0 (0 -0)
Eristalis taeniops wied. 0.3 0.2 (0 -2) 0.0 0.0 (0 -0)
Eristalis aeneus (Scopol.) 0.7 0.4 (0 -4) 0.2 0.1 (0 -1)
Megaselia Scalaris (Loew) 0.6 0.3 (0 -3) 0.0 0.0 (0 -0)
Table 2: Dipterous flies from Matruh and South Sinai

Families
Matruh South Sinai
Genus Species Samples Genus Species Samples
Calliphoridae 3 3 516 2 2 98
Muscidae 7 9 20564 3 5 3520
Anthomyidae 1 1 2 0 0 0
Otitidae 1 1 6 0 0 0
Piophilidae 1 1 275 1 1 9
Sarcopagiedae 1 1 26 0 0 0
Sphaeroceridae 4 7 27379 2 2 22
Milichiidae 1 1 184 1 1 18
Chloropidae 1 1 366 1 1 26
Drosophilidae 1 2 125 1 2 11
Sepsidae 1 3 356 1 1 2
Syrphidae 1 2 11 1 1 2
Phoridae 1 1 7 0 0 0
Total 24 33 49817 13 16 3708
Table 3: Flies collected at South Sinai from January to December 2009
Species 1 2 3 4 5 6 7 8 9 10 11 12 13
Calliphora vicina 1 1 1 2 0 0 0 0 1 2 1 0 9
Chrysomyia albiceps 1 2 1 5 0 0 24 11 18 15 11 1 89
Lucilia sericata 0 0 0 0 0 0 0 0 0 0 0 0 0
Musca domestica 199 177 279 245 401 190 370 402 440 380 260 131 3474
Musca sorbans 2 4 2 12 11 0 0 0 2 1 1 1 36
Muscina stabulance 0 1 0 0 0 0 0 0 1 1 0 0 3
Fannia canicularis 0 0 0 0 0 0 0 0 0 0 0 0 0
Stomoxys calcitrans 0 0 0 0 0 0 0 0 0 0 0 0 0
Hydrotaea meteorica 0 0 0 0 0 0 0 0 0 0 0 0 0
Synthesiomyia nudiseta 0 0 0 0 0 0 0 0 0 0 0 0 0
Limnophora variegata 1 0 1 0 0 0 0 1 0 0 0 0 3
Limmophora multipunctata 0 0 0 1 0 0 1 0 1 0 1 0 4
Adia cinerella 0 0 0 0 0 0 0 0 0 0 0 0 0
Physiphora demandata 0 0 0 0 0 0 0 0 0 0 0 0 0
Piophila casie 0 1 0 2 0 0 1 0 2 1 2 0 9
Parasarcophaga hirtipes 0 0 0 0 0 0 0 0 0 0 0 0 0
Coproica vagans 2 3 2 2 0 0 0 0 3 4 2 1 19
Cop. Ferruginata 0 0 0 0 0 0 0 0 0 0 0 0 0
Cop. Digitata 0 0 0 0 0 0 0 0 0 0 0 0 0
Ceroptera algira 0 1 0 0 0 0 0 0 1 1 0 0 3
Copromyza costalis 0 0 0 0 0 0 0 0 0 0 0 0 0
Limosina brivicostata 0 0 0 0 0 0 0 0 0 0 0 0 0
Limosina bifrons 0 0 0 0 0 0 0 0 0 0 0 0 0
Meoneura vagans 0 9 0 0 0 0 0 0 2 5 2 0 18
Hippelate pusio 0 10 0 5 0 0 0 0 3 6 2 0 26
Drosophila melanogaster 1 1 2 0 0 0 0 0 2 0 1 0 7
Drosophila histrioides 0 0 0 2 0 0 0 0 1 0 1 0 4
Sepsis thoracica 0 0 0 0 0 0 0 0 0 0 0 0 0
Sepsis lateralis 0 0 0 2 0 0 0 0 0 0 0 0 2
Sepsis fissa 0 0 0 0 0 0 0 0 0 0 0 0 0
Eristalis taeniops 0 0 0 0 0 0 0 0 0 0 0 0 0
Eristalis aeneus 0 0 0 1 0 0 0 0 0 1 0 0 2
Megaselia Scalaris 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 4: Flies collected at Matruh Governorate from January to December


2009
Species 1 2 3 4 5 6 7 8 9 10 11 12 13
C. vicina 0 0 0 0 0 1 0 0 0 0 0 0 1
Ch. albiceps 0 0 0 2 2 1 23 63 68 76 62 0 297
L. sericata 4 7 1 42 17 26 2 8 10 44 54 3 218
M. domestica 201 278 295 1213 1036 1863 850 1910 3787 4163 3911 231 19738
M. sorbans 0 0 0 3 1 0 3 28 26 51 41 0 153
M. stabulance 0 0 1 0 13 2 2 0 1 2 0 0 21
F. canicularis 0 0 0 0 1 0 0 1 1 2 0 0 5
S. calcitrans 0 0 3 2 6 0 2 25 3 4 2 0 47
H. meteorica 0 1 1 34 159 365 3 0 11 7 3 0 584
S. nudiseta 0 0 0 0 0 0 0 0 1 1 0 0 2
L. variegata 0 0 0 1 3 2 0 2 0 1 0 0 9
L. multipunctata 0 0 0 0 0 0 1 3 0 1 0 0 5
A. cinerella 0 0 1 0 0 1 0 0 0 0 0 0 2
Ph. demandata 0 0 0 0 0 0 1 1 2 2 0 0 6
P. casie 41 64 0 0 44 0 6 27 16 21 12 44 275
P. hirtipes 0 0 0 0 2 0 2 6 11 4 1 0 26
Co. vagans 1410 1644 1950 2400 5796 903 112 336 31 3670 2810 4710 25772
Co. ferruginata 81 111 10 8 6 2 2 5 4 12 9 91 341
Co. digitata 302 407 0 0 63 0 6 27 3 18 12 351 1189
Ce.algira 18 23 0 0 0 0 0 0 0 10 2 12 65
C. costalis 0 0 0 0 0 0 0 0 6 0 0 0 6
L. brivicostata 0 2 0 0 1 0 0 1 0 1 0 0 5
L. bifrons. 0 0 0 0 1 0 0 0 0 0 0 0 1
M. vagans 17 21 0 0 20 0 11 31 12 32 19 21 184
H. pusio 26 30 0 0 8 0 32 134 2 71 26 37 366
D. melanogaster 1 1 0 0 7 0 1 4 24 14 10 2 64
D.histrioides 0 0 0 0 3 0 4 19 6 9 19 1 61
S. thoracica 2 4 0 0 13 0 24 187 37 26 32 3 328
S. lateralis 0 0 0 0 3 0 0 0 2 1 3 0 9
S. fissa 0 0 0 0 0 0 2 12 0 3 2 0 19
E. taeniops 0 0 0 0 0 0 0 2 0 1 0 0 3
E. aeneus 0 0 0 0 1 0 0 4 0 2 1 0 8
M. scalaris 0 0 0 0 1 0 0 2 0 3 1 0 7
Discussion
The survey yielded a total 49817
flies belonging to 33 species, 24
genera and 13 families, Calliphori-
dae, Muscidae, Anthomyiidae, Ot-
titdae, Piophilidae, Sarcophagidae,
Sphaeroceridae, Millichiidae, Chlo-
ropidae, Drosophilidae, Sepsidae,
Syrphidae, and Phoridae in Matruh
Governorate were collected during
the period of investigation, compa-
rable to 3708 flies belonging to 16
species, 13 genera and 9 families,
Calliphoridae Muscidae, Piophi-
lidae, Sphaeroceridae, Millichudae,
Chloropidae, Drosophilidae, Sep-
sidae and syrphidae in South Sinai
Governorate. Among the collected
species Musca domestica L. was the
most abundant in two Governorates.
The results agreed with Amin et al.
(1997a,b;1998). Lucilia sericata
(Meig), Fannia canicularis (L.),
Stomoxys calcitrans (L.), Hydrotaea
meteorica (L.) Syn-thesiomyia
mudiseta (Van.) Adia cinerella
(Fall.) Physiphora demandata (Fab)
Para-sarcophaga hirtipes Wied.,
Coproica ferruginata (Stemh.), C.
digitata (Duda), Copromyza costalis
Zetter, Limosina brivicostata Duda,

L. bifrons Stenh., Sepsis thoracica


(Rob.-Dec.), S. fissa Becker, Eris-
talis taeniops Wied. and Megaselia
scalaris (Loew) were found only in
Matruh Governorate.
Concerning the total number of
species of each family in discerning
order in Matruh Governorate was:
Muscidae (20564) >Sphaeroceridae
(27379) Calliphoridae (516) > Chlo-
ropidae (366) > Sepsidae (356) >
Piophilidae (275) >Milichuiidae
(184) Drosophilidae (125) >Sar-
cophagidae (26) >Syrphidae (11) >
Phoridae (7). However, in thede-
scending order in South Sinai Gov-
ernorate was: Muscidae (3520) >
Calliphoridae (98) > Chloropidae
(26)> Sphaeroceridae (22) > Milich-
iidae (18)>Drosophilidae (11)> Pi-
ophilidae (9)>Sepsidae (2)>Syrph-
idae (2).
The total number of Musca do-
mestica L. and Coproca vagans
(Haliday) were significantly higher
in Matruh than South Sinai (t = 6.8,
P < 0.001 and t = 10.8 P < 0.001
respectively). This may be attribut-
ed to the accumulation of garbage,
decaying fishes, human and animal
excreta which are the most attrac-
tive matter of this species. M. do-
mestica L. was the most abundant in
two governorates. This agreed with
Morsy et al. (1991); Amin et al.
(1997) and Abdel Halim et al.
(2005).
Conclusion
The out come of this survey of the
dipterous flies in the two ecological-
ly different Egyptian governorates
pave the way for good understand-
ing of the zoonotic diseases trans-
mission and prevalence in each
governorate. Thus, feasible control
measures can be proposed.
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009

J. Egypt. Soc. Parasitol., 39 (3), 2009: 811- 820

APOPTOSIS AND HISTOPATHOLOGICAL LESIONS IN PARASITE-
INFECTED SPECIES OF BIVALVE MOLLUSCS

By
AHMED ABDEL AZIZ
Department of Zoology, Faculty of Science, Cairo University, Egypt

Abstract

Apoptosis (programmed cell death) is an important mechanism for preserva-
tion of a healthy and balanced immune system in vertebrates. Little is known,
however, about how apoptosis processes regulate invertebrate immune system.
Thus, the present histopathological study was performed to search for the level
of apoptosis on hemocytes of three bivalve species namely, Macoma edentula,
Hiatula rupelliana and Gastrana fraglis in the Timsah Lake infected with
branchial Rickettsia-like parasites and ciliated parasites in the digestive gland.
Microscopically, special to elongated intracytoplasmic Rickettsia-like colonies
were observed in the base of gill filaments of the clams. Histologically, the dis-
tribution and shapes of apoptotic cells were classified into three main types.
The aggregation of apoptotic cells were observed in the apical and the basal
parts of the ciliated cells lining the gill epithelium. These results provide a first
insight into apoptotic processes in mollusc immune cells.
Key words:Histopathology,apoptosis,Bivalvia,branchial Rickettsia-like parasi-
tes,Ultrastructure, Macoma edentula, Hiatula rupelliana , Gastrana fraglis.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------


Introduction

All living organisms are confront-
ed with multiple and simultaneous
threats in the natural environment.
Physiological and immunological
responses to these threats are at-
tempts by the animals to maintain
homeostasis of anatomy and func-
tion. In bivalve molluscs, after
physical and chemical variations in
tem-perature, salinity, and dissolved
oxygen, parasitic, pathogenic, and
toxic micro-organisms represent
some of the more persistent chal-
lenges to animal health. Pathogenic
bacterial and protozoan parasites are
often cited as factors limiting pro-
duction of bivalves species harvest-
ed for human consumption (Lee,
2000; Formiga-Cruz et al., 2002;
Potasman et al., 2002; Karin, 2009).
Also, harmful and toxic microalgae
are increasingly recognized as
stressors of the bivalve populations
(Shumway, 1990; Landsberg, 2002).
Moreover, an increase in harmful
algal blooms has been listed as one
188
of the growing concerns that may
contribute to increased impact of
diseases and parasites on important
marine resource species and the
food webs that support them
(Landsberg, 1996; Harvell et al.,
1999). Accordingly, studies on
physiological and immunological
changes induced by parasites and
harmful algal provide clues about
the importance of these stressors in
population biology and thus, pro-
duction of commercially important
bivalves.
Parasitic diseases can have lethal
effects on bivalves depending upon
the severity of infection. The proto-
zoan parasites have been shown to
affect the hemocytes of bivalves;
effects described include direct in-
fection of hemocytes by several
Bonamia ostreae cells resulting ul-
timately in lysis of hemocytes
(Coehennec-Laurean et al., 2003;
De Silva and Villalba, 2004). Simi-
lar cells damage also occurs in other
bonamiosis-affected oyster species.
Symptoms of Dermo disease,
caused by Perkinsus marinus in
Crassostrea virginica (Mackin et
al., 1950), include repressed oxida-
tive burst in oyster hemocytes in-
fected with P. marinus meronts
(Anderson et al., 1995). Moreover,
P. marinus affect phagocytosis, oxi-
dative burst, and apoptosis of hemo-
cytes in C. virginica (Yee et al.,
2005; Goedken et al., 2005). By
contrast, P. olseni does not affect
clam hemocytes, but inhabits con-
nective tissues within different or-
gans of Ruditapes sp., provoking
intense hemocytic infiltration and
parasite encapsulation (Casas, 2002;
Choi et al., 2005).
Thus, hemocytes provide an ex-
cellent biological material to study
host pathogen interactions because
the internal defense system of bi-
valves relies on these cells. Com-
parative studies of different host and
pathogen pairs with differing re-
sistance and virulence, respectively,
provide additional insight into why
particular hosts do or do not develop
disease when challenged, or why
certain microbes do or do not cause
disease. R. philippinarum is highly
susceptible to V. tapetis and hemo-
cytes of this clam could be killed by
contact with the pathogen (Allam et
al., 2000). In contrast, Mercenaria
mercenaria and C. virginica are not
susceptible to the pathogen (Maes
and Paillard, 1992; Paillard et al.,
1996; Allam et al., 2006). To ex-
plain the difference, hypothesis that
following contact with branchial
Rickettsia-like organism, cell apop-
tosis of three bivalve species could
be impaired to a different degree
was tested.
This work is a survey study, with
emphasis on morphology, pathogens
and involvement of branchial Rick-
ettsia-like organism in three bi-
valves; Macoma edentula, Hiatula
rupelliana and Gastrana fraglis,
and the morphological distinction
represented functional differences.

Materials and Methods

Specimens of Macoma edentula,
Hiratula rupelliana and Gastrana
188
fraglis were collected from the
heavily polluted areas of Timsah
Lake at the Western side of the Suez
Bay in an area closed to all fishing.
The animals were cleaned of fouling
organisms, transferred to the labora-
tory and maintained in aquaria sup-
plied with filtered sea water under
constant aeration. The green algae
were added as food.
About 20 clams from each species
were randomly chosen and pro-
cessed for analysis of pathological
conditions. These clams were
shucked and gill lamella was exised
from every specimen and processed
according to the Ray (1966) method
for detection of Perkinsus-like para-
site, which basal on incubation of
bivalve tissue in fluid thioglycolate
medium.
Apoptosis: Tissues intended for
light microscopy were fixed in al-
coholic Bouin's fluid, dehydrated in
ethanol series, cleaned in xylene,
and embedded in paraffin wax. Par-
affin sections (3-4 m thick) were
stained with hematoxylin and eosin.
EM: Small pieces of examined
tissue from non-infected and para-
site-infected clams were fixed in
2.5% buffered glutaraldhyde with
phosphate-buffer solution (pH
=7.2). After over night fixation,
small pieces of the tissue were post-
fixed in 1% osmium tetraoxide, then
gradually dehydrated in ascending
series of alcohols and tissues were
embedded in low viscosity epoxy
resein. Ultrathin sections were
maintained on uncoated copper
grids and double-stained with aque-
ous uranyl acetate and lead citrate
and examined using trans-mission
electron microscopy.

Results

The parasitic ciliate organism is
tear-drop shaped with a prominent
basophilic lobulated nucleus (Fig.
1A). These cells were situated with-
in the cytoplasm of the digestive
epithelial cells of the digestive
gland (Fig. 1B). No ciliates were
observed in any organs of the snail,
or in any other part of the digestive
tract. Moreover, no conjugation or
dividing cells were observed. On
transverse section, the ciliates ap-
peared round with faint eosinophilic
cilia (Fig. 1C), a silver stained cili-
ate, depicting the oblique rows of
cilia running a long the length of the
cells (Fig. 1D). The visible cilia
were of considerable length. The
nucleus appeared spherically lobular
and centrally positioned. On the an-
terior tip, a densely staining rod ex-
tends into the cytoplasm, possibly
composing part of the cytopharyn-
geal structure. Fig. 1D & E are two
photos of the silver stained ciliates
depicting the rod structure on the
anterior tip of the cell. Fig. (1F)
shows a ciliate in transverse section,
with the rod clearly visible in the
centre of the anterior end indicating
the rod extend into the cell. The
shape of the nucleus confirmed that
the lobules were nuclei material,
which preferentially stains chromo-
somal material or DNA.
Ciliates were only observed with-
in the digestive cells of pear snail
188
digestive gland. The basophile cells,
with their abundant RER, directly
adjacent to infected digestive cells,
appeared unaffected (Fig. 2A). In
contrast, absorptive digestive cells
inhabited by ciliates had disrupted
cytoplasm, with vacuolation. Cyto-
plasmic membrane was ill-defined
(Fig. 2B). Nucleus of the infected
cells showed degenerative changes.
In some sections, segments of a sin-
gle memb-rane were between the
ciliate and host cell, with the ab-
sence of double membrane or host
cytoplasmic material within this
area, indicates that ciliate may be
surviving within a phagocytic vacu-
ole inside the host cells (Fig. 2C). In
cases where up to three ciliates in-
habited a single cell, each was sepa-
rated by host cytoplasmic material
(Fig. 2D).
Histologically, Rickellsia-like sp.
parasitized nearly all of the intesti-
nal tissues, but more heavily and
frequently found in mantle, gonad,
bases of primary gill filaments and
interstitium tissues between the di-
gestive diverti-culua and around
digestive tubes. The structure of gill
filaments were some-what damaged
in the three tested species. Apopto-
sis of hemocytes in M. edentula and
H. repelliana revealed an aggrega-
tion of the apical and basal part of
the ciliated, non-ciliated and endo-
thelial cells lining the gill epitheli-
um (Fig. 3A-D). Apoptosis were
more numerous in the frontal epi-
thelium of the gill filaments in G.
fraglis (Figs. 3 E&F). Apoptotic
cells could be indicated by the pres-
ence of round or oval bodies (apop-
totic bodies) typically intensely dark
purple-blue masses (2.5 per cell)
and variable in size (Fig. 3 A-F).
Ultrastructurally, the apoptotic
cells in the gill filaments of the
three tested species were classified
into three main types; with many
possible inter-mediates. A cluster of
densely packed type I cells was
found in M. eduntula and identified
by the highest nuclear (N) / cyto-
plasmic (C) ratio situated at the pe-
riphery of the gill filaments (Figs. 4
A & B). The more detached type II
found in H. rupeliana containing up
to 10 relatively large homogenous
electron dense granules per section
and observed in the epithelial com-
ponent of the gill filaments (Figs. 4
C&D). Type III, containing up to 30
granules per section, which vary in
size and electron density was gener-
ally found towards the center of the
gill filaments of G. fraglis (Figs. 4
E&F).
Generally, the cytoplasm of the
type II is more electron dense then
of the type III (Figs. 4 A-F).








N
V
N
188








































Fig. 2: (A) Non infected cell with normal nucleus (N). (B) Infected cell show-
ing degenerate nucleus (N) and numerous vacuoles (V). (C) Nucleus (N) of
digestive cell with numerous lysosme (L). (D) Parasites within segment of
membrane (M) within cytoplasm of a digestive cell. X 2400.

A B
C D
N
V
N
N
N L
M
188
















Fig. 4: Gill filaments of M. edentula (A, B), H. rupelliana (C, D) and Gastrona
frglis (E, F) infected with Rickettsia-like parasite. Note varied number of elec-
tron dense granules of apoptotic cells (arrows). X2400.

A B
C D
E
F
188
Discussion

This is the first histopathological
study of three species of bivalves par-
asitized by Rickettsia-like organism.
Ciliates were the second commonest
parasite, found in the studied species.
Except the Rickettsia-like organisms,
which were reported in Hippopus
hippopus (Norton et al., 1993), Vene-
rupis rhomboidies (Villalba et al.,
1999), Ostrea puelchana (Kroeck and
Montes, 2005) and Argopecten pur-
puratus (Karin, 2009). In the present
study, the ciliate parasites constituted
the first record for their host and for
Egypt. Both host responses character-
ized by hemocytic encapsulation and
induction of homogenous esinophilic
substances were very similar to those
of the previous reports from other
species of clams (Sagrista et al., 1995;
McLaughlin and Faisal, 1998; Lee et
al., 2001). Montos et al. (1996) stated
that the eosinophilic substances origi-
nate from hemocytes and mainly con-
sist of a defense molecule, so-called
polypeptide 225. Tissue lysis ob-
served in oysters infected with P.
marinus (McLaughlin and Faisal,
1998) was also observe in the present
study.
In the present study, different lev-
els of apoptosis were demonstrated
in the hemocytes distributed in the
gill filaments of the three tested
species of molluscs. This improve-
ment in the cellular integrity was
demonstrated by the light and elec-
tron microscopical analysis and re-
sults from two major processes:
DNA repair and apoptosis. The re-
pair of DNA strand breaks in mod-
erately damaged cells, and the elim-
ination of critically injured cells or
with low capacity for DNA repair
(i.e. aging cells), probably act to
protect the snails from further toxic
effect of stress and parasites in natu-
ral environment (Christl et al.,
2004; Cermonte et al., 2005; Jokela
et al., 2005; Galimany et al., 2008).
The plasma protein and human
fibronectin strongly induced C. gi-
gas hemocytes to undergo apoptosis
(Lacoste et al., 2002). In the present
study, the increase in the number
and distribution of dead hemocytes
(i.e., decrease in the percentage of
hemocyte survival) occurred in par-
allel with the degree of hemocytes
with the morphological features of
apoptosis. Recent studies also
showed that the normal responses of
hemocytes of different species of
snails to a wide variety of parasitic
infections have the potential to
damage the genetic materials or in-
terfere with the process of cell divi-
sion (Piger et al., 2004; Goodken et
al., 2005; Pearce et al., 2005; He-
garet et al., 2007).

Conclusion

Classic hypothesis would be that
such a process participates in the
maintenance of the immune system
homeostasis by controlling hemocyte
longevity and clearance, by eliminat-
ing abnormal or infected cells or by
promoting the removal of hemocytes
from sites of inflammation, as record-
ed in vertebrate (Osborn, 1996). Fur-
181
ther work is needed to clarify the ba-
sis of this phenomenon and to deter-
mine the precise function of hemo-
cyte apoptosis in the different snail
species.

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Explanation of Figures

Fig. 1:Digestive gland of M. edentula containing parasitic ciliates (arrows) (A), H. rupelliana
containing parasitic ciliates (arrows) (B) and Gastrana fraglis containing parasitic ciliate (arrow)
(C), stained with hematoxylin and eosin .(D) A silver stained parasites(arrows). (E) A silver
stained parasite (arrow).(F) A silver stained parasite in transverse section(arrow).Scale bar: 2
m.
Fig. 3: Gill filaments of M. edentula (A, B), H. rupelliana (C, D) and Gastrana fraglis (E, F)
infected with Rickettsia-like parasite(R). Note frequent distribution of apoptotic cells (arrows).
(A, C and E X60), (B, D and F X110).
8



8


821
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009

J. Egypt. Soc. Parasitol., 39 (3), 2009: 821 - 826

ELISA-SEROPREVALENCE OF TOXOPLASMA GONDII IN
DRAUGHT HORSES IN GREATER CAIRO, EGYPT
By
FOUAD M. HARIDY
1
, NAHLA M. SHOUKRY
2
, ALY AWAD HASSAN
3

AND TOSSON A. MORSY
4
General Organization for Veterinary Services (formerly)
1
, Department of
Zoology, Faculty of Science, Suez Canal University
2
and Departments
of Parasitology
3,4
, Faculty of Veterinary Medicine, Cairo University
3
, and
Faculty of Medicine, Ain Shams University, Cairo 11566
4 ,
Egypt

Abstract

Toxoplasma gondii is one of the important zoonotic parasites of worldwide.
In this paper the seroprevalence of T. gondii in draught horses (3-15 years) in-
cluding 90 males and 10 females in the first half of the year 2009 was studied.
The result showed that the overall ELISA-T. gondii antibodies were 25% of the
horses in Greater Cairo, 50% (females) and 22.2% (males).
Keywords: Egypt, ELISA, Toxoplasma gondii, draught horses.
Present as a poster ISC International Conference Emerging Zoonotic Diseases,
Cairo, Egypt, 14 17 October 2009
----------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------

Introduction

Toxoplasma gondii is an intracel-
lular protozoan parasite of world-
wide distribution (Strickland, 2000).
T. gondii infects most tissues in
mammals and various tissues in avi-
an species and horses (Aganga et
al., 1983; Uggla and Nilsson, 1985;
Marsh et al., 1996; Fenger et al.,
1997; Kisthardt and Lindsay, 1997).
Rinaldi and Scala (2008) stated that
T. gondii is one of the most com-
mon parasites of man and other
warm-blooded animals. The public
health organizations repeatedly en-
courage the accurate data of animals
and humans due to its importance as
a source of zoonosis. Infection dis-
play subclinical or chronic course in
equines and serological technique
for antibodies against T. gondii has
great diagnostic value (El Shazly et
al., 1996). Non-human protozoa
parasites including toxoplasmosis
were successfully diagnosed with
serological methods (El Shazly et
al., 2001) as ELISA (Uggla and
Nilsson, 1985: Haridy et al., 2007;
Elsheikha and Morsy, 2009).
In Egypt, toxoplasmosis was re-
ported in man (Rifaat and Morsy,
1968; Elsheikha et al., 2008), in
particular children (Wishahy et al.,

822
1973) and women (Eissa et al.,
1990) and edible animal (Rifaat et
al., 1977) and birds (Rifaat et al.,
1969) and stray animal (Khalid et
al., 1982) as well as rodents (Morsy
et al., 1987).

Materials and Methods

This study was carried out in the
first half of the year 2009 in Greater
Cairo. Blood samples without anti-
coagulant were collected from 100
draught horses (90 males & 10 fe-
males) with ages from 3-15 years.
Blood samples were allowed to clot
on lab bench at room temperature in
an upright position for few hours,
centrifuged at 3000 rpm for 20
minutes to separate sera which were
stored at -20C until tested.
Sera were diluted 1: 200 in PBS
pH 7.2, and analyzed in a conven-
tional ELISA system carried out
principally (Voller et al., 1976). The
micro-titration plates (Nunc, Roskil-
de, Denmark) were coated overnight
at 4C with a soluble antigen prepa-
ration consisting of a sonicated de-
tergent extract of purified freeze
thawed T. gondii trophozoites (Ug-
gla et al., 1990). The antigen used
was at a protein concentration of
1g/ml of 50mM carbonate buffer
pH 9.6. After washing in PBS with
0.05% tween 20, 100l of the dilut-
ed serum was added to each well
and incubated at 37C for 2 hours.
After repeated washing with PBS-
Tween, 100 l of anti-horse IgG
conjugated with alkaline phosphates
(Sigma) in PBS was added and in-
cubated for 3 hours at room temper-
ature after which the plates were
again washed and shaken dry, then
50l of p-nitro phenyl phosphate
was added at concentration of 1mg/
ml substrate buffer (Sigma) were
added to all wells and the plate were
incubated at 37C for 30 minutes.
The enzymic hydrolysis of substrate
was stopped by the addition of 50 l
2M NaOH. Reading was carried out
at 400 nm using ELISA reader. Ab-
sorbance values over 0.3 were con-
sidered positive.

Results

A total of 25/100 sera of the
draught horses were positive to T.
gondii (25%) by ELISA. They were
five females (50%) and twenty
males (22.2%).

Table 1: ELISA-prevalence of T. gondii in horses carts in Greater Cairo.

Sex No of horses Positive cases Negative cases
Males 90 20 (22.2%) 70 (77.8%)
Female 10 5 (50%) 5 (50%)
total 100 25 (25%) 75 (75%)



823


Fig. 1: Three sick horses
824
Discussion

In the present study, ELISA-
prevalence of T. gondii in Egyptian
draught horses was 50% (females)
and 22.2% (males) with an overall
prevalence of 25%. In Nigeria,
Aganga et al. (1983) using IHA re-
ported 37.1%. In China, Ling and
Wan (1984) using IHA reported
4.5%. In Brazil, Leite-Larangeria et
al. (1985) by IFAT reported 32.8%.
In Germany, Erb (1986) by IFAT
reported 55%. In Turkey, Akca et
al. (2004) in Kars Province reported
39% in local horses, and Gl et
al. (2007) in Ankara Province re-
ported 28% in sport horses. The dif-
ferences in the prevalence of anti-
Toxoplasma antibodies might be
due to the habitat, geographic, or
climatic conditions or due to differ-
ence in the serologic techniques
used. In general, T. gondii causes
subclinical infection in horses, and
thus diagnosis was performed using
various serological tests to detect
antibodies against this infection.
Kijlstra and Jongert (2008) report-
ed recent calculations of the disease
burden of toxoplasmosis rank this
foodborne disease at the same level
as salmonellosis or campylobacteri-
osis. The high disease burden in
combination with somewhat bad
results of the currently available
treatment options have led to a plea
for more effective prevention. They
added that Toxoplasma as a hazard
associated with the consumption of
undercooked meat or meat products
and provides an analysis of the vari-
ous options to control the risk of
human toxoplasmosis via this
source.
In Egypt, the consumption of
horse meat is prohibited by law and
unwanted horses being culled.
However, the horse meat is accepted
in some countries abroad. Undoubt-
edly, the horses are herbivores ani-
mals they acquire T. gondii infec-
tion when ingesting food contami-
nated with oocysts.
Generally speaking, T. gondii is a
serious threat particularly to preg-
nant, non-immune women and their
babies. Cat feces and meat are the
most important sources of infection.
The horse is not a direct source of
infection for human, but may indi-
rectly spread the infection to human
and other herbivores via the cats
which consumes infected horses
carcass and then shed oocysts in the
environment. Monitoring and sur-
veillance programs may be imple-
mented for pre-harvest control of
Toxoplasma infection of farm ani-
mals, with the reduction of the envi-
ronmental oocystic load as the most
important milestone.
No doubt, the livestock manage-
ment influences the parasites risk.
The extensive management of
parasitosis increases the health of
the animals and consequently hu-
man welfare, but may increase the
infection risk by an arthropod vector
825
and/or an intermediate host trans-
mitting the zoonotic parasites.

Conclusion

In the epidemiological studies of
toxoplasmosis for recommendation
of the feasible control measures
non-human hosts as horses and
donkeys, as well as stray animals
must be considered. This is particu-
larly true in the developing and un-
der development countries were eq-
uines are one of the main vehicles
of transportation or source of meat
according to social habits in differ-
ent regions in the world.

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2:371-84
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Herez, H.A.; Younis, T.A.; Morsy,
T.A., 1990: Placental villous matu-
ration in patients with repeated
abortions and chronic toxoplasmo-
sis. J. Egypt. Soc. Parasitol., 20, 2:
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1997: Epizootic of equine protozoal
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Gl, Z.; Karaer, Z.; Babr, C.;
Kili, S., 2007: Investigation of To-
xoplasma gondii Antibodies in sport
horses in Ankara Province. Trkiye
Parazitol. Dergisi, 31:264-7.
Haridy, F.M.; Morsy, G.H.;
Abdou, N.M.I.; Morsy, T.A.,
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1

Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 827 836
MOTHER KNOWLEDGE, ATTITUDES, PERCEPTION REGARDING
INTESTINAL PARASITES AND DIARRHOEA IN THREE REGIONS
OF GAZA STRIP, PALESTINE
By
BASIL J. KANOAAND ADNAN I. AL-HINDI
Department of Biology, Faculty of Science, The Islamic University of
Gaza, P.O.Box 108, Gaza, Palestine
E-mail:ahindi@iugaza.edu.ps, Fax:00970-8-2860800
Abstract
The knowledge, attitudes and practice (KAP) among mothers towards intesti-
nal parasites and diarhoea in three regions in Gaza strip were studied. A total of
659 mothers of children attended a primary health care centre (PHCC) for med-
ical services were selected. Data were obtained through self administered ques-
tionnaire which distributed to each mother attending the PHCC. The question-
naire included some sociodemographic, economical information and imple-
mented in year 2006. In the present study age group ranged between 15 and
more than 35 years. It was found that children belonging to mothers in the age
groups 15-25 years and >35 years old were found infected with intestinal para-
sites and diarrhea and had similar prevalences (37.3 & 37.1%). Mother educa-
tion had a positive effect for the decreasing of parasitosis among children. The
variation in the prevalence of intestinal parasites due to region was noted where
the south of Gaza Strip had the high prevalence (40.6%) with a significant dif-
ference (p=0.004). Children living in houses with sandy yards was infected
with intestinal parasites more those living in houses with tiles (p=.02).
Key words: Intestinal parasites, diarrhea, knowledge, perception
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Intestinal parasites are widely
prevalent worldwide and were re-
ported in different studies in the
community of Gaza strip. Diarrhoea
predispose to childhood morbidity
and mortality in developing world
(WHO, 1997).
The intestinal parasites in Gaza
strip was first reported by Yassin et
al. (1999). Then many studies were
done in the last decade and found
different prevalence's of intestinal
parasites ranged between (24-70%).
Yassin et al. (1999) found a rate of
27.6%, Shubair et al. (2000) found
24.5%, while Al-Hindi (2002) re-
ported a rate 36.3%. Al- Wahaidi
(1997) reported overall prevalence
48.0% & 12.0% in both Beach camp
and Rimal area. Studies of intestinal

parasites showed prevalence rate of


53.0% in Yemen, 18.4-27.8% in
Saudi Arabia, 74.0-93.0% in Ethio-
pia, 14.3% in Bahrain, 23.1% in
United Arab Emirates (UAE) and
58.0-85.0% in Lebanon (Araj et al.,
1996). The knowledge is very im-
portant on the prevalence of intesti-
nal parasites worldwide. Udonsie et
al. (1996) found that improvement
of health education reduced the re-
infection rate of intestinal nema-
todes. In the Gaza strip the role of
health education in decreasing the
prevalence of intestinal parasites
was statistically significant (Kanoa
et al., 2006). Knowledge, attitudes,
and practices towards intestinal par-
asites were studies by many authors
(Kamunvi et al., 1993) reflected
relatively poor comprehension of
causes, treatments and methods of
prevention towards intestinal hel-
minthes in Kenya. In upper Egypt,
mothers considered worms to be
very harmful to health and the ma-
jority of them agreed that worms
should be treated in addition good
level of knowledge was observed
for ways of preventing infection by
washing hands and vegetables (Cur-
tale et al., 1998).
In Gaza strip the only study re-
garding KAP on intestinal parasites
and diarrhea did not represent all
Gaza Strip but focused on the Al-
Nussirat refugee camp, Ab-Murad
(2004) reported that prevalence of
intestinal parasites was 24.1%
among children aged 1-4 years; and
diarrhea was 10.1% among children
less than 1 year.
The present study aimed to test
the KAP among mothers towards
intestinal parasites and diarhoea in
three regions in Gaza strip.
Subjects, Materials and Methods
The present study was a clinic
based investigation through a cross-
sectional survey on 659 mother's of
children who attended a primary
health care centres (PHCC) seeking
for medical services in three regions
of Gaza Strip. Data were collected
from three regions representing all
Gaza strip including North gover-
norate (Jabalia, Beit-lahia and Beit
Hanoun), Gaza and South gover-
norate included (Rafah,
Khanyounis, Garara and Abassan).
Data were obtained through self
administered questionnaire distrib-
uted to each mother attending
PHCC. The questionnaire included
some sociodemographic, economi-
cal as age, education of mother, in-
come, residence, occupation, num-
ber of children per family, house-
hold characters, number of rooms/
house, house ground, house playing
yard, and environmental health data
as source of water, sewers type, dis-
posal of garbage, breeding animals
indoors, bathroom enclosed to the
kitchen.
Knowledge, attitude and practices
the KAP for intestinal parasites and
diarrhea were also collected.
Ten health educators from the
Ministry of Health kindly shared in
the data collections.

Data analysis: Data were comput-


erized and analyzed statistically us-
ing SPSS (version 8). Frequency,
cross tabulation and chi-square test
were used.
Results
In the present study, age group
ranged from 15 to >35 years, major-
ity was in age group 26-35 (40.1%).
This study included three regions;
Gaza 224 (34.0%); while South
Governorate represented 218
(33.1%) and the North was 217
(32.9%). Most of mothers were
married 594 (90.1%). 251/659
women (38.1%) had seco-ndary
school certificate. The working
women were 27.3% (Tab. 1).
Most of the mothers were from
the city 364 (55.2%), and most chil-
dren were in family with 3-6 per-
sons (48.6%), other variables like
house ground, house playing yard
and family income differed (Tab. 2).
The main source of drinking water
was the tap of municipality 397
(60.2%). The most common type of
sewers was the closed one 515
(78.1%). For disposing of garbage,
most mothers were used nylon bags
555 (84.2%), while less used open
containers. Eighteen percent of the
mothers breed animals indoors.
Most kitchens were found closed to
the bathroom 407 (61.8%).
Most of the mothers (Tab. 4) said
that their children had diarrhea 510
(77.4%) with different number of
episodes. High percentage of inter-
viewed mothers 611 (92.7%) know
how to deal with diarrhea. They re-
ported that watery stool gave a
prevalence of 55.2% as symptoms
during diarrhea.
High percentage of the mothers
(Tab. 5) knows intestinal parasites
595 (90.3%), and 44.6% of them
said that contaminated food and
drinks was the main infection route.
Cleaning food and drinking water
was the most preventative methods
adopted by mothers against intesti-
nal parasites 462 (70.1%).
Misuse of medication was obser-
ved among the mothers, but using of
antibiotics in the treatment of intes-
tinal parasites was in 180 (27.3%).
Table 1: Household characters in three regions in Gaza Strip.
% Frequency variability
55.2
31.4
13.4
364
207
88
Residency: City
Residency: Village
Residency: Refugee camp
28.1
48.6
23.4
185
320
154
Number of children/family: <3
Number of children/family: 3-6
Number of children/family: >6
6.2
75.6
18.2
41
498
120
Rooms/house: 1 room
Rooms/house: 2-4 room
Rooms/house: >4 room
19.4
80.6
128
531
House ground: Sandy
House ground: Cemented
35.5
64.5
234
425
House playing yard: Yes
House playing yard: No

Table 2: Environmental health status in three regions in Gaza Strip


% Frequency variability
15.8
60.2
24.0
104
397
158
Water source: Wells
Water source: Tap of Municipality
Water source: Filtered water
21.9
78.1
144
515
Sewers opened
Sewers closed
84.2
15.8
555
104
Disposal of garbage: In nylon bags
Disposal of garbage: Open containers
17.9
82.1
118
541
Breeding animals in doors: Yes
Breeding animals in doors: No
61.8
38.2
407
252
Bathroom enclosed to kitchen: Yes
Bathroom enclosed to kitchen: No
Table 3: Mothers, socio-economic, demographic characters, education and job.
% Frequency Mothers variability
30.5
40.1
29.4
201
264
194
Age-group (year):15-25
Age-group (year):26-35
Age-group (year):>35
34.0
33.1
32.9
224
218
217
Residence: Gaza
Residence: South
Residence: North
90.1
6.1
3.8
594
40
25
Married
Widow
Divorced
45.4
31.3
23.4
299
206
154
Monthly income: <200 $ (low)
Monthly income:200-500$ (Middle)
Monthly income: >500 $ (High)
5.9
9.1
19.9
38.1
27.0
39
60
131
251
178
Education: Illiterate
Education: Primary school
Education: Preparatory school
Education: Secondary school
Education: University degree
27.3
72.7
180
479
Working: Yes
Working: No
72.1
26.1
1.8
475
172
12
Mother: Housewife
Mother: Worker
Mother: Employee
Table 4: Infection of children with relation to environmental health.
With diarrhea
No. %
With intestinal parasites
No. %
Variability
82 (78.8) 47 (45.2) Water source: Well (n=104)
304 (76.6) 143 (36) Water source: Municipality (n=397)
124 (78.5) 61(38.6) Water source: Filter (n=158)
(
2
=0.38; p >0.05) (
2
=2.96; p >0.05)
428 (77.1) 208 (37.5) Garbage: Plastic bag (n=555)
82 (78.8) 43 (41.3) Garbage: Open container (n=104)
(
2
=0.15; p >0.05) (
2
=0.55; p >0.05)
324 (79) 165 (42.2) Bathroom enclosed to kitchen (n=410)
186 (74.7) 86 (34.5) No ( n=249)
(
2
=1.65; p >0.05 (
2
=2.13; p =0.08

Table 5: Mothers ages of infected children.


Table 6: Mother's knowledge about intestinal parasites infection
% Frequency Variable of mothers knowledge
90.3
9.7
595
64
Intestinal parasites: Yes
Intestinal parasites: No
8.2
44.6
15.2
4.6
27.5
54
294
100
30
181
Intestinal parasites transmission: No answer
Intestinal parasites transmission: Contaminated food & drink
Intestinal parasites transmission: Contaminated water
Intestinal parasites transmission: Contaminated food
Intestinal parasites transmission: Using of patient's materials
19.1
42.8
18.5
4.9
11.2
3.5
126
282
122
32
74
23
Symptoms of intestinal parasites: No answer
Symptoms of intestinal parasites: Diarrhea and fever
Symptoms of intestinal parasites: Refuse eating
Symptoms of intestinal parasites: Stool with blood and mucus
Symptoms of intestinal parasites: Vomiting
Symptoms of intestinal parasites: Itching
21.5
12.7
35.5
19.7
5.2
5.3
142
84
234
130
34
35
Complications of intestinal parasites: No complications
Complications of intestinal parasites: Anaemia
Complications of intestinal parasites: Dryness of mucus membranes
Complications of intestinal parasites: Weakness
Complications of intestinal parasites: Weight loss
Complications of intestinal parasites: Intestine clogging
18.5
70.1
6.7
4.7
122
462
44
31
Preventative methods: No answer
Preventative methods: Cleaning of food and water
Preventative methods: Cleaning of hands
Preventative methods: Consultation of physician
6.4
30.0
24.0
27.3
12.0
42
200
158
180
79
Treatment type: Don't know the type
Treatment type: Giving purgative
Treatment type: Giving Flagyl
Treatment type: Giving antibiotics
Treatment type: Giving purgative and Flagyl
49.6
50.4
327
332
Participation in health education: Yes
Participation in health education: No
14.6
85.4
96
563
Types of intestinal parasites: One type
Types of intestinal parasites: More than one type
With diarrhea
No. %
With intestinal parasites
No. %
Age group
152 (75.6) 75 (37.3) 15-25 y (n=201)
216 (81.8) 104 (39.4) 26-35 y (n=264)
142 (73.2) 72 (37.1) >35 y (n=194)
510 (77.4) 251 (38.1) Total
(
2
=5.26; df=2, p =0.07 (
2
=3.45; df=4, p >0.05)
29 (74.4) 18 (46.2) Mother: Illiterate (n=39)
44 (73.3) 24 (40.0) Mother: Primary (n=60)
94 (71.8) 44 (35.1) Mother: Preparatory (n=131)
211 (84) 102 (40.6) Mother: Secondary (n=251)
132 (74.2) 61 (34.3) Mother: University (n=178)
(
2
=10.59; p =0.03) (
2
=3.45; p >0.05)
236 (46.3) 115 (45.8) Income: Less than 200$
160 (31.4) 79 (31.5) Income: 200-500$
114 (22.4) 57 (22.7) Income more than 500$
(
2
=1.41; p >0. 05) (
2
=0.099; p >0. 05)
470 (79.1) 227 (38.2) Married (n=594)
34(85.0) 22 (55.0) Widow (n=40)
6 (24.0) 2 (8.0) Divorced (n=25)
(
2
=43.07; p =0.001 (
2
=14.45; p =0.001
126 (24.7) 59 (23.5) Mother work (n=180)
384 (75.3) 192 (76.5) Not work (n=479)
(
2
=7.72; p =0.004) (
2
=2.96; p =0.05)

Table 7: Infection of children and sanitation and house characters


With diarrhea
No. %
With intestinal parasites
No. %
Residence
184 (36.1) 79 (31.5) Gaza (n=224)
170 (33.3) 102 (40.6) South (n=218)
156 (30.6) 70 (27.9) Gaza North (n=217)
(
2
=6.68; p =0.03) (
2
=10.88; p =0.004)
134 (26.3) 60 (23.9) Family < 3 individual (n=185)
264 (51.8) 126 (50.2) Family 3-6 individual (n=320)
112 (22.0) 65 (25.9) Family >6 individual (n=154)
(
2
=9.28; p =0.01) (
2
=3.84; p >0.05)
106 (82.8) 59 (46.1) Sandy house ground (n=128)
404 (76.1) 192 (36.2) Or Tile (Balat) (n=531)
(
2
=2.66; p =0.06) (
2
=4.31; p =0.02)
30 (5.9) 19 (6.7) House: 1 room (n=41)
406 (79.6) 197(78.5) 2-4 room (n=498)
74 (14.5) 35 (13.9) >4 room (n=120)
(
2
=22.24; p =0.001 (
2
=5.69; p =0.05
98 (83.1) 49 (41.5) Animal indoors (n=118)
412 (76.2) 202 (37.3) No (n=541)
(
2
=2.63; p =0.06 (
2
=0.72; p >0.05
Discussion
The age of the investigated women's
was in the reproductive age who
attended the primary health care
centres, where their age ranged be-
tween 15->35 years old. The num-
ber of females in Gaza Strip at the
age group of 15-49 years old was
estimated to 295,480 (43%) from all
Gaza strip females (MOH, 2005).
The three studied regions were
found to represent the Gaza Strip;
the north governorate (including
Jabalia, Beit-lahia and Beit Hanoun)
was the highly dense populated area
as estimated by 281. 727 people
especially in Jabalia refugee camp
as a highly crowded area and side
by side homes. Followed by Gaza
516.882 person where it represents
the city as urban community and
high socio-economic status. The
south governorate included (Rafah,
Khanyounis, Garara and Abassan)
where some areas considered as a
village 460.667 person (PCBS,
1999). This showed a variation in
knowledge of mothers between the
three studied regions. In the study
(45.4%) of women were of low in-
come level. This reflected the pov-
erty in the Palestinian community
reported by UNRWA emergency
Appeal (2008). Abu-Murad (2004)
reported that the Israeli siege and
closure has had a devastating impact
on the economic situation of the
Palestinians in general and the refu-
gees in particular.
The education level of the Pales-
tinian was improved highly and the
present study supports this urge
where the university educated
women recorded (27%).

Table 8: Mothers (n=659) Knowledge and practice towards diarrhoea


% Frequency Mothers knowledge variability
77.4
22.6
510
149
Diarrheic child: Yes
Diarrheic child: No
23.2
30.7
33.4
12.7
153
202
220
84
Number of episodes: Non
Number of episodes: 1-3 times
Number of episodes: 4-7 times
Number of episodes: >7 times
92.7
7.3
611
48
Mothers know steps: Yes
Mothers know steps: No
14.3
44.0
28.2
13.5
94
290
186
8
Steps taken with diarrheic child: Oral rehydration salt
Steps taken with diarrheic child: Breast feeding
Steps taken with diarrheic child: Giving potato to child
Steps taken with diarrheic child: Going to Physician
38.8
13.1
34.0
14.1
256
86
224
93
Reasons of diarrhea: Cold
Reasons of diarrhea: Food type
Reasons of diarrhea: Playing outdoors
Reasons of diarrhea: Artificial feeding
12.1
8.8
55.2
23.8
80
58
364
157
Diarrhea symptoms: Increasing times of defecation
Diarrhea symptoms Stool bulk
Diarrhea symptoms Watery stool
Diarrhea symptoms Dryness of mucous membrane
3.3
26.4
7.9
62.4
22
174
52
411
Diarrhea complications: No answer
Diarrhea complications: Constipation
Diarrhea complications: Refuse eating
Diarrhea complications: Refuse eating & tired
95.8
4.2
631
28
Opinion of laboratory examination: Yes
Opinion of laboratory examination: No
80.9
19.1
533
126
Receiving health education: Yes
Receiving health education: No
18.2
42.2
10.6
29.0
120
278
70
191
Health education place: No answer
Health education place: Primary health care center
Health education place: Media
Health education place: Pharmacy
5.2
33.1
30.0
24.3
7.4
34
218
198
160
49
Importance of stool examination: No answer
Importance of stool examination: for diarrhea reasons
Importance of stool examination: for parasite type
Importance of stool examination: for bacteria type
Importance of stool examination: To determine treatment
Gaza Strip is a high dense popu-
lated area, (48.6%) had children 3-
6/family. The crowding index re-
flected the problem of crowdness in
small a limited area, (75.6%) lived
in house with 2-4 room. This agreed
with Abu-Murad (2004).
Houses grounds were cemented
(80.6%) and (19.4%) with sandy
grounds. This may pose the trans-
mission of infective stages in sand.
Municipality tap for drinking was
60.2% regular controlled by chlo-
rination (Gaza Municipality, 2008).
Al-agha and Mortaja (2005) found
in parts of Gaza the chloride and
nitrate contents of domestic water
exceeded WHO guideline. Drinking
water must be monitored regularly
to avoid. Al-Sharif (2003) reported
that the main source of contamina-
tion of the wells water in Beit-lahia

was waste water treatment facility


(WWTF). Improvement in the infra-
structure was observed in the last
decade where (78.1%) of women
whose houses connected to closed
sewers. The awareness was highly
towards the garbage disposal where
(84.2%) women used plastic bags.
A considerable percent of the inves-
tigated women was found breeding
animals (17.9%), as zoonotic para-
sites can't be excluded. Also, the
close distance between kitchen and
bathroom was high (61.8%). This
reflected the high possible transmis-
sion and contamination inside the
houses in case of intestinal parasitic
infection and diarrhea causing or-
ganisms. Also, due to the increas-
ing in the population in small area
of land put more pressure on sewer
system and seeping from pipes and
contact to drinking water occurred.
The regular sewers flooding are
very common in Gaza Strip streets.
The women (77.4%) reported that
their children had diarrhea. This
phenomenon indicated the difficult
health situation of the children and
risk factors related to diarrhea. In-
testinal parasitic diseases were re-
ported in many studies in Gaza Strip
in the last decade (Al-Hindi and Al-
Zain, 2008; Kanou et al. 2006; AL-
Wahaidi, 1997). Artificial feeding
was common. The prevalence of
diarrhea was similar to 62% (Jouda
and Abu-Ayada, 1998) and differed
from 54% (Jouda and Abu-Ayada,
1999) with variant episodes number.
High knowledge (92.7%) was re-
garding diarrhea. The oral rehydra-
tion solution (ORS) was of low use
(14.5%) compared to continuous
breast feeding (44%). The results
agreed with Al-Whaidi (2006) wo
found that most women do not stop
breast feeding and ORS was poorly
used by mothers with different edu-
cated levels. In the present study
cold and playing with dirties were
the commonest causes of diarrhea
(38.8%) and (34%) respectively. Te
agreed with Jouda (1999) who re-
ported 42.1%, while Al-Wahaidi
(2006) found that the commonest
causes of diarrhea were cold and
teething. So, cold represented the
main cause of diarrhea (Jouda and
Abu-Ayada 1998, 1999; Al-Wahai-
di, 2006). Again, physician consul-
tation in diarrhea was high (93.6%).
Watery stool was the most symp-
toms of them (55.2%).
The present study showed that
80.9% mothers received health edu-
cation in primary health care cen-
ters. Varied responses were reported
regarding the importance of stool
examination. Al-Wahaidi (2006) re-
ported that treatment of diarrhea
was practiced on the expense of
ORS.
The highest children with intesti-
nal parasites and diarrhea (39.4%)
and (81.8%) of mothers with age
26-35 years (P<.0.05 & p=0.07)
respectively. No doubt, early mar-
riage affected mothers health.
Children of Illiterate mothers was
the most infected (46.2%), while
there was similar prevalence for
both primary and secondary (40%)
and (46%). This brings that similar

chances and sucibtability for para-


sites for all. This was due to the fact
that most of the mothers had sec-
ondary education. The group with
parasites and diarrhea had family
income < 200$ (45.8%-46.3%), re-
flecting the socioeconomic differ-
ence. The children of widow mother
had highest intestinal parasites
(55%) and higher diarrhea (85%).
This may be correlated to the moth-
er care and reflect the care of each
parent towards their children. Chil-
dren whose mother work had lower
parasites (23.9%) compared to those
of none working one (76.5%). The
working mother had a chance of
education as116/180 (64.4%) had
barcarole degree P=0.05, P=0.04).
The highly infected children were
from south (40.6%), those with diar-
rhea were from Gaza (36.1%).
South is an agricultural area with
poor ignored sewers where
khanyounis had cesspool sewage
system. The viral and bacterial en-
teritis were common in children es-
pecially in summer. Abu Elamreen
(2007) reported that infections with
bacterial pathogens peak during the
summertime when there are suitable
conditions such as humidity and
high temperature which facilitate
bacterial growth and dissemination.
A total of 27.3% of mothers gave
antibiotics. Abu Elamreen (2007)
reported that misuse of antibiotics
resulted in increased resistance to
drugs. Family member had crucial
effect on prevention of infection,
50.2% of children with intestinal
parasites belong to family size (3-6)
and 51% of diarrhea.
Conclusion
Prevalence of intestinal parasites
and diarrhea was found in children
in spite of the knowledge and per-
ception of their mothers. Improve-
ment of health conditions is a must
side by side with mother education.
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837
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 837 - 847
FASCIOLA GIGANTICA: LARVAL PRODUCTIVITY OF
SHEEP-DERIVED MIRACIDIA IN RADIX NATALENSIS
AND GALBA TRUNCATULA
By
Y.D. DAR
1/2
, D. RONDELAUD
2
and G. DREYFUSS
2
Department of Zoology, Faculty of Science, University of Tanta, Tanta
1
,
Egypt, and UPRES EA no. 3174, Faculties of Medicine and Pharmacy,
87025 Limoges
2
, France
Abstract
Experimental infections of Egyptian Radix natalensis and French Galba
truncatula with miracidia of Fasciola gigantica of sheep were carried out to
determine the larval productivity of this parasite. Rediae and cercariae were
thus counted in snails dissected at regular intervals from day 21 to day 49 post-
exposure (p.e.) at 24C, while cercarial shedding was studied in other two
groups of snails after day 30 p.e. At day 49, the total number of free rediae and
that of cercariae-containing rediae in R. natalensis (shell height, 10.0 mm)
were 71.5 and 44, respectively, whereas mean values in G. truncatula (shell
height, 5.7 mm) were 57.3 and 33 rediae, respectively. The life span of cercar-
ia-shedding snails, the prepatent period, the patent period, and the total number
of cercariae shed showed insignificant differences between both snail species.
Compared to the data already obtained with a cattle isolate of parasite, the
number of live rediae was significantly greater in G. truncatula and significant-
ly lower in R. natalensis when exposed to sheep-originating miracidia. In cer-
cariae, the differences between cattle- and sheep-derived infections were insig-
nificant, whatever snail species. The results may be explained by the existence
of an interpopulation of snail infection with F. gigantica, probably due to var-
iations in frequency of natural encounters between this snail population and the
parasite isolate. However, the better production of rediae and cercariae in G.
truncatula might be due to the origin of snails used for this study because allo-
patric snails produced more larvae than sympatric congeners when they are
subjected to experimental infections.
Key-words: Fasciola gigantica, Galba truncatula, Radix natalensis, larval
burden, sheep-derived miracidia.
Correspondence: Dr. Y. Dar, Department of Zoology, Faculty of Science, Universi-
ty of Tanta, Egypt. Fax: (20) 20403350804. E-mail: yaser_disoky@yahoo.com
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
838
Introduction
Numerous factors may influence
on the success of a digenean infec-
tion in the snail host. According to
Smyth and Halton (1983), there are
three types: those which depend on
the mollusc, those which come from
the parasite, and environmental
factors. The necessity of a favoura-
ble temperature and that of slow
running water are known from a
long time for Fasciola hepatica
(Taylor, 1965). However, the effects
of some biotic factors, and particu-
larly that of the definitive host spe-
cies from which the eggs of the
adult fluke are obtained, are not still
known accurately. In F. hepatica,
the greatest cercarialproduction in
Galba truncatula was obtained with
cattle- or nutria-derived miracidia,
while the lowest was that of snails
infected with miracidia of lago-
morphs (Rondelaud and Dreyfuss,
1995; Vignoles et al., 2004). Be-
sides, more rediae and cercariae of
F. hepatica were found in infections
of Pseudosuccinea columella with
cattle-derived miracidia than in
those subjected to exposures by
marmoset-derived miracidia
(Mendes et al., 2008). In contrast,
the results are more conflicting in
the case of F. gigantica. Indeed, Al-
Kubaisee and Altaif (1989) found
that snails (Radix auricularia) in-
fected with buffalo-originating mi-
racidia produced fewer cercariae
than those exposed to a sheep-
derived isolate. Mohamed et al.
(1998) reported an inverse finding
was noted because snails (Radix
natalensis) infected with sheep-
derived miracidia produced a great-
er number of cercariae than those
exposed to isolates coming from
buffaloes or cattle.
The discrepancy noted in the lar-
val production of F. gigantica in
their snail hosts raised the question
of whether the species of the defini-
tive host would not have an effect
on the subsequent larval develop-
ment of the parasite in the interme-
diate host. The redial development
of F. gigantica had not yet well
studied (Al-Kubaisee and Altaif
1989; Mohamed et al., 1998).
Dar et al. (2004) reported the ex-
perimental infections of two
lymnaeid species (R. natalensis &
G. truncatula) with cattle-derived
miracidia of F. gigantic, which was
noticed in other experimental infec-
tions by using the same populations
of snails and sheep-originating.
The present work aimed to study
redial and cercarial production of
this digenean in two species of snail
hosts to answer: How many rediae
of F. gigantica did the snail develop
within its body by using isolate
from cattle or sheep? How did the
dynamics of cercarial shedding oc-
cur over time during the patent pe-
riod? The outcome results were
compared with that obtained by the
same authors to analyze the influ-
ence of the definitive host on the
subsequent larval production of F.
gigantica in the snail host.
Materials and Methods
839
R. natalensis was living in an
irrigation canal at El Mansoria, Giza
Governorate. Snails measuring 4
mm in height originated from a
strain maintained in the laboratory
since 2002. Owing to the scarcity of
G. truncatula populations in Lower
Egypt (Dar et al., in press), the
snails used in the present study
came from a small pool at Veyrac,
department of Haute Vienne, central
France. Snails measuring 4 mm in
height were collected from this site
in September-October 2008. Eggs
of F. gigantica originated from the
abattoir of Tanta of Gharbia gover-
norate. They were collected from
the gall-bladders of infected sheep.
They were incubated at 20C for 20
days in the dark for the eggs of F.
hepatica (Ollerenshaw, 1971).
Two groups of 50 R. natalensis
were exposed to individual bimira-
cidial exposures with these miracid-
ia. A similar protocol was used for
two groups of 50 G. truncatula. The
first group of each snail species was
used to study redial burden in dis-
sected snails from day 21 post-
exposure (p.e.) up to day 49, where-
as the two others were utilized to
follow the dynamics of cercarial
shedding over time. After exposure,
the R. natalensis were reared in
covered aquaria with 5 snails per
liter of permanently-oxygenated
water, while the G. truncatula were
bred in 14-cm Petri dishes accord-
ing to the method by Rondelaud et
al. (2007). Water originated from a
spring head located on calcareous
soil so that the dissolved calcium
contents ranged from 60 to 73
mg/L. Pesticide-free lettuce was
given ad libitum as food for snails.
These aquaria and dishes were
cleaned every week. Besides, water
present in Petri dishes was changed
every day. Aquaria and dishes were
placed in an air-conditioned room
under the following conditions: a
constant temperature of 241C, a
diurnal photoperiod of 12 hours
with a 3,000-4,000 lx light intensity.
To study the development of lar-
val burden, samples of 3-5 snails
each were collected from each
group and were dissected at days
21, 28, 35, 42, and 49 p.e. under a
stereomicroscope. The numbers of
live rediae, cercaria-containing re-
diae, and free cercariae present
within the snails body were count-
ed in the course of infection. At day
30 p.e., surviving snails from the
other two groups were individually
isolated in 35-mm Petri dishes, with
3-4 mL of spring water and a piece
of lettuce. These recipients were
placed in the same air-conditioned
room as above and water and let-
tuce, if necessary, were changed.
Metacercariae of F. gigantica were
daily enumerated and removed from
dishes. This surveillance continued
up to the death of snails.
The parameters noted in dissected
snails were the height of their shells,
the total number of live rediae, that
of cercaria-containing rediae, and
that of free cercariae. Those record-
ed for cercariae-shedding snails
were the survival of snails at day 30
840
p.e., the frequency of each snail
category: cercariae-shedding snails
(CS snails), infected snails died
without shedding (NCS snails),
uninfected snails, and the life span
of each category. For CS snails,
parameters were the length of the
prepatent period, that of the patent
period, and the total number of shed
cercariae. The percentages of fixed
and floating metacercariae were
also calculated. Individual values
noted for each parameter were aver-
aged and standard deviations were
established taking into account snail
species and snail group. A Pearson
correlation test, a Student t-test, a
Chi-square test, and one-way analy-
sis of variance were used to deter-
mine levels of significance (Stat-
Itcf, 1988).
Results
Redial burden: In the first two
groups, the shell height of snails
significantly increased (R. na-
talensis: r = 0.96, P < 0.01; G. trun-
catula: r = 0.99, P < 0.01) with the
length of infection until day 49 p.e
(Fig. 1). At day 49, R. natalensis
mean height (a mean of 10 mm) was
significantly greater (t = 9.471, P <
0.01) than in G. truncatula (5.7
mm). The total number of rediae in
R. natalensis significantly increased
(r = 0.84, P < 0.01) during the ex-
perimental infection, as did that of
cercariae-containing rediae (r =
0.94, P < 0.01) until day 49 p.e.
(Fig. 2). Also, the numbers of rediae
in G. truncatula showed significant
augmentations (live rediae: r = 0.91,
P < 0.01; cercariae-containing redi-
ae: r = 0.97, P < 0.01) during the
same period. At day 49 p.e., the
mean numbers of rediae were 71.5
in R. natalensis and 57.3 in G. trun-
catula, whereas numbers of cercari-
ae-containing rediae were 44/snail
for the former species and 33/snail
for the latter. The mean numbers of
free cercariae (Fig. 3) inside both
snail species reached pick values at
day 42 p.e. (160.5 cercariae in R.
natalensis and 254.3 in G. truncatu-
la) before decreasing at day 49 to
66.8 & 123, respectively.
Cercarial shedding: In the other
two groups, the survival rate of G.
truncatula at day 30 p.e. (Tab.1)
was significantly higher (
2
= 42.86,
P < 0.01) than in R. natalensis.
Among survivors, the percentage of
CS snails was significantly higher
(
2
= 9.54, P < 0.01) in G. truncatu-
la (96%) than in R. natalensis
(70%). The life span of CS snails,
the prepatent period, and the patent
period in both snails did not differ
significantly. No significant differ-
ence between the numbers of meta-
cercariae from both species was
noted. The floating cysts were 9.8%
in G. truncatula and 4.2% in R.
natalensis.
In R. natalensis, four peaks at
days 2, 8, 12 and 32 (a mean of
24.7, 26.8, 26.5, and 30 cysts/snail,
respectively) were noted (Fig. 4).
However, between days 20 and 30,
the number of cercariae per day per
snail only varied from 1 to 9. In G.
truncatula, the mean number of
841
cercariae peaked at day 5 (70
cysts/snail) before gradually de-
creasing until day 17 (4.3
cysts/snail). Two other peaks were
at days 20 & 28 (21 and 20
cysts/snail, respectively). After
wards, the values decreased until
day 43.
Table 1: Characteristics of the experimental infection of Radix natalensis and
Galba truncatula with Fasciola gigantica originated from sheep.
Snail species R. natalensis G. truncatula
No. of snails at the onset of experiment 50 50
No. of surviving snails at day 30 p.e. (%) 20 (40.0) 50 (100)
Snail categories (%):
- Uninfected
- NCS snails
- CS snails
4 (20.0)
2 (10.0)
14 (70.0)
2 (4.0)
0
48 (96.0)
Survival times (days)*:
- Uninfected
- NCS snails
- CS snails
70.0 13.0
49 5
68.3 13.5
66.5 17.5
0
59.0 10.0
Prepatent period (days)* 38.3 5.8 31.5 3.9
Patent period (days)* 30.0 9.0 27.5 10.9
No. of metacercariae per snail*. 200.0 77.8 285.0 100.6
Fixed metacercariae (%) 95.8 90.2
Floating metacercariae (%) 4.2 9.8
* Mean value S.D.
Discussion
In the present study, the shell
height of R. natalensis at day 49 p.e.
was greater than in G. truncatula
(10 mm instead of 5.7 mm). This
agreed with that of Hubendick
(1951) and Brown (1994) who
found that adults were 22 and 12
mm, respectively. This difference
can explain the redial burdens noted
in these snails (Fig. 2), as there was
a positive relationship between the
number of rediae and the size of
snail host (Zischke, 1967; Dar et al.,
2003, 2004). How-ever, the role of
snail species on the larval develop-
ment of F. gigantica cannot be ex-
cluded (Boray, 1978).
In the present study, the larval
burdens were compared with those
reported by Dar et al. (2004) for
experimental infections of both
snail species with miracidia origi-
nating. Number of live rediae at day
49 p.e. was significantly greater (F
= 10.21, P < 0.05) in R. natalensis
infected with cattle-derived miracid-
ia (92.7 8.1 rediae/snail) than in the
present study using sheep isolate of
parasite. An inverse finding was in
G. truncatula, with a significantly
842
higher (F = 9.20, P < 0.05) redial
burden in sheep-derived infections
compared with that by using cattle
isolate (42.3 5.4 rediae/snail). In
shed cercariae, the differences be-
tween cattle- and sheep-derived
infections were insignificant. How-
ever, the present study did not allow
affirming an effect of the definitive
host on the subsequent larval devel-
opment of F. gigantica in R. na-
talensis. This may be explained
(Mohamed et al. 1998) by the hy-
pothesis of admitting existence of
an inter-population variability of
snail infection with F. gigantica in
R. natalensis, and reported by Ron-
delaud (1993) in another model
snail/parasite (G. truncatula/F. he-
patica). He found that the frequency
of natural encounters between a
population of G. truncatula and the
parasite had an effect on the success
of experimental infections of this
snail. Consequantly, in the present
population of R. natalensis it would
be little susceptible to infection with
sheep-originating miracidia, proba-
bly owing of a few contact between
both partners in the field, while the
greater susceptibility of this snail in
cattle-derived infections (Dar et al.,
2004) resulted from more frequent
natural contacts between R. na-
talensis and cattle-coming miracidia
of F. gigantica. In G. truncatula,
the above hypothesis may explain
the difference noted in the redial
burden. However, another assump-
tion based on a greater redial and
cercarial production of F. gigantica
when allopatric snails (instead of
sympatric congeners) were expo-
sured to miracidia cannot be ex-
cluded in the G. truncatula/F. he-
patica. (Gasnier et al., 2001;
Goumghar et al., 2001)
The significantly lower number of
live rediae at day 49 p.e. in cattle-
derived infections of G. truncatula
might be due to snail breeding tem-
perature; 24C in the present study
and 20C (Dar et al., 2004). Ken-
dall (1953), Gold and Goldberg
(1979) found that a lower tempera-
ture lengthened the prepatent period
of larvae within their snail host and,
as a consequence, allowed a slower
differentiation of redial and cercari-
al over time. Despite variation in
breeding temperature, no difference
in the numbers of cercariae between
both groups of G. truncatula was
noted indicating the independence
of this environmental factor from
the number of cercariae shedding
from the snail.
In the present study, owing to the
scarcity of G. truncatula popula-
tions in the districts surveyed, fur-
ther studies on the susceptibility of
Egyptian lymnaeids to F. gigantica
must be considered by exposing
several populations to R. natalensis
to the same isolate of miracidia
coming from cattle or sheep. How-
ever, the present study showed the
existence of variations in the atti-
tude of Egyptian populations of R.
natalensis to sustain the larval de-
velopment of F. gigantica according
to the origin of the used miracidia.
Conclusion
843
There is a still incomplete adapta-
tion between Egyptian populations
of R. natalensis and sympatric mi-
racidia of F. gigantica, at least for
several isolates.
Acknowledgements
The authors are grateful to Dr. P.
Vignoles, University of Limoges,
France, for his kind input in the
realization of figures.
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efficient than sympatric ones. Int. J.
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Temperature effect on susceptibility
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Goumghar, M.D.; Dreyfuss, G.;
Rondelaud, D.; Benlemlih, M.;
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rived from cattle and marmoset
infections. J. Helminthol., 82: 81-4.
Mohamed, S.H.; Mostafa, O.M.;
Mohammad, A.H.; 1998: Suscep-
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Legend of figures
Fig. 1: Shell heights of R. natalensis and G. truncatula during experimental infec-
tion with F. gigantica.
Fig. 2: Redial development of F. gigantica in two lymnaeid snails infected with
sheep-coming miracidia.
Fig. 3: Number of free cercariae of F. gigantica counted in body of two lymnae-
id snails infected with sheep-coming miracidia.
Fig. 4: Number of F. gigantica cercariae per day per snail during patent period
in R. natalensis and G. truncatula.
845
0
2
4
6
8
10
12
21 28 35 42 49
Days post-exposure
S
h
e
l
l

h
e
i
g
h
t

(
m
m
)

R. natalensis
G. truncatula
Figure 1
Radix natalensis
0
20
40
60
80
100
120
21 28 35 42 49
Days post-exposure
N
u
m
b
e
r

o
f

r
e
d
i
a
e
Rediae
Rediae with cercariae
846
Galba truncatula
0
20
40
60
80
21 28 35 42 49
Days post-exposure
N
u
m
b
e
r

o
f

r
e
d
i
a
e
Figure 2.
0
100
200
300
400
21 28 35 42 49
Days post-exposure
N
u
m
b
e
r

o
f

f
r
e
e

c
e
r
c
a
r
i
a
e
R. natalensis
G. truncatula
Figure 3
847
Radix natalensis
0
20
40
60
80
1 5 9 13 17 21 25 29 33 37 41
Patent period (days)
N
u
m
b
e
r

o
f

c
e
r
c
a
r
i
a
e
Galba truncatula
0
20
40
60
80
1 5 9 13 17 21 25 29 33 37 41
Patent period (days)
N
u
m
b
e
r

o
f

c
e
r
c
a
r
i
a
e
Figure 4

Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 849 - 864
A NON OPIOID FAST TRACK ANESTHETIC REGIMEN FOR
COLONIC RESECTION
By
SOHAILA H. OMAR, KHALDA G. RADWAN, MAHA A. YOUSSIF,
HANAN F. KHAFAGY, NABAWEYA M. KAMAL, HOSSAM H. EL-
SABAE, AND HEND H. KAMEL
Department of Anesthesiology, Theodor Bilharz Research Institute, Im-
baba P.O.B. 30, Giza, Egypt
Abstract
Fast-tracking implies a preoperative patient care paradigm that reduces time
to recovery and discharge. The current study adopted fast-track anesthetic
techniques, comparing outcome of a multimodal non-opioid and another opioid
regimen, on recovery profiles after colonic surgery, with standard anesthetic
practice. Seventy five ASA II colectomy patients were randomly assigned to
one of three groups. Control group for conventional general anesthetic tech-
nique and two fast-track anesthesia groups using combined light general anes-
thesia and epidural techniques. Epidural maintenance was by infusion cocktail
of bupivacaine-fentanyl in opioid-based group, while in non-opioid group by
bupivacaine-ketamine which were both continued postoperatively for pain in
lower doses and concentrations. Postoperative analgesia in control group was
achieved by morphine. Supplemental ketorolac and acetaminophen were added
only to non-opioid group. Early and intermediate recovery profiles were com-
pared among the three groups together with recorded side effects. All patients
in fast-track groups had significant shorter times to: awakening, extubation,
orientation, both PACU arrival and discharge, hospital stay with a significant
lower mean VAS for pain at rest, and rescue analgesia, compared to control
group. Control group had a significant higher rate of postoperative nausea &
vomiting, drowsiness and pruritis. Non-opioid fast-track regimen had a signifi-
cant shorter PACU and hospital stay with lower side-effects rate than opioid
one. Fast-track anesthesia enhanced recovery profile. Non-opioid regimen was
superior to opioid-based, having a better recovery profile and a lower rate of
side-effects.
Key words: Fast-track- fast-track eligibility score system-multimodal anal-
gesia-recovery profile.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction
For several years, the concept of
fast-tracking in cancer colon sur-
gery has been developed. Fast-
tracking implies implementation of
a preoperative patient care paradigm
that reduces the time to discharge
home and resumption of normal
activities after both major (inpa-
tient) and minor (outpatient) surgi-
cal procedures (White et al., 2007).
The modality is based on a multi-
disciplinary approach provided by
surgeons, anesthesiologists, nurses
and physiotherapists, aiming at de-
creasing response to patho-
physiological changes induced by
surgical trauma (Roig et al., 2007).
The pivotal role played by the an-
esthesiologist in facilitating recov-
ery process has assumed increased
importance. The choice of anesthet-
ic drugs and concomitant medica-
tion can influence fast-tracking pro-
grams (Kranke et al., 2008). Such
programs were introduced success-
fully with enhanced recovery profile
in several surgical procedures, as
cardiac (Lena et al., 2008), colonic
(Roig et al., 2007; Mrat et al.,
2007) , abdominal aortic bypass
(Brustia et al., 2007), thoracic sur-
geries (Gregor et al., 2008; Mueh-
ling et al., 2008) and nephrectomy
(Recart et al., 2005). But, what is
the relative importance of individu-
al, surgical, or anesthetic features in
the enhancement of recovery
(Brustia et al., 2007).
This study aimed to focus on an-
esthesiologist's contribution in facil-
itating recovery, designed fast-track
modalities tailored for colonic re-
section surgeries, known for a great
degree of trauma and stress. These
involve short acting anesthetic
drugs, adjuvants and techniques.
The first was to confirm the influ-
ence of fast-track anesthetic tech-
nique, on recovery profile, fast-
track eligibility and patient out-
come, in such major procedures.
The second was to compare out-
come of two fast-track anesthetic
regimens, opioid-based and a mul-
timodal non-opioid regimen.
Subjects, Material and Methods
After obtaining institutional ethical
committee approval and written,
informed patient consent, 75 ASA
physical statuses II patients sched-
uled for elective major colonic re-
section surgeries were randomly
assigned to one of three anesthetic
treatment groups of 25 patients
each. Randomization to 3 groups
was performed using sealed enve-
lopes opened by anesthesiologist in
the operating room. Patients with a
history of unstable cardiovascular,
pulmonary, hepatic, renal, neuro-
logic, psychiatric, or metabolic dis-
eases were excluded. Patients re-
ceiving chronic benzodiazepine or
tricyclic antidepressant therapy
were also excluded. Groups com-
prised 1 group for conventional
general anesthetic technique (Con-
trol), and two fast-track anesthesia
(FTA) groups (using combined light
general and epidural techniques

with fast-track concept), with opi-


oid-based fast-track anesthesia reg-
imen in one group (OFTA), and a
non-opioid fast-track anesthesia reg-
imen in other group (NOFTA). All
preoperative data were collected by
a research assistant blinded to the
different anesthetic groups.
Blinding of patients and health
care providers was ensured. Upon
arrival at the operating room, rou-
tine monitors (non-invasive blood
pressure, electrocardiogram & pulse
oximeter) and end-tidal carbon di-
oxide monitoring were applied for
recording heart rate (HR), mean
arterial pressure (MAP), oxygen
saturation (SpO
2
) and end-tidal car-
bon dioxide (ETCO
2
). An arterial
catheter was inserted in the radial
artery under local anesthetic infiltra-
tion to monitor invasive arterial
blood pressure (Drager Infinity
Kappa, Monitor version VF-5W,
Germany). After insertion of two
large bore venous cannulae in the
forearms, all patients received intra-
venous midazolam 0.02 mg/ kg,
metoclopramide 10 mg and
ranitidine 50 mg as premedication.
A fluid pre-load with 500 ml of
crystalloid solution was also given.
Prior to induction, all patients were
given 100% oxygen, and baseline
hemodynamic variables were rec-
orded. Only in the two fast-track
groups lumbar epidural catheters
were inserted. Before induction of
general anesthesia, patient back was
sterilized and wrapped with identi-
fication of the interspace between
L1-L2 or L2-L3 where an epidural
catheter G20 was introduced
through Touhy needle G18 (B
Braun, Melsungen Germany) and
advanced 4-6 cm into the epidural
space using a midline approach.
After a test dose of 3 ml xylocaine
2% with adrenaline 1:200000, both
groups were given a 10 ml bolus
dose of a cocktail of (5 ml Bupiva-
caine 0.25% + 5ml Xylocaine 1%).
Group (OFTA) had a 25 g fentanyl
added to the cocktail, while
(NOFTA) group had Ketamine 0.5
mg/ kg added to the cocktail. The
level of somatosensory blockade
was determined by pinprick test.
Induction of general anesthesia
was achieved in all groups with in-
travenous administration of
propofol 2mg/kg. Fentanyl 2g/kg
(IV) was co-administered only in
the control group. Laryngoscopy
and tracheal intubation were facili-
tated with atracurium 0.5 mg/kg
(IV). After tracheal intubation, a
temperature probe, central venous
pressure (CVP) and urinary cathe-
ters were inserted for recording
body temperature, CVP and urine
output. Control group (C) anesthesia
was maintained by the standard
clinical practice, with general anes-
thesia: isoflurane 1-2% in combina-
tion with nitrous oxide 65% in oxy-
gen, supplemental doses of fentanyl
2550 g (IV) and top-up doses of
atracurium (0.1 mg/kg) to maintain
adequate analgesia and neuromus-
cular blockade. Both FTA groups
anesthesia were maintained with
combined light general and epidural
techniques. Light general anesthesia

was administered by subMAC


isoflurane 0.4 % in combination
with nitrous oxide 65% in oxygen,
together with propofol infusion 50-
100 g/kg/min guided by the hemo-
dynamic changes intraoperatively.
Intermittent top-up doses of atracu-
rium (0.1 mg/ kg) IV were used to
maintain adequate neuromuscular
blockade. Epidural maintenance
was achieved by an epidural infu-
sion cocktail that comprised in opi-
oid-based group (OFTA) 20 ml bu-
pivacaine 0.25% + fentanyl 1 g/
kg, while (NOFTA) group cocktail
comprised 20 ml bupivacaine 0.25%
+ ketamine 0.5 mg/ kg, at a rate of
4-6 ml /hr. Mechanical ventilation
was adjusted in all groups to main-
tain end-tidal carbon dioxide (CO
2
)
concentration of 32-36 mmHg. All
surgeries were performed by the
same surgical team.
Hemodynamic parameters were
recorded at 5-min intervals from
induction of anesthesia until the pa-
tient was discharged from PACU.
Increases in HR, or mean arterial
blood pressure (MAP) more than
20% of the pre-induction (baseline)
recordings were treated intra-
operatively by an increase of the
inspired isoflurane concentration in
the control group or the propofol
infusion rate for the two (FTA)
groups. Persistently increased HR or
MAP values not responding to in-
creasing the dose of the primary an-
esthetic were treated in control
group by supplemental doses of fen-
tanyl 2550 g (iv), while in the
(FTA) groups were treated by in-
creasing rate of epidural infusion.
Esmolol (0.2-0.5 mg/ kg) was given
as a second line of treatment for
such problem in all groups. Pre-
operative hypotension was defined
as a MAP reduction by more than
20% of baseline value and treated
with either 500ml hydroxyeth-
ylstarch 6% (Voluven, Fresenius
Kabi, Germany) when CVP value
was low, or bolus doses of ephed-
rine 6 mg incrementally. Bradycar-
dia was defined as reduction in
heart rate (HR) less than 50 beat/
min and treated by atropine 0.01
mg/ kg. Fluid administration was
adjusted to 10 ml /kg/h (5 ml /kg/h
for crystalloids and 5 ml/ kg/h for
colloids) in all groups. Blood loss
was substituted by colloid solution
and a hemoglobin value below 7
mg/ dl was considered as a trigger
for transfusion of packed red blood
cells. The active temperature control
included intravenous warm fluid,
total temperature management sys-
tem (Bair Hugger 750, USA), hu-
midified inspired gases, besides
maintenance of the operating room
temperature at 24C in all groups.
Thirty minutes before end of the
procedure, all patients were given
ketorolac 30 mg (IM). At end of
procedure all anesthetics were
turned off and residual muscle re-
laxant was reversed with intrave-
nous neostigmine 50 g/kg and at-
ropine 2g/kg. Patients were extu-
bated when they demonstrated satis-
factory return of motor function and
responsiveness. Response to com-
mands and orientation were as-

sessed in all patients prior to


transport to the post anesthesia care
unit (PACU).
Patients were not transferred to
hospital ward except after reaching
fast-track eligibility score (FTES)
12 in the PACU (Appendix I)
(White and Song, 1999).
Postoperative analgesia in both FTA
groups was provided by an epidural
infusion of 0.125% bupivacaine at a
rate of 4-5ml/ h. Fentanyl 25g, was
added to epidural infusion in OFTA,
while ketamine 0.3mg/kg was added
to epidural infusion in NOFTA
group. NOFTA group only received
ketorolac 30 mg( iv) every 6 hours,
and acetaminophen 1 gm infusion
every12 hours for the first 24 hour
postoperatively. Postoperative anal-
gesia for control group (C) was pro-
vided by morphine 0.1mg/ kg at end
of the procedure. Protocol for res-
cue medication in all groups was
morphine 0.1 mg/ kg (IV) for pain
when VAS >3, ondansetron 4 mg
(iv) for vomiting and nalbuphine 3
mg (iv) for pruritis. Visual analogue
scale (VAS) was recorded for pain
(0= none to 10 = maximal). A VAS
3 was considered mild, 4-7 rep-
resented moderate, and 8 was
considered severe.
The following data were investi-
gated and recorded: ASA (Ameri-
can Society of Anesthesiologists),
type and duration of surgery and
anesthesia, postoperative recovery
parameters (defining the end of sur-
gery as time zero), postoperative
pain evaluation at rest every hour
with a Visual Analogue Scale
(VAS), demand for additional res-
cue analgesia, postoperative com-
plications, length of stay in post-
anesthesia care unit (PACU) and in
hospital ward. The incidence of he-
modynamic abnormalities and re-
admission were also recorded. Re-
covery process, is divided into three
phases: early recovery, from the
discontinuation of anesthetic agents
until recovery of protective reflexes
and motor function; intermediate,
when the patient achieves criteria
for discharge; and late, when the
patient returns to his/her preopera-
tive physiological state (Awad and
Chung, 2006). The early recovery
end points recorded included times
to awakening (from discontinuation
of anesthetics until eye opening),
extubation (from discontinuation of
anesthetics until endotracheal tube
(ETT) removal), and orientation
(from discontinuation of anesthetics
until patients were able to correctly
state their name, or oriented to place
and time). Time to PACU admis-
sion was recorded and PACU dis-
charge was judged using the new
fast-track eligibility scoring system
(FTES). They both represent the
intermediate recovery phase. Pa-
tients were considered fast-track
eligible if they achieved a score of
12 (with no score <1 in any individ-
ual category). FTES was recorded
every 5 min from patient admission
to the PACU, until the patient
achieved fast-track eligibility12.
Supplemental oxygen was adminis-
tered only if the SpO
2
<96% on ad-
mission to the PACU. The need for

therapeutic interventions (supple-


mental oxygen, analgesic, or anti-
emetic rescue medications) in the
PACU hospital stay was recorded.
Epidural, CVP catheters and arterial
lines were removed before transfer
to the ward.
Appendix I: Fast-track eligibility score (FTES) and criteria (White and Song, 1999).
Level of consciousness:
Awake and oriented
Arousable with minimal stimulation
Responsive only to tactile stimulation
2
1
0
Physical activity:
Able to move all extremities on command
Some weakness in movement of extremities
Unable to voluntarily move extremities
2
1
0
Hemodynamic stability:
Blood pressure < 15% of baseline MAP value
Blood pressure 1530% of baseline MAP value
B1ood pressure > 30% below baseline MAP value
2
1
0
Respiratory stability:
Able to breathe deeply
Tachypnea with good cough
Dyspneic with weak cough
2
1
0
Oxygen saturation status:
Maintains value > 90% on room air
Requires supplemental oxygen (nasal prongs)
Saturation < 90% with supplemental oxygen
2
1
0
Postoperative pain assessment:
None, or mild discomfort
Moderate to severe pain controlled with IV analgesics
Persistent severe pain
2
1
0
Postoperative emetic symptoms:
None, or mild nausea with no active vomiting
Transient vomiting or retching
Persistent moderate to severe nausea and vomiting
2
1
0
Total possible score: 14
MAP = mean arterial pressure, IV= intravenous.
A minimal score of 12 (with no
score < 1 in any individual catego-
ry) would be required for a patient
to be fast-tracked after general anes-
thesia.
Statistical analysis: Based on dif-
ference in hospital stay in fast track
regimen compared with traditional
anesthesia technique (Mrat et al.,
2007), sample size of 25 patients in
each group gave 90% power to de-
tect difference p<0.05 using two
tailed and confidence Level 95%
(Norman and Streiner, 2008). Re-

sults were expressed as mean


standard deviation, or number (%).
Comparison between numerical data
of different parameters in all groups
was done by one way ANOVA with
post hoc LSD. Categorical data
were compared using Chi square
test. Data were considered signifi-
cant if p= 0.05, highly significant
if p< 0.01 and extremely significant
if p< 0.001. Analysis was carried
out by SPSS computer program
(version 16 windows).
Results
Patient characteristics and dura-
tion of surgery and anesthesia was
comparable (Tab. 1). All parameters
of recovery profile (Tab. 2) were
shorter in both fast-track anesthesia
groups when compared to the con-
trol group. Comparing NOFTA
group data to the control group
showed significantly shorter times
to: awakening, extubation, orienta-
tion, PACU arrival and PACU dis-
charge (P<0.001). OFTA group also
showed significantly shorter times
to: PACU arrival and PACU dis-
charge (P<0.05) and times to:
awakening, extubation and orienta-
tion (P<0.01) compared to control.
Hospital ward stay was significantly
shorter in NOFTA group (P<0.01)
and OFTA group (P<0.05) when
compared to the control group.
There was no significant difference
between FTA groups in recovery
profile except for time for discharge
from PACU and hospital ward stay,
which were shorter in NOFTA than
OFTA (P<0.05 and P<0.001 respec-
tively).
As regards postoperative side-
effects (Tab. 3), incidence rate of
drowsiness, nausea and vomiting
and pruritis was significantly lower
in NOFTA (P< 0.001) and in OFTA
group (P< 0.05) when compared
with control group, while headache
was significantly lower in NOFTA
(P< 0.01) and OFTA (P< 0.05),
when compared with the control
group. Constipation showed no sig-
nificant difference in incidence rate
in the three groups. Between FTA
groups there was a significantly
lower incidence rate of nausea &
vomiting, pruritis (P< 0.05) and
drowsiness (P< 0.01) in NOFTA
group compared to OFTA. Mean
VAS during first 24 hours was sig-
nificantly lower in both OFTA and
NOFTA groups compared with con-
trol (P<0.001), but there was no sta-
tistical difference between both
FTA groups. No patient required
rescue analgesia (morphine IV) dur-
ing the hospital stay in NOFTA
group. Consequently, NOFTA
group showed a statistically signifi-
cant difference in total morphine
consumption in the postoperative
period, when compared to both con-
trol (C) and OFTA group (P<
0.001). Also, there was a statistical
significant difference between
OFTA and Control (P< 0.001) (Ta-
ble III). No patient had hypertensive
episode intra-operatively that re-
quired medical interference. Hypo-
tensive episodes were dealt with by
administration of colloid and turn-

ing down propofol infusion rate and


isoflurane concentration in FTA and
control groups respectively. No
patient had respiratory depression in
the postoperative period or required
oxygen supplementation after dis-
charge from PACU. Only two pa-
tient were excluded from the study
as they were readmitted to surgery
same day for internal bleeding, and
they were replaced by two more
cases to fulfill data.
Table 1: Demographic characteristics and duration of surgery and anesthesia in groups.
Variables Control (n= 25) OFTA (n= 25) NOFTA (n= 25)
Age (yr) 50.1 5.6 51.4 5.2 52.6 3.4
Weight (kg) 91.4 6.5 88.9 7.4 87.6 8.2
ASA (I/II) 12/13 11/14 13/12
Gender (male/ female) 11/14 12/13 10/15
Duration of surgery (min) 204.7 27.9 190.6 40.3 183.4 50.4
Duration of anesth (min) 255.1 32.6 245.3 27.9 238.6 22.4
Data expressed as mean SD or number, OFTA = Opioid-based Fast-Track Anesthesia, NOFTA =
Non-Opioid Fast-Track Anesthesia, Anesth. = Anesthesia
Table 2: Recovery profile in groups.
Recovery Profile Variables Control OFTA NOFTA
Time to awakening (eye opening) (min) 13.9 8.1 8.9 3.2** 6.7 1.6***
Time to tracheal extubation (min) 14.6 5.8 9.8 4.8** 7.5 2.1***
Time to orientation (min) 16.7 6.2 11.6 5.2** 8.2 4.3***
Time to PACU arrival (min) 18.6 8.2 13.9 6.4* 10.4 4.7***
Time to PACU discharge (Time to Fast-
track eligibility score (FTES) 12 ( min)
93.6 32.8 73.9 24.8* 52.9 14.7***#
Hospital Ward stay (days) 7.3 1.2 3.5 0.6*** 2.1 0.5***###
*p< 0.05; **p< 0.01; ***p< 0.001 relative to control group,
#
p< 0.05; ###p<0.001 relative to OFT group,
OFTA = Opioid-based Fast-Track Anesthesia, NOFTA = Non-Opioid Fast-Track Anesthesia , FTES = fast-
track eligibility score, PACU = post-anesthesia care unit

Table 3: Postoperative variables and total rescue analgesic requirement


throughout hospital stay in groups
Variables Control OFTA NOFTA
Mean VAS first 24 hours 4.3 1.6 1.6 0.5*** 1.3 0.2***
Nausea & vomiting 18 (72%) 9 (36%)* 2 (8%)***,
#
Drowsiness 15 (60%) 7 (28%)* 0 (0%)***,
# #
Pruritis 13 (52%) 5 (20%)* 0 (0%)***,
#
Headache 12 (48%) 4 (16%)* 2 (8%)**
Constipation 3 (12%) 2 (8%) 0 (0%)
Rescue iv Analgesic Consumption (Morphine mg) 35.0 14.0 14.0 4.0*** 0.0 0.0***,
###
*p< 0.05; **p< 0.01; ***p< 0.001 relative to control group,
#
p< 0.05;
##
p< 0.01;
###
p< 0.001 relative to OFT
group, VAS = Visual analogue scale
Discussion
The present study succeeded in
providing a shorter and better re-
covery profile in fast-track groups
when compared with control group.
This was demonstrated by signifi-
cant earlier achievement of fast-
track eligibility, shorter length of
stay in PACU and hospital ward.
Remarkably, NOFTA group had a
shorter PACU stay than OFTA.
This may be attributable to absence
of opioid-induced adverse side-
effects that delay recovery process.
The lack of analgesic and antie-
metic requirements in NOFTA
group postoperatively might be at-
tributed to the use of a multimodal
"little of everything" combination of
neuroaxial NMDA antagonist keta-
mine with bupivacaine, the non ste-
roidal anti-inflammatory drugs
(NSAID) ketorolac and the non-
narcotic analgesic acetaminophen
IV. This multimodal analgesic ap-
proach appears to be superior to
opioid-based analgesia, in terms of
efficacy, but more so in terms of
outcome, since most adverse side-
effects incidence rates in NOFTA
group were significantly lower than
OFTA group. It was apparent that
the delayed recovery times and dis-
charge in the control group were
related to the higher incidence of
pain and morphine-induced side-
effects in that group during the early
postoperative period. Fast-track an-
esthesia incorporates early tracheal
extubation, decreased length of ICU
and hospital stay, and avoidance or
reduction of complications. These
complications will have a much
greater adverse effect on hospital
stay and healthcare costs. A growing
body of evidence from randomized
trials has identified many anesthetic
interventions that can improve out-
come after surgery. This includes
new short-acting hypnotic, opioid,
and neuromuscular blocking drugs.

Ideally, the process begins in the


preoperative period and extends into
the post-discharge period (Myles
and David, 2005).
The fast-track principles shorten
duration of hospitalization and re-
duce morbidity (Mrat et al., 2007).
One of the pioneers of multimodal
fast-track program was by Kehlet
and Holte (2001). Such fast-track
programs have already been intro-
duced in several surgical procedures
successfully, e.g. colonic surgeries
(Hjort Jakobsen et al., 2004; Wind
et al., 2006; Mrat et al., 2007; Roig
et al., 2007), thoracic surgery
(Gregor et al., 2008; Muehling et
al., 2008), abdominal aortic by-
pass(Fleron et al., 2003; Marret et
al., 2006; Brustia et al.,2007), ne-
phrectomy (Recart et al.,2005) and
cardiac surgery (Lena et al.,2008).
In the current study, a non-opioid
fast-track anesthesia regimen was
designed and targeted at avoiding
the cumbersome side-effects well
known for opioids, that delay re-
covery and prolong ICU stay and, in
general hospital stay. We quite well
succeeded in reducing PACU stay
in non-opioid group when compared
to opioid-based regimen. Of the
limitations of the current study is
that our data lack statistics for pa-
tient satisfaction, in spite of being
insinuative from our results as
NOFTA group had a rather smooth
uneventful recovery and patients
actually were verbally quite satis-
fied.
The anesthesiologist expanded to
involve responsibility for providing
rapid emergence from anesthesia
and avoiding postoperative side ef-
fects and early complications. The
evaluation of clinically meaningful
outcomes (quality of recovery, re-
sumption of normal activities of
daily living) has increasingly be-
come a focal point of anesthesia-
related clinical research involving
new drugs and techniques (White et
al., 2007).
Wind et al. (2006) listed many
factors in fast-track programs in-
cluding preoperative education of
patients, prevention of preoperative
fasting, application of glucose solu-
tions 2 hours preoperative, regional
anesthesia, short term anesthetics,
adequate preoperative fluids, pre-
vention of hypothermia, non-opioid
analgesics and early mobilization .
The ability to deliver a safe and ef-
fective anesthetic with minimal side
effects and a rapid recovery is man-
datory for fast-tracking patients
after surgery. Interest in facilitating
the recovery process following an-
esthesia has led to controversies
regarding the optimal anesthetic
technique (local vs. regional vs.
general), as well as the best types of
anesthetic drugs (volatile, intrave-
nous, muscle relaxant, local anes-
thetic, sympatholytic). Intravenous
drugs remain popular for sedation,
as well as induction of anesthesia,
because of their ease of administra-
tion, rapid onset of action and re-
covery, and high patient acceptance.
However, volatile (inhaled) anes-
thetics are more popular for mainte-
nance of anesthesia because of the

ease in titrating to an adequate


depth of anesthesia during surgery.
In addition, early recovery after
general anesthesia can be facilitated
by using a combination of nitrous
oxide (N
2
O), volatile anesthetics
with low blood: gas partition coeffi-
cients (desflurane or sevoflurane),
and short-acting sympatholytic
drugs (esmolol and dexmedetomi-
dine). The pre-emptive use of non-
opioid analgesics for prevention of
pain, and antiemetic drugs for
prophylaxis against postoperative
nausea and vomiting is also critical
to the success of a fast-tracking
general.
The present fast-track regimen
used combined light general anes-
thesia with epidural anesthesia, and
continued postoperative epidural
infusion for analgesia. Attenuation
of the catabolic component of the
stress response, by provision of ex-
cellent pain relief which facilitates
mobilization, reduction of pulmo-
nary, cardiac and thrombotic com-
plications, faster recovery of intesti-
nal function, decrease of postopera-
tive protein breakdown and preser-
vation of tissue protein, are the most
important advantages of epidural
anesthesia and analgesia, already
acknowledged by literature (Wind
et al., 2006; Zonca et al., 2008) .
Epidural analgesia could improve
postoperative immediate and long-
term outcome (White et al., 2007).
Nevertheless, epidural anesthesia
and postoperative analgesia per se
do not reduce morbidity after major
colonic resection surgeries (Carli et
al., 2002; Mrat et al., 2007). Epi-
dural analgesia can be a valuable
adjuvant to fast-track anesthesia
techniques for major surgery (Block
et al., 2003). The benefits of epidur-
al analgesia are most apparent when
used as part of a multimodal analge-
sic regimen (White et al., 2007).
Multimodal (or "balanced") anal-
gesia involved the use of more than
one modality of pain control to ob-
tain additive (or even synergistic)
beneficial analgesic effects while
reducing opioid-related side effects
(Wind et al., 2006). In some con-
texts, multimodal analgesia refers to
systemic administration of analgesic
drugs with different mechanisms of
action (Coloma et al., 2001) while
in others it refers to concurrent ap-
plication of analgesic pharma-
cotherapy and regional analgesia
(Ashburn et al., 2004). The use of
non-opioid analgesics [ non-
steroidal anti-inflammatory drugs
(NSAIDs) ketorolac, cyclooxygen-
ase-2 (COX-2) inhibitors, aceta-
minophen, ketamine, and local anes-
thetics] as part of a multimodal an-
algesic regimen minimized postop-
erative pain and opioid-related side
effects (White et al., 2007).
The current study fulfilled all the-
se aspects of multimodal context, by
administration of pharmacologic
analgesic drugs having different
mechanisms of action (fentanyl,
ketorolac, acetaminophen and ket-
amine) together with epidural anes-
thesia and analgesia. Continuous
epidural infusion provides better

static and dynamic pain relief than


IV opioid-based regimen (Wu et al.,
2005). Epidural local analgesia,
compared to intravenous patient
controlled analgesia (IV-PCA), re-
duced postoperative pulmonary
complications after thoracic or up-
per abdominal surgery (Wu et al.,
2005), improved preoperative nutri-
tional profiles and health-related
quality of life scores, while better
preserving exercise capacity after
colon surgery (Carli et al., 2002).
These factors can facilitate the
achievement of postoperative mile-
stones (e.g., earlier tracheal extuba-
tion, discharge from the intensive
care unit, and shorter ambulation
time) but there was little evidence
that epidural analgesia actually re-
duced mortality or hastens hospital
discharge even after major surgery
(Wu et al., 2005). The impact of
specific short-acting anesthetics, use
of multimodal analgesic and antie-
metic therapies facilitated more rap-
id recovery and discharge (Twersky
et al., 2008).
Midazolam was primarily given to
provide sedation, reduce anxiety,
optimize intraoperative hemody-
namic stability, and decrease post-
operative side effects. Benzodiaze-
pines remain the most commonly
used premedication because even
small doses of these compounds
(midazolam 20 g kg
-1
IV) can im-
prove the perioperative fast-tracking
process by reducing anxiety and
anxiety-related complications, as
well as improving patient comfort
and satisfaction (White et al., 2007).
In the current study, using a multi-
modal approach for analgesia in
NOFTA group, comprising the use
of NSAID ketorolac, epidural ad-
ministration of NMDA antagonist
ketamine and the non-narcotic anal-
gesic acetaminophen infusion in the
immediate postoperative period,
yielded a satisfactory analgesic ef-
fect, superior to OFTA group, and
evaded the unfavorable opioid-
related side-effects. The current ap-
proach is consistent with others
(White et al., 2007; Twersky et al.,
2008), who acknowledge the need
for multimodal strategies to improve
perioperative management and out-
comes. Curatolo and Sveticic
(2002) evaluated multimodal anal-
gesic therapy for the treatment of
acute postoperative pain found that
adding a NSAID (or ketamine) to
morphine was advantageous, and
that combination of acetaminophen
and a NSAID was superior to either
drug alone. Unfortunately, most
multimodal analgesia studies have
focused on the combination of an
opioid with a single non-opioid
drug. Ideally, multiple non-opioids
(NSAIDs, acetaminophen, COX-2
inhibitors and gabapentin) could be
combined to achieve more optimal
pain relief and, perhaps ultimately,
an opioid-free environment (White
et al., 2007). So, multimodal anal-
gesia represents a key element for
successful fast-track surgery by
minimizing postoperative pain, opi-
oid-related organ dysfunction and
facilitating the recovery process was
managed in this trial.

Newer fast-tracking criteria recog-


nize the importance of controlling
not only pain, but also opioid-
related side effects (post-operative
nausea and vomiting PONV). In
addition to the administration of
antiemetic drugs, multimodal strate-
gies to reduce the risk of PONV
were adopted, including use of
propofol and local anesthetic-based
analgesic techniques, adequate hy-
dration, as well as minimizing pre-
operative opioid use (White et al.,
2007). In the current study, the rate
of incidence of PONV was lower in
the NOFTA group, presumably re-
lated to the multimodal antiemetic
strategy we adopted in that group,
since all three groups were given
routine preoperative antiemetic,
metoclopramide. Early clinical in-
vestigations pointed out in that as-
pect the importance of using short-
acting intravenous (propofol), as
well as minimizing the total dose of
opioid analgesic medication admin-
istered during the preoperative peri-
od (Latham et al., 2000). In order to
minimize the adverse effects of opi-
oid analgesics, postoperative anal-
gesia was provided in our study by
neuroaxial-administered small dose
opioids, adjunct to local anesthetic
in OFTA group, and intravenous
non-opioid analgesics in NOFTA
group, whose benefits over opioid
are well acknowledged (Latham et
al., 2000; Myles and David, 2005).
Neuroaxial-ketamine in sub-
anesthetic dose not only is effective
in reducing morphine requirements
in the first 24 hours after surgery,
but also reduces postoperative nau-
sea and vomiting (Bell et al., 2006).
This clarifies why we administered
epidural ketamine adjunct to local
anesthetic bupivacaine in NOFTA
group both intra- and post-
operatively in a dose of 0.5 mg/kg
and 0.3 mg /kg respectively.
Preoperative hypothermia can
have a wide range of detrimental
effects, which may include in-
creased rates of wound infection,
morbid cardiac events, blood loss,
and even prolong the hospital stay
(Nesher et al., 2003; White et al.,
2007). Throughout the procedure
temperature was monitored and hy-
pothermia was reduced by using
warming irrigation and IV fluids as
well as using total temperature
management system (Bair Hugger
750) besides maintenance of the
operating room temperature at 24C
in all groups. Although fast-tracking
was first introduced for ambulatory
anesthesia several years ago, data
about how often occur is still being
collected and refined. Additionally,
no universally accepted practice
guidelines or selection criteria have
been developed. Type of surgery is
likely to influence fast-track eligi-
bility (Twersky et al., 2008). The
colonic resection surgery is well
known for a great degree of trauma
and stress. Although they were
comparable in duration, and per-
formed by same surgical team, yet
still they could have influenced the
present results, especially with vari-
ations in surgical approaches.

Unfortunately, data collection was


discharged without follow-up or
patient satisfaction measurements to
be part of assessing fast-track pro-
grams. This was due to lack of
communication with patients here-
after. Another limitation was the
absence of bispectral index (BIS)
guided monitoring of depth of anes-
thesia, whereas improving the titra-
tion of both intravenous and inhaled
anesthetics by using cerebral moni-
toring devices may facilitate the
fast-tracking process. A
cost/efficacy analysis was not in-
cluded, and would have been com-
plementary to results. Ultimately,
the choice of anesthetic technique
and pharmacological agents should
be tailored to the needs of the pa-
tient as well as the type of proce-
dure, being performed as an integral
component of a fast-track program.
The universally applicable goals
valid for every class of intervention
are that they should be easy to use,
have minimal side effects, maintain
homeostasis, allow for a predictable
on- and offset, and give minimal
impairment of recovery and func-
tion (Kranke et al., 2008).
Conclusion
The implementation of fast-track
anesthesia technique in major co-
lonic resection surgeries may en-
hance recovery profile and reduce
the length of PACU and overall
hospital stay. Data imply that non-
opioid fast-track regimen outcome
is better than opioid-based regimen.
Its evading the unfavorable opioid-
related side-effects that influence
recovery negatively may alone justi-
fy its adoption in clinical practice.
Further large-scale cost/benefit
analysis studies, involving variable
types of surgeries, are needed to
elicit contributing role of anesthesi-
ologists as part of a multidiscipli-
nary fast-track program involving
surgeons, nursing staff and physio-
therapists as well, for a better layout
of the ideal "fast-track program".
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865
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 865-880
CHARACTERIZATION, CLONING AND EXPRESSION OF NS3
PROTEIN GENE OF HEPATITIS C GENOTYPE 4A
By
SALWA F. SABET
1
, MAGED M. AL-SHERBINY
2
,
ESSAM H. IBRAHIM
3
AND PAUL HAGAN
4
Department of Zoology, Faculty of Science, Cairo University
1, 2
National Organization for Drug Control and Research (NODCAR)
51 Wezaret El Zeraa street, Cairo, Egypt
3
, Division of Infection and
Immunity, Joseph Black Building (B4-09d), Institute of Biomedical and
Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
4
Abstract
Clone and express NS3 gene of the Egyptian strain ED43 of HCV genotype
4a in E. coli was studied. Gene and protein sequences of NS3 gene of the ED43
strain were first analyzed using PC/GENE program. DNA homology was 89%
the homologies and that of the protein was 78.8% indicating that NS3 gene of
the genotype 4a is different from those isolated from other strains.
DNA of NS3 region of genotype 4a was amplified from HCV_ED43/PUC19
plasmid. The PCR product was cloned and expressed in E. coli M15 using
pQE-30 vector. Fusion protein containing the peptides coded by HCV NS3
(NS3_4a) was expressed by Escherichia coli. The specific HCV antigenicity
of the NS3_4a fusion protein was identified by western blotting.
Keywords: HCV, Genotype 4a, Egyptian strain ED43, NS3_4a, Cloning, Ex-
pression
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Hepatitis C virus (HCV) infects
more than 10% of the general popu-
lation in Egypt when intravenous
injection with an antimony com-
pound for endemic schistosomiasis
in the past was implicated (Tanaka
et al, 2004). Egypt could be consid-
ered a candidate country for per-
forming trials of prophylactic and
therapeutic vaccines as it has higher
rates of HCV than neighboring
countries as well as other countries
in the world with comparable socio-
economic conditions and hygienic
standards for invasive medical, den-
tal, or paramedical procedures (La-
vanchy and McMahon, 2000). Little
is known about genotype 4a which
is the most widely distributed of
type 4 sequences, being the princi-
pal genotype in the Middle East and
Egypt, and accounts for a major
866
proportion of cirrhosis and hepato-
cellular carcinoma in these popula-
tions this genotype (Dusheiko et al.,
1994; Ray et al., 2000).
The NS3 protein has approximately
70 kd and involved in critical events
of viral replication, thus making it
an attractive target for antiviral ther-
apy (Hong et al., 2000, Sillanp et
al., 2009). Analysis showed motifs
characteristic of RNA helicase en-
zymatic function (Tai et al., 1996).
Genomic analysis of the NS3 pro-
tein could be characterized as a ser-
ine protease (De Francesco and
Steinkhler 2000) as well as an
ATP-driven motor activity (Penin et
al., 2004).
In the present study, NS3 gene of
the Egyptian strain ED43 of HCV
genotype 4a was chosen to be
cloned and expressed in E. coli.
Gene and protein sequence of NS3
gene of was first analyzed using
PC/GENE program, and then ampli-
fied from HCV_ED43/PUC19
plasmid. The PCR products were
then cloned and expressed in M15
strain of E. coli using pQE-30 vec-
tor. The expression and antigenicity
of the NS3 proteins in E. coli was
evaluated by SDS-PAGE and West-
ern blotting.
Materials and Methods
Analysis of NS3 gene of HCV
genotype 4a genome: PUC19 vector
con-taining HCV genotype 4a DNA
genome of Isolate ED43
(HCV_ED43) was provided by Dr.
Richard M. Elliott (Institute of Vi-
rology, University of Glasgow,
Glasgow). The complete nucleotide
sequence of HCV_ED43 which is
9355 nucleotide long is deposited in
the EMBL database under accession
number Y11604.
Analysis of the nucleic acid se-
quence and the deduction of amino
acid sequences of the NS3
(NS3_ED43) gene of HCV_ED43
were performed by PC/GENE soft-
ware programs (Intelligentics, Inc.).
These programs have been used to
determine the open reading frame,
restriction enzymes cleavage sites as
well as the primary, secondary
structure analysis of the deduced
amino acid sequences. The nucleo-
tide sequence of the NS3_ED43 was
subjected to similarity search using
standard nucleotide-nucleotide
BLASTN option of the internet
(http://www.ncbi.nlm.gov/BLAST)
(Altschul et al., 1997). Multiple
DNA and protein alignments of
NS3_ED43 were done with some of
the isolates which showed a high
degree of similarity using PC/GENE
program.
Cloning Strategy of the NS3_ED43
gene
The original DNA concentration of
the HCV_ED43 genome was 0.98
g/l and was diluted to a final con-
centration of 0.1 g/l by adding 10
l of DNA to 90 l sterile distilled
water (dH
2
O).
The cloning strategy of the
NS3_ED43 and gene was done
(Sambrook et al., 1989) and the
QIAexpressionist kit instruction
manual as: amplification of DNA
867
region of interest using whole
PUC19/HCV_ED43 plasmid as a
template. A restriction enzyme tar-
get site was introduced into each of
the PCR primers. The restriction
sites of the enzymes that were not
found to cut inside the target se-
quence and at the same time are pre-
sent in the multiple clonal site
(MCS) of the appropriate vector,
were chosen to be added to the pri-
mers. The resulting PCR product
and cloning vector were digested
with the restriction enzymes to gen-
erate complementary ends at the
PCR product and the vector which
were then ligated and transformed
into E. coli.. DNA sequences coding
for the whole NS3 protein was am-
plified from PUC19/HCV_ED43.
The sequence of primers used was:
NS3_ED43F primer: 5'-
CGCGGATCCGCCC
CCATCACAGCATACGC-
3'.NS3_ED43R primer: 5'-
GACGTCGAC TGTCA CTACC
TC GAGATCAGC -3'. The ampli-
fied region was cloned into pQE30
cloning and expression vector be-
tween the BamHI and SalI Sites.
Expression of NS3 (NS3_4a) fu-
sion protein: Recombinant M15
cells were grown overnight at 37C
in 20 ml of LuriaBertani (LB)
broth (1% tryptone, 0.5% yeast ex-
tract, 1% NaCl) containing 100
g/ml ampicillin, 25 g/ml kana-
mycin (LB
kan,amp
). The 20 ml culture
was inoculated into 1 litre of
LB
kan,amp
media and cultured at 37C
with vigorous shaking for 1 h. Ex-
pression of recombinant protein was
induced by adding isopropyl b-D-
thiogalactoside (IPTG) to a final
concentration of 1 mM. After 4 h of
induction, cells were harvested by
centrifugation 4000 rpm for 20 min
& frozen overnight at -20C until
needed for purification.
Preparation of pure lysates was
done under denaturing conditions on
Ni2+-nitrilotriacetate (NTA)
agarose (Qiagen) according to the
manufacturers instructions. The
cell pellet was thawed for 15 min on
ice and resuspended in buffer B
(100 mM NaH
2
PO
4
, 10 mM TrisCl,
8 M urea, pH to 8.0) at 5 ml/g wet
weight (25 ml buffer was added to 5
g cells). The cells were gently vor-
texed for 20 min at RT until the so-
lution became translucent. The ly-
sate was centrifuged at 10,000 rpm
for 30 min at RT to pellet the cellu-
lar debris. The supernatant (cleared
lysate) was saved and the cell debris
was discarded. Five ml of the 50%
Ni-NTA slurry was added to 25 ml
lysate and mixed gently by shaking
(200 rpm on a rotary shaker) for 60
min at RT. The lysateresin mixture
was carefully loaded into an empty
column with the bottom cap still
attached. The bottom cap was re-
moved and the flow-through was
collected. The column was washed
twice with 4 ml buffer C (100 mM
NaH
2
PO
4
, 10 mM TrisCl, 8 M urea,
pH to 6.3). The recombinant protein
was eluted by adding 2.5 ml buffer
D (100 mM NaH
2
PO
4
, 10 mM
TrisCl, 8 M urea, pH to 5.9) 4 times
and followed with 2.5 ml buffer E
(100 mM NaH
2
PO
4
, 10 mM TrisCl,
868
8 M urea, pH to 4.5) 4 times. A 10
l 5X SDS-PAGE sample buffer
was added to 40 l of each elution
and stored at 20 C for SDS-PAGE
analysis. The samples were analysed
in 12% SDS-PAGE. The protein
content of purified samples was
measured using BioRad protein as-
say reagent (BioRad)
For Western-blot analysis, fusion
protein was electro-transferred on to
nitrocellulose membrane (Watt-
man). The membrane was blocked
with 1% bovine serum albumin in
Tris-buffered saline (TBS, 20 mM
Tris-Cl, pH 7.5, 500 mM NaCl) for
2 h at RT with shaking.. The mem-
brane was then incubated with anti-
HCV pooled human sera (from
Egyptian blood donors infected with
hepatitis C), washed, then incubated
with second antibody [horseradish
peroxidase (HRP)-labelled Protein
A (Sigma)]for 1 h at 37 C. After
washing, the membrane was devel-
oped using the enhanced chemilu-
minescent (ECL2) kit from Amer-
sham. The same steps were repeated
using sera from healthy Egyptian
donors to ensure the proteins recog-
nition specify.
Results
HCV Sequence, NS3_ED43 gene
of the HCV_ED43 is 1893 bp and
lies between nucleotide bases 3358
and 5250 of the genome. Amino
acid translation of the NS3_ED43
gene using PC/GENE program re-
vealed a protein that consists of 631
aa and have a molecular weight of
67.5 kDa.
Amino acid analysis of NS3_ED43
protein showed threonine (11%),
glycine (9.3%), valine (8.5%), leu-
cine (8.2%) and alanine (8.2%)
(Tab. 1).
Table 1: Absolute and % of amino acid residues of NS3_ED43 deduced pro-
tein.
Code Nb % Code Nb %
Thr 70 11 Glu 22 3.4
Gly 59 9.3 Lys 20 3.1
Val 54 8.5 Gln 19 3
Leu 52 8.2 Phe 19 3
Ala 52 8.2 Cys 18 2.8
Ser 43 6.8 His 14 2.2
Pro 43 6.8 Asn 13 2
Asp 31 4.9 Met 10 1.5
Arg 30 4.7 Trp 7 1.1
Ile 30 4.7 Xaa 1 0.1
Tyr 24 3.8
The estimated half-life is consid-
ered to be 4.4 hour in mammalian
reticulocytes, in vitro, more than 20
hour in yeast, in vivo, and more
than 10 hour in E. coli, in vivo.
869
The isoelectric point (pI) of the
NS3_ED43 deduced protein was
found to be 6.54
The three highest points of hydro-
philicity of the NS3_ED43 protein
were determined. The first was lo-
cated from amino acid 371 to 376
(Lys-Lys-Lys-Cys-Asp-Glu) with
an average hydrophilicity (Ah) of
2.33, second was located from ami-
no acid 117 to 123(Arg-Arg-Arg-
Gly-Asp-Thr-Arg) with an Ah of
1.93 and third was from amino acid
457 to 462 (Ser-Arg-Ser-Gln-Arg-
Arg) with an Ah 1.63.
Sites annotated analysis of the
deduced NS3_ED43 protein showed
one N-glycosylation site, one tyro-
sine sulfatation site, 11 protein ki-
nase C phos-phorylation sites, 10
casein kinase II phosphorylation
sites, 16 N-myristoylation sites, one
gram-positive cocci surface proteins
`anchoring' hexapeptide, one cell
attachment sequence and one
ATP/GTP-binding site motif A (P-
loop).
The BlASTN (for DNA) program
was used to search the similarity
between NS3_ED43 DNA sequence
and other DNA sequences, using
non redundant Gen-
Bank+EMBL+DDBJ+PDB se-
quences database. Three isolates
were chosen for comparison with
NS3_ED43. BLASTN showed sim-
ilarities of 89% with isolate 25 of
genotype 4a (Is. 25), 82% (homolo-
gy search covered 90% only of
NS3_ED43 sequence) with isolate
24 of genotype 4d (Is. 24), and 87%
(covered 45% of NS3_ED43 se-
quence) with isolate MD4-2 of gen-
otype 4 (Is. MD4-2). DNA of the
isolates was multiple aligned with
NS3_ED43 DNA sequence using
PC/GENE program.
Amino acid translation of the nu-
cleotide sequences of the three iso-
lates that were aligned with
NS3_ED43 gene was done and mul-
tiple protein alignment of
NS3_ED43 protein with the de-
duced proteins were done using
PC/GENE program (Fig. 1). Align-
ment revealed an identity of 78.8%
between the NS3_ED43 and the
three proteins.
DNA sequence coding for
NS3_ED43 was amplified from
PUC19/ HCV _ED43 and the ex-
pected size (1.9 kb) of PCR product
was obtained (Fig.2).
PCR product of the amplified
NS3_ED43 DNA was purified from
gel by High Pure PCR Purification
Kit (Roche), double digested with
the restriction enzymes BamHI and
SalI., and purified from solution.
The purified double digested DNA
concentration was 20ng/l for each
band (Fig. 3). PCR product cloned
to pQE30, miniprep took place and
electrophoresis of the double digest-
ed DNA with BamHI and SalI
showed a band of around 1.9 kb
which is the size of the NS3_4a in-
sert (Fig. 4). 5 DNA sequence
analysis of NS3_4a region of re-
combinant pQE-30/NS3_4a plasmid
showed that NS3_4a insert was li-
gated in frame with the 6xHis-
tagged DNA of pQE-30 vector (Fig.
5) and alignment search with origi-
870
nal HCV_ED43 by BLAST pro-
gram showed that the sequence of
the insert is similar to that of the
original NS3_ED43 (Fig. 6).
M15 bacterial cells containing
recombinant plasmids pQE
30/NS3_4a was induced with 1mM
IPTG. Four hours after induction,
the cells were harvested, samples of
whole-cell lysates were prepared
and analysed by SDS-PAGE. A
band of approximately 70 kDa was
in good agreement with predicted
molecular mass (Fig. 7). Nearly all
of the expressed fusion protein was
found in the insoluble fraction after
sonication (Fig. 8). NS3_4a protein
was solubilized with 8 M urea and
was purified on Ni2+-NTAagarose
under denaturing conditions. After
washing, bound proteins were eluted
at low pH, and fusion proteins of
high purity were obtained (Fig. 9).
The yield was about 8.44 mg/2litre
culture.
Western blot analysis checked the
NS3_4a protein antigenicity. Ex-
pressed NS3_4a protein was recog-
nized by the anti-HCV antibodies
present in the pooled human sera of
Egyptian patients infected with
HCV and a band was approximately
at 70 kDa corresponding to NS3_4a
(Fig. 10). Specificity by using sera
from healthy donors showed no
specific recognition of the NS3_4a
protein.
Discussion
Egypt could be considered a candi-
date country for performing trials of
preventive and therapeutic vaccines
as it has higher rates of HCV than
neighboring countries as well as
other countries in the world with
comparable socioeconomic condi-
tions and hygienic standards for in-
vasive medical, dental, or paramedi-
cal procedures (Lavanchy and
McMahon, 2000). Although Egypt
has a very high prevalence of HCV
and a high morbidity and mortality
from chronic liver disease, cirrhosis,
and hepatocellular carcinoma, not
much is known about genotype 4a
which is the most widely distributed
of type 4 sequences, being the prin-
cipal genotype in the Middle East
and Egypt (Dusheiko et al., 1994;
Ray et al., 2000).
NS3 protein was chosen in the
present study for its importance as a
multifunctional virus-specific pro-
tein that possesses multiple enzy-
matic activities (Misialek et al.,
2009). NS3 contains serine protease
activity in its N-terminal region and
accounts for processing of the viral
polyprotein at four cleavage sites,
NS3/ 4A, NS4A/4B, NS4B/5A, and
NS5A/5B. The serine proteinase
activity of NS3 is an attractive tar-
get for new drugs that could block
viral replication efficiently (Lordini
et al., 2003; Penin et al., 2004). Al-
so, helicase and nucleic acid-
stimulated nucleoside triphospha-
tase activities are found in its C-
terminal region (Bartenschlager,
1997; Kwong et al., 1998). The NS3
helicase NTPase domain probably
has multiple functions, including
RNA-stimulated NTPase activity,
871
RNA binding, and unwinding of
RNA regions with extensive sec-
ondary structure by coupling un-
winding and NTP hydrolysis. This
enzyme acts as an ATP-driven mo-
tor and is thought to switch between
alternative conformations during
active unwinding of double-stranded
RNA (Penin et al., 2004).
In the present study, prior to clon-
ing, the DNA sequence of the NS3
region (NS3_ED43) of the HCV-
ED43 was analyzed using
PC/GENE program. NS3
(NS3_ED43) gene of the HCV-
ED43 is 1893 bp and lies between
nucleotide bases 3358 and 5250 of
the genome. The DNA sequence
analysis revealed that it has a rela-
tively higher proportion of GC ba-
ses (56.7%) than AT bases (43.2%)
with a base percentage of 27.3 % G,
29.4 % C, 22.8% A and 20.3% T.
Amino acid translation of the
NS3_ED43 gene using PC/GENE
program revealed a protein that con-
sists of 631 aa and has a molecular
weight of 67.5 kDa. Amino acid
composition analysis of the
NS3_ED43 protein revealed the
presence of a high percentage of
threonine (11%), glycine (9.3%),
valine (8.5%), leucine (8.2%) and
alanine (8.2%). The N-terminal of
the NS3_ED43 protein sequence is
A (Ala).The estimated half-life is
considered to be 4.4 hour in mam-
malian reticulocytes, in vitro, more
than 20 hour in yeast, in vivo, and
more than 10 hour in E. coli, in vi-
vo.
The three highest points of hydro-
philicity of NS3_ED43 protein were
determined. The first point was lo-
cated from amino acid 371 to 376
with an average hydrophilicity (Ah)
of 2.33, second point was located
from amino acid 117 to 123 with an
Ah of 1.93 and third was from ami-
no acid 457 to 462 with an Ah 1.63.
No previous data of the antigenic
determinants of the NS3 proteins of
the other HCV genotypes was avail-
able. Sites annotated analysis of the
predicted protein encoded by
NS3_ED43 revealed the presence of
one N-glycosylation site. It is
known that potential N-
glycosylation sites are specific to
the consensus sequence Asn-Xaa-
Ser/thr. The presence of the consen-
sus tripeptide is not sufficient to
conclude that an asparagine residue
is glycosylated, due to the fact that
the folding of the protein plays an
important role in the regulation of
N-glycosylation (Pless and Lennarz,
1977). No glycosylation was men-
tioned for NS3 in the previous stud-
ies which suggest that NS3 folding
might have depressed this site activ-
ity. Also, one Tyrosine sulfatation
site, 11 protein kinase C phosphory-
lation sites, 10 Casein kinase II
phosphorylation sites that might be
responsible for interactions with
protein kinases A and C. Numerous
NS3 interactions with cellular com-
ponents have been reported, includ-
ing protein kinases A and C, p53,
and histones H2B and H4, but their
significance is unclear (Tellinghui-
sen and Rice, 2002). One Gram-
872
positive cocci surface protein `an-
choring' hexapeptide has been re-
ported as well as one Cell attach-
ment sequence and one ATP/GTP-
binding site motif A (P-loop) which
might be responsible for the NS3
helicase NTPase domain interac-
tion with ATP. 16 N-myristoylation
sites were also found along the se-
quence, Myristoylation is essential
for the biological function of most
proteins. As attachment of the
myristoyl residue to glycine resi-
dues provides hydrophobicity and
promotes protein-protein interac-
tions (Johnson et al., 1994), it might
have other vital functions in the
NS3 protein other than providing
hydrophobocity as NS3 is not
known to have hydrophobic regions.
The BlASTN (for DNA) program
gave the similarity between
NS3_ED43 DNA sequence and oth-
er DNA sequences, Three NS3 iso-
lates from those which showed sig-
nificant similarity were chosen for
multiple alignments with
NS3_ED43. BLASTN Homology
search revealed similarities of 89%
with isolate 25 of genotype 4a (Is.
25), 82% (homology search covered
90% only of NS3_ED43 sequence)
with isolate 24 of genotype 4d (Is.
24), and 87% (covered 45% of
NS3_ED43 sequence) with isolate
MD4_2 of genotype 4 (Is. MD4_2).
These results support the findings
that suggested that the Egyptian
genotype 4a is different from those
isolated from other parts of the
world (Stuyver et al., 1994; Bukh et
al., 1995; Alfonso et al., 2001).
Amino acid translation of the nu-
cleotide sequences of the three iso-
lates that were aligned with
NS3_ED43 gene and multiple pro-
tein alignment of NS3_ED43 pro-
tein with the deduced proteins were
done using PC/GENE program.
Alignment revealed an identity of
78.8% between the NS3_ED43 and
the three proteins. Amino acid com-
position comparison between
NS3_ED43 and the proteins of the
three isolates was done and revealed
that the amino acid composition is
almost conserved in the four iso-
lates.
Computer analysis of NS3_ED43
was followed by cloning and ex-
pression of NS3_ED43 DNA region
in pQE-30/M15 E.coli. In previous
studies, Fragments Vishnuvardhan
et al., 1997; Jiao et al., 2004, Frick,
2007), whole (Poliakov et al., 2002,
Cheng et al., 2002), as well as fu-
sion proteins from different ligated
gene fragments such as NS3-NS4a
(Du et al., 2002; Thibeault et al.,
2004).
Amplification of NS3_ED43
DNA began by cloning of
NS3_ED43 DNA via
PUC19/HCV_ED43 plasmid as a
template. Core insert into pQE30
vector, t BamHI and SalI restriction
sites were introduced to the forward
and reverse primers respectively.
pQE-30 vector with PCR product of
the core region (designated as
NS3_4a) was used to transform
M15(pREP4) bacteria supplied with
the kit. Successful transformation
was confirmed by plasmid miniprep
873
and gel electrophoresis showing the
DNA bands of the expected size
(1.9kb).
Optimized conditions for expres-
sion of NS3_4a protein was done
according to the manufacturers
manual of QIAexpressionist kit with
the only exception that before in-
duction, the bacterial cultures of
NS3_4a was grown at 37C, SDS-
PAGE of the whole induced cultures
before purification showed the
bands of NS3_4a fusion protein of
69 kDa (Jiao et al., 2004). The solu-
bility of expressed NS3_4a protein
was checked before preparing the
proteins on large scales, and it was
found that the NS3_4a protein was
expressed in the insoluble form.
Thus, large scale preparations of the
NS3_4a protein was done under
denaturing conditions and Core_4a
protein content was estimated to be
around 4 mg/liter. Reactivity of
NS3_4a against human sera in
Western blot was analysed. The ex-
pressed NS3_4a protein was recog-
nized by the anti-HCV present in
the pooled human sera of Egyptian
patients infected with hepatitis C
and a band appeared approximately
at 69 corresponding to the NS3_4a.
Specificity of this recognition was
confirmed by using sera from
healthy donors which showed no
specific recognition of the two pro-
teins. So, this E. coli-derived
NS3_4a protein displayed specific
antigenicity.
Further studies are recommended
in experimental animals to specify
these proteins antigenicity. Moreo-
ver, characterization of more HCV
isolates, extracted from the blood of
Egyptian patients, will be important
for the improvement of diagnostic,
epidemiological and clinical treat-
ment regimens as well as the devel-
opment of a candidate vaccine
against HCV genotype 4a.
Conclusion
Analysis of NS3 region of the iso-
late ED43 revealed that the Egyp-
tian genotype 4a is different from
other genotypes. Construction of
pQE-30/NS3_4a recombinant plas-
mid and expression of NS3_4a gene
in E. coli (M15) was successful and
have immunogenicity in Western
blot useful in vaccine development
studies against the Egyptian geno-
type 4a. Further studies of NS3 re-
gion are recommended in experi-
mental animals and cell cultures to
explore its role in diagnostic, epi-
demiological, treatment and devel-
opment of a candidate vaccine
against HCV genotype 4a.
Acknowledgment
The authors would like to thank
the staff, the Institute of Virology,
Glasgow University for kind co-
operation, especially Prof. Dr. Rich-
ard Elliot for his valuable assistance
and advices.
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876
To be continue
877
Fig. 1: Multiple alignments between NS3_ED43 protein and proteins of different HCV isolates. Consensus length:
631, Position in alignment perfectly conserved: '*' Position well conserved: '.', Identity: 497 (78.8%), Similarity: 08
(17.1%)
Fig. 2 Fig. 3
Fig. 2: Agarose gel electrophoresis of PCR product after amplification of NS3_ED43 DNA from
HCV_ED43 genome. Lane M: 1 kb ladder marker. Lane 2: control sample. Lane 3: PCR product of
NS3_ED43 gene.
Fig. 3: Agarose gel electorophoresis of PCR product of NS3_ED43 DNA digested with BamHI and SalI and
purified from solution. Lane M:1 kb ladder marker. Lane V: pQE-30 DNA digested with BamHI, Lane 1:
pure double digested (BamHI/SalI) NS3_ED43 PCR product.
878
Fig. 4: Double digestion of miniprep samples of recombinant pQE-30/NS3_4a plasmid with BamHI/XhoI.
Lane M: 1 kb Ladder marker. Lane V: pQE-30 digested with BamHI/SalI. Lane 1-5: DNA resulted from
minipreps digested with BamHI/XhoI. V: vector. I: insert.
Fig. 5: DNA sequence analysis of NS3_4a insert (NS3-1) inside pQE-30 by ABI PRISM model
310 DNA automated sequencer. Bases 1-50 are pQE-30 vector sequence, bases starting from 51
~ end partial NS3_4a sequence, Sequence of 6xHis, BamHI site, Beginning of NS3_4a
sequence.
879
Fig. 6: Similarity search result between sequences of NS3_4a and NS3_ED43 genes. Query: DNA Sequence of NS3
_4a gene. Subject: sequence of original NS3_ED43 DNA. Identities: 609/619 (98%). Gaps: 1/619 (0%). Strand:
Plus/Plus.
Fig. 7: SDS-PAGE (12% gel) of bacterial cultures resulted from small expression of NS3_4a protein in M15
bacteria. Lane M: prestained wide range MW marker. Lane 1: uninduced culture. Lane 2: induced culture.
Lane 3: flow-through. Lane 4: wash.
880
Fig. 8: SDS- PAGE (12% gel) of bacterial cultures resulted from expression of Core_4a and NS3_4a proteins
in M15 bacteria to determine solubility. Lane M: prestained wide range MW marker. Lane 1: uninduced
culture of NS3_4a protein. Lane 2: soluble fraction of induced culture of NS3_4a protein. Lane 3: insoluble
fraction of induced culture of NS3_4a protein.
Fig. 9: SDS- PAGE (12% gel) of the NS3_4a protein after purification from large-scale culture. Lane M:
prestained wide range MW marker. Lane 1: elution 1 of NS3_4a protein. Lane 2: elution 2 of NS3_4a pro-
tein.
Fig. 10: Western blot of purified NS3_4a protein recognized by pooled sera of Egyptian patients with HCV.

Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 881 - 905
EMERGING CUTANEOUS LEISHMANIASIS IN SIRTE-LIBYA:
EPIDEMIOLOGY, RECOGNITION AND MANAGEMENT
By
FOUAD M FATHY
1
, FATHI EL-KASAH
2
AND
ABDULLA M. EL-AHWAL
3
Department of Parasitology, Faculty of Medicine Alexandria University,
Egypt
1
and Departments of Dermatology,
2
and Tropical Medicine
3
, Fac-
ulty of Medicine, Al-Tahady University, Sirte, Libya.
Abstract
The present work aimed to determine the risk factors, lesion pattern and
effective therapy of emerging ZCL in Sirte-Libya. The study was carried out on
163 patients referred to health centers of Al-Gadaheya and Al-Hisha villages in
the years 2006 & 2007. Methods consisted of a predesigned questionnaire (per-
sonal and demographic data), clinical examination of lesions, and parasitologi-
cal examination by slit smear, treatment and follow up. Results showed an an-
nual incidence of 0.95%, with onset peak during autumn months. Important
local risk factors included: increased occupational exposure of farmers and
construction worker to infection from fat sand rat burrows, facilitated by lack
of prevention knowledge and prophylactic measures; close association of bad-
ventilated animal shelters to houses, and increased soil moisture by warm
spring ponds. The majority of lesions were multiple (73%) located on legs,
arms, and face 66.8%, 52.1% and 41.1%. Most lesions were active 1-2 month
duration and 1-3 cm size, ulcerative type (77.3%), and papulo-nodular (21.5%).
Giemsa slit smear proved quite reliable for active lesions, confirmed 79.5% of
lesions. The majority of lesions (60.1%) were treated by intra-lesional Pento-
stam. Systemic route was restricted to facial, over-joint, multiple or large le-
sions producing good response in 31.9%. Cryotherapy and oral Fluconazole
gave satisfactory response in 5.5% & 2.5% of cases.
Keywords: Libya, ZCL, epidemiology, recognition, management.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Leishmaniasis is a universal dis-
ease which represents an increasing
problem in many areas worldwide.
The disease is endemic in 88 coun-
tries, with a world prevalence of 13
million cases, incidence of two mil-
lion new cases each year and about
380 million people at risk (WHO,

2006). Old-world cutaneous leish-


maniasis is particularly endemic in
the Middle East (Talari et al., 2006;
Anwar et al., 2007) and North-
African countries (Ashford et al.,
1976; Saleh et al., 2007).The zoono-
tic Leishmania major (Mon 25) is
endemic almost all-over North-West
Libya except Tripoli- area (El-Buni
et al., 1993) and is spreading east-
wards, with a total of about 40,000
cases since 1971 (Annajar, 2008).
The disease is transmitted via sand-
fly (fig.4), introducing pro-
mastigotes to be engulfed and trans-
formed into amastigotes inside skin
macrophages (Neva and Brown,
1994). El-Buni et al. (2000a) identi-
fied Phlebotomus papatasi as the
main insect-vector in Libya and
Ashford et al. (1977) identified the
large fat sand rat (Psammomys obe-
sus, fig.1) and the small field rat
(Meriones libycus, fig.3) as the res-
ervoir hosts. Sirte governorate, to
the North at the middle of Libya,
started to become endemic after re-
cording the first cases at the West-
ern village of Al-Gadaheya in year
2002. About 1,600 cases were rec-
orded since then (Annajar, 2008).
The fluctuating annual incidence
since then, without a steady sharp
decline indicates that the disease is
not on its way to eradication. The
reason for the increased incidence is
not clear, but it might be related to
some human and/or environmental
emerging factors. In the last four
years, the disease was considered an
urgent health problem, after the oc-
currence of the first ZCL epidemic
in Taurgha area, involving 7,000
cases (Annajar, 2008); a town locat-
ed only about 70 kilometers to the
North of Al-Gadaheya and Sirte
border. Ulceration is the rule in all
ZCL lesions, spontaneous healing
takes months and usually ends in a
depressed pigmented scar with pos-
sible secondary infection, chronicity
or disfigurement (Hepburn, 2003).
Physicians in new or non-endemic
areas and even countries may not be
familiar with the emerging disease
pattern (Lapierre et al., 1975; Morsy
et al., 1991-b and El-Hajj et al.,
2004). Therefore, since the disease
is potentially epidemic and deep
scar-forming, physicians particular-
ly in rural areas with limited re-
sources must be prepared to recog-
nize the disease at an early stage and
proceed directly with its proper
management.
The present study aimed to: 1-
Determine the personal and envi-
ronmental risk factors of ZCL. 2-
Define the clinical and parasitologi-
cal features of the prevailing skin
lesion pattern. 3- Select the suitable
and effective therapeutic regimens.
Subjects, Materials and Methods
The present study was carried out
on 163 patients, attending the health
centers of two nearby villages to the
North-West of Sirte Governorate:
Al-Gadaheya and Al-Hisha (fig.28),
over two years (2006 and 2007).
Each case was subjected to the
following: 1-A comprehensive ques-
tionnaire form completed for every

patient, including personal and de-


mographic data about: age, sex, na-
tionality, residence, occupation,
health education, presence of nearby
rodent burrows or animal shelters,
etc 2- Clinical examination was
done to record the features of skin
lesions, as location on skin, number,
type, size and duration, etc... 3- Par-
asitological examination of skin slit
smear taken from lesion margin af-
ter consent of the patient, and done
properly according to the following
procedure (Ramirez et al., 2000):
For multiple lesions the more recent
one was chosen. Sample was taken
from the most advancing, indurated
and therefore, the most active part
of the lesion margin. The site was
thoroughly cleaned with 70% etha-
nol. Pressure was applied to the
margin site by the fore-finger and
thumb to achieve haemostasis. With
no.15 sterile surgical blade, a slit 3
mm long by 3 mm deep was done
perpendicular to the lesion circum-
ference and oozing blood was re-
moved by sterile gauze. Once bleed-
ing had stopped, the blade was used
to scrape the white dermal tissue
from both inner walls of the slit and
smeared onto a clean glass slide. A
total of 4 scrapings were smeared on
two slides to increase the chance of
amastigote detection. Slides were
then air-dried, fixed in 100% meth-
anol and stained with Giemsa (Gar-
cia, 2001). After proper staining, the
cytoplasm appeared light blue, the
nucleus and kinetoplast stained red
to purple. The amastigotes were in-
tracellular in intact macrophage or
close to a disrupted one. The smears
were examined microscopically by
the oil immersion lens to detect at
least one amastigote with clear in-
tracellular kinetoplast. Local anes-
thetic was used whenever indicated
to reduce the patient discomfort and
anti-leishmanial drug was not given
before diagnosis.4- Pentostam (Gal-
axo-Wellcome, England) was given
locally or systemically according to
the situation and the response was
estimated and followed up. Intrale-
sional infiltration with Pentostam
was done after Blum et al. (2004).
The basic aim was to fill the infect-
ed part of the dermis with the drug
to achieve maximal concentration;
which required careful and complete
infiltration of the lesion area includ-
ing the base and the whole margin,
until the lesion surface was
blanched. A fine gauge needle
(25G) was inserted at three points
around the margin under the edge.
Infiltration from each insertion point
was directed in a V-shaped pattern
towards the centre and the margin
covering together the bed of the en-
tire lesion. The drug was continu-
ously injected under pressure as the
needle advanced in the dermis and
not in the subcutaneous tissue. The
process was repeated twice per
week, but it was rather painful and
requires experience. Cryotherapy
was done by topical application of
liquid nitrogen via cotton tipped
applicator and moderate pressure to
the lesion and up to 2 mm outside
the margin. The freezing time did
not exceed 15-20 seconds. Adequate

application was reflected in whiten-


ing of the skin at 2-3mm outside the
lesion. All cases were followed up
for three months after the end of
treatment. Patients were warned
about the signs of relapse including
scar thickening, crusting or ulcera-
tion.
Results
The annual incidence of the dis-
ease among village population was
0.95%. The majority of cases had
lesion onset during autumn season.
As shown in (Tab.1), the major-
ity of patients were: 1-belong to the
active ages of 12-40 years (75.5%),
children formed (13.5%). 2- Males
(66.2%) and females (33.8%). 3-
Mostly Libyans, however foreigners
formed (28.3%), mainly Egyptian
land-workers. 4- Resident in the
village and did not leave for 2
months before lesion onset (100%).
5- Have outside work (81%), in
land-work as agriculture and con-
struction (39.9%); non-working
housewives and children (19%). 6-
Living or working (72.3%) near
rodent burrows (Fig. 1, 2, 3). 7-
have non-hygienic animal shelters
(57%) around houses (Fig.5). 8-
Mostly sleep near ground level on
rubber cushion, in ground-floor
house, and may sleep outside in
summer (27%). 9- Family history
for ZCL was positive in (19.6%),
especially among housewives and
children. 10- All had depression and
anxiety from fear of deep scar and
disfigurement. 11- Lacked health
education about ZCL (58.3%). Be-
sides: stagnant ponds formed by
natural warm spring or deep artifi-
cial well were noticed on field visit,
which were used for irrigation and
animal drinking.
Table 1: Personal and demographic data
Factor Case No. (n=163) %
Age Less than 12 22 13.5
12 - 40 123 75.5
More than 40 18 11
Sex male 108 66.2
female 55 33.8
Nationality Libyan 117 71.7
Foreigner 46 28.3
Occupation Land work 65 39.9
others 67 41.1
non-worker 31 19
Residence - in village 163 100
Rodent burrow-near house 118 72.3
Animal shelter 93 57
Sleeping outside 44 27
Family cases 32 19.6
Scar fear 163 100
Health education 68 41.7

Table 2: Lesion clinical features:


Clinical feature Case No. (n=163) %
Location on skin Face & neck 67 41.1
Arm & hand 85 52.1
Leg & foot 109 66.8
others 14 8.5
Number single 44 27
multiple 117 73
Five,or more 40 24.5
Type ulcerative 126 77.3
Papulo-nodular 35 21.5
Rare forms 2 1.2
Size 1 - 3 cm 108 66.2
Less than 1 cm 28 17.2
More than 3 cm 27 16.6
Duration 1-2 month 128 78.5
less than 1 month 19 11.7
more than 2 month 16 9.8
Giemsa slit smear
(for L major
amastigotes)
positive 120 79.5
negative 31 20.5
Not done 12 7.3
Local pain 21 12.8
Secondary bacterial infection 24 14.7
Enlarged local lymph nodes 16 9.8
Joint mobility limitation 18 11
The distribution of cases accord-
ing to prevailing lesion features was
shown in (Tab. 2). The majority of
examined lesions were: 1-Located
on exposed unclothed body parts;
leg, arm and face (66.8%, 52.1% &
41.1%, respectively). The face was
frequently affected (fig. 9, 10, 12),
especially in children, and in fe-
males (21.4%). 2- Multiple (73%) -
(fig. 9,12,16,24), five or more
(24.5%), range: 1-32 lesions. 3- Ul-
cerative type (more than 1 cm,
covering crust, 77.3%), early pap-
ulo-nodular (21.5%, fig.7,8). Rare
types (1.2%): sporotrichoid (fig.17)
and empetigenous (fig.18), one case
each.
4- Small to moderate size 1-3 cm
(66.2%); large ulcers 5 cm or more
(fig.13, 14) formed (13.4%), range:
0.5-12cm. 5- Mostly active with
short duration 1-2 months from on-
set till presentation (78.5%), range:
1week-4 months. 6- Painless
(87.2%) , unless secondary infected,
but minor itching was common.7-
Positive for Leishmania amastigotes
(79.5% of 151examined cases), by
Giemsa skin slit smear (fig.22,23).
8- Clinically and by medical history,
most lesions had characteristic form
development or sequence. They
started at the bite site (recalled by
few patients) in less than two
months, as a small red papule (less

than 5mm)-(Fig.6,7) red-brown


nodule (less than 1cm), covered
with white scales (fig.8,9) centre
became necrotic and exudative
ulcer covered with loose brown
crust formed of dried exudates
(fig.10), which when falls deep
overt ulcer with raised rolled edge
and purple indurated margin
(fig.11,12) heals by depressed
usually pigmented scar (fig.21).
Secondary bacterial infection was
observed in (14.7%) of lesions
(fig.19,20), with enlarged draining
lymph nodes (9.8%). Over-joint le-
sions (fig. 15,16) constituted (11%),
associated with some degree of
movement limitation.
Pentostam (Sodium stibogluconate)
was the mainstay drug used success-
fully by different routes to treat
(92%) of cases (Tab. 3).
Table 3: Effective Pentostam regimens
Criteria Intra-lesional Systemic
No. of treated cases 98 (60.1%) 52 (31.9)
Recovery period 3-4 weeks 2-3 weeks
Dosage 1-2 ml (100mg/ml), 2/week 10-20 mg/kg/day, I.M. or I.V.
Systemic Pentostam was given
daily, with tolerable side effects,
required hospitalization and was
restricted to facial, multiple, large,
over-joint, old and slow healing le-
sions. It was used successfully in
(31.9%) of cases (fig. 24,25). In-
tralesional Pentostam (fig. 26) had
less frequent dosage without hospi-
talization, and was suitable for all
other types of lesions and effective-
ly treated (60.1%) cases.
Oral antifungal Fluconazole gave
acceptable response in 4 cases at a
dose of 200mg/day for 6 weeks.
Local hypertonic saline (10%) injec-
tion stimulated granulation tissue
formation in slow-healing or deep
lesions. Cryotherapy (fig. 27) was
used alone, twice per week, with
good result in 9 cases with early,
small, papulo-nodular, non-infected
lesions; but also in combination
with other therapies as required.
Clinical criteria of therapeutic re-
sponse included reduced lesion size
and margin induration with flatten-
ing and smoothening of the edge
and start of re-epithelialization.
Secondary infected or exudative
lesions were treated first with a dry-
ing agent (Potassium permanganate
solution), local antibiotic (Gen-
tamycin cream) together with sys-
temic antibiotics (Ciprofloxacin) for
10 days, before Pentostam therapy.
All cases were followed up for 3
months after completion of treat-
ment without evidence of relapse.
Discussion
In the present study, all ZCL
cases were diagnosed on clinical

and parasitological basis together


with positive therapeutic response to
specific anti-leishmanial drugs.
They had residence in the village
without travel to endemic areas at
least for 2 months before the onset
of lesion, which is the maximal in-
cubation period (Harman, 1992).
Hence, all cases were indigenous
and contracted the infection in the
village proving ZCL endemicity in
Sirte. Since moist oriental sore gives
life-long immunity (Markell et al.,
1992), this endemicity should be a
recent one as patients of different
age groups and particularly adults
were affected. This coincides with
the first discovery of ZCL cases in
Sirte only 7 years ago (Annajar,
2008). The annual incidence was
0.95%. Taking into consideration,
the possibility of under-estimation
as some natives still prefer tradi-
tional treatment, this figure never-
theless, reflects the common occur-
rence and possible endemo-
epidemic nature of the disease. The
majority of cases had lesion onset
during autumn season of the years
2006 & 2007, which is due to the
emergence of the second generation
of adult sandflies from pupae in late
summer (El-Buni et al., 1997a).
Therefore, the disease in Sirte is
recently endemic with a seasonal re-
emerging nature. In accordance, El-
Buni et al. (1997b) identified a total
of 190 ZCL cases from 24 localities,
all presented to Tripoli central hos-
pital from September to December.
Also, at Sidi Bouzid focus of ZCL
in Tunisia the highest density of P.
papatasi was in late summer (July
to August). Furthermore, Fichet-
Calvet et al. (2003) found that L.
major infection reached its peak in
sand rats (70%) in September.
Concerning risk factors, in the
present study the majority of pa-
tients (72.3%) were living or work-
ing near burrows of Psammomys
obesus, the main reservoir of ZCL
in Libya and North Africa, which
lives in colonies in non-
mountainous salty soil desert, feed-
ing on the plant roots of family
Chenopodiaceae (Morsy et al.,
1996; El-Zurghany et al., 2006).
This rodent does not tend to change
place and its burrow provides a mi-
croclimate consisting of shade,
moisture and organic nitrogenous
matter quite suitable for sandfly
multiplication (Petrisceva, 1971).
Human invasion of its territory by
the new developmental settlement
projects involving ground clearance,
can results in partial opening of the
burrow tunnel, exposing the agricul-
ture or construction workers to bites
of disturbed clusters of possibly in-
fected sandflies (Faris et al., 1988;
Markell et al., 1992; El-Buni et al.,
1993). This fact explains why the
adult males were more prone to in-
fection as about half of the 81%
working cases were agriculture and
construction male workers. Kubey-
inje et al. (1997) in Northern Saudi
Arabia reported that ZCL was more
frequent and severer in expatriates
like farm-workers from non-
endemic areas. Therefore, proximity
to rodent burrows proved to be a

major risk factor which dictates a


solution. Periodic mechanical plow-
ing and proper destruction of rodent
burrows together with their feeding
plants for a minimal distance of 1
Km is a sure sand rat control meth-
od (Kamhawi, 1993).
The second observed environmen-
tal risk factor was the presence of
domestic animal shelters inside or
close to houses (57%). These were
under-ventilated, non-exposed to
sun with densely covered sides and
unclean with accumulated animal
waste, thus forming another excel-
lent shady moist sandfly breeding
site and an optimal feeding shelter
for the small field rat (Meriones
libycus), also defined as an im-
portant reservoir of ZCL in Libya
(Ashford et al.,1977). Contrary to
sand rat, it is highly mobile and fast
spreading in search of a variety of
food and shelter inhabiting low or
high mountainous land (El-
Zurghany et al., 2006). M.libycus
can adapt itself to live and feed
around human habitation (Ne-
ouimine, 1996). It can invade ani-
mal shelter or grain store present
inside or close to house and infect
sandflies present there. This can
possibly explain the present obser-
vation that non-working housewives
and children, formed (19%) of pa-
tients. In accordance, Rassi et al.
(2007) in South Iran observed that
M.libycus was the chief reservoir of
ZCL forming 75.4% of collected
rodents with 8.4% infected by L.
major. Also, P.papatasi formed
75% of female sandflies collected
from indoor locations with 2.7% of
them infected by L. major. Morsy et
al. (1991-a) in Egypt, could isolate
L.major strain (London-1), which is
equal to (Mon-25, the Libyan strain)
from Meriones crassus at a limited
focus in North Sinai. Therefore, the
present authors emphasize the im-
portance of separating animal shel-
ters from houses, open their sides
for adequate exposure to sun and
clean them periodically.
The third local predisposing factor
in the examined villages was the
presence of stagnant surface lakes
originating from natural warm water
spring or from deep artificial wells
and used for irrigation of cultivated
land and animal drinking. The in-
creased humidity and moisture of
soil around them together with vast-
ly scattered animal manure makes
the soil ideal for sandfly egg deposi-
tion and larval feeding. In accord-
ance, Neouimine (1996) reported
that environmental modifications
such as construction of dams can
change the temperature and humidi-
ty of soil and vegetations with re-
sulting increased density of sandfly
and rodent populations and that the
increased incidence of CL among
non-immune settlers was observed
in water irrigation schemes in Libya
Saudi Arabia, Syria and other coun-
tries. Saleh et al. (2007) in central
Tunisia reported that the first out-
break in 1982 followed the con-
struction of Sidi Saad dam. Schlein
et al. (1982) in Jordan noticed a
high infection rate of P.obesus
(93%), with increased density of

P.papatasi which correlated with


high humidity in P.obesus burrows.
Interestingly, Neva and Brown
(1994) reported that sandflies be-
come especially active on warm
humid nights as they tend to ovipos-
it after blood meal optimally in a
warm humid soil. Stagnation of ob-
served lakes can be reduced by re-
moving the growing obstructive
plants. Residual insecticides should
be applied to such possible sandfly
breeding or resting sites, including
animal shelters and houses which
should also be well-ventilated.
Considering the well-known re-
ported observation of continued
eastward spread of ZCL in Libya
(Annajar, 2008), starting in 1971
(Kadiki and Ashraf, 1971) by re-
cording the first 40 cases in Nalut
town in the west mountain -near
Tunisian border- and reaching re-
cently at the west border of Sirte,
the present authors are inclined to
attribute this extension in distribu-
tion of disease to coincidental
changes in world climate in the last
4 decades. The disease may have
arrived from Tunisia-containing the
same parasite strain and reservoir
(Helal et al., 1987) via M.libycus
migration along the extension of the
west mountain there and perhaps is
slowly on its way to Egypt. Increas-
ing global warmth and humidity can
affect the ecology and biology of
both vector and reservoir (Shope,
1991; Patz et al., 1996). It may in-
crease reproduction and biting activ-
ity of sand fly, prolong its breeding
season and adult survival and short-
ens the maturation period of the
parasite inside the vector. It can also
increase reservoir adaptation and
capacity for migration. Cross et al.
(1996) observed increased geo-
graphical and seasonal distribution
of P.papatasi so that disease trans-
mission could be extended through-
out the 12 months of the year in en-
demic locations with a 3 rise in
temperature. According to Fichet-
Calvet et al.(2000) working in south
Tunisia, rainfall can increase the
multiplication of sand rat by in-
creasing the growth and availability
of its feeding plant; its infection
with L.major was increased 4.5 fold
in wet season compared to dry one.
Alternatively, disease transmission
can increase during heavy rainfall
by driving possibly infected rodents
out of their burrows. Neva and
Brown (1994) reported that heavy
rainfall in 1986 resulted in a large
epidemic of L. major infection in
El-Khartoum, with10,000 cases ,
peak incidence in September and
both sexes and all age groups were
equally affected (El-Safi et
al.,1991). Weather change, there-
fore, is an important precipitating
risk factor which can affect the tim-
ing and intensity of disease outbreak
or epidemic .It may also partially
explain the observed fluctuation of
annual incidence in Sirte. In favor of
a universal climate effect, increased
incidence with appearance of new
foci have been recently reported
from different areas around the
world in Algeria (Mihoubi et al.,
2006), Morocco (Cuernaoui et al.,

2005), Pakistan (Anwar et al.,


2007), Iran (Talari et al., 2006;
Razmjou et al., 2009) and in Sri
Lanka (Nawarantna et al., 2007).
The climate factor is difficult to
control, however, the WHO is look-
ing forward for a climate-based ear-
ly warning system in trial to predict
epidemics in climate-related diseas-
es including arthropod-born diseases
(El-Naiem et al., 2003).
Comparing risk factors in Sirte
and Taurgha (the near famous epi-
demic site), the present authors sug-
gest that the increased incidence of
ZCL in Sirte is -on one hand- relat-
ed to the ongoing development of
new land for housing and agricul-
ture with increased occupational
exposure to the infected nearby sand
rat burrows .The continued influx of
more non-immune, non-health-
educated dwelling and working
population further aggravated the
condition. The importance of this
risk factor was reflected by the find-
ing of a clear-cut increased sex ratio
2:1, with land-workers forming
about half of the working cases. On
the other hand, domiciliary infection
facilitated by close association of
infested animal shelters to houses is
the second risk factor that should
not be under-estimated. Increased
soil moisture by natural pond is a
contributing precipitating factor
while a wetter season with increased
rainfall can be via increasing the
numbers of infected vectors and
reservoirs - the timing factor decid-
ing the first emergence of the dis-
ease in autumn 2002, and contrib-
uting to its subsequent seasonal re-
emergence in the following years. In
contrast, during Taurgha epidemic
in 2005, both sexes and all age
groups were almost equally affected
(Annajar, 2008), so domestic infec-
tion rather than occupational expo-
sure is more likely. This could be
facilitated by close animal shelters -
rich in M.libycus and sandflies- to-
gether with over-crowding of hous-
es by big families of African cast
and the presence of a real large
warm water lake from a natural
spring. The precipitating factor- alt-
hough unknown yet- could be due to
imported pottery containing infected
sandflies from Gharian, a known old
endemic town in the west mountain
(as the inhabitants believe) , and/or
arrival of migrating infected
M.libycus from Benwalid ( a nearby
old focus) - (fig.28) or again started
by an exceptionally warm moist
season.
As regards the observed pre-
vailing lesion pattern, the majori-
ty of lesions were located on ex-
posed body parts because sandfly
cannot bite through ordinary
clothes (Harman, 1992); hence
the importance of wearing com-
plete protective clothes. Lesions
were more frequent on legs and
feet probably related to the local
traditional type of clothing with
long sleeves and no trouser, or
due to the low level flight range
of sandfly (Schmidt and Roberts,
2000). Minority of lesions

(8.5%) was distributed on the


trunk; this may be due to the
habit of sleeping with minimal
clothes particularly in summer.
Likewise, El-Buni et al. (1997-b)
observed that most lesions were
distributed on the leg followed
by the arms, 49.3% and 41.7%
respectively. Neva and Brown
(1994) reported that skin lesions
produced by L.major occur pri-
marily on the lower limbs, are
moist and tend to ulcerate very
early. In the present study, the
face was found to be frequently
targeted (41%), because it is the
most body part which is kept un-
covered especially in children.
About half of these facial lesions
occurred in females which is
cosmetically unacceptable. Simi-
larly, El-Dwebi et al. (2008), in
Tripoli, studying CL in children,
observed facial lesions in 43.6%
out of 473 cases. Talari et al.
(2006), reported affection of the
face in 47% of examined 117
cases. Insecticide-treated bed net
(ITN) is highly recommended at
home, it can protect the face and
repellants can be used above the
age of two years (Alexander and
Maroli, 2003). The observed in-
creased frequency of multiple
lesions (73%) can be caused by
single bites of several infected
sandflies or otherwise by multi-
ple interrupted bites of a single
blocked -by L.promastigotes in
pharynx- infected sandfly, which
is more likely if the lesions ob-
served were close and isophoric
passing the same stage of evolu-
tion (Moschella and Hurley,
1992). This type was frequently
seen in infants and young chil-
dren infected at home (fig.9,16).
Multiple lesions all-over the
body up to 32, were observed
particularly in non-immune land-
workers, who used to sleep out-
side in the farm at a ground level
with minimal clothes in summer;
they were most probably exposed
to multiple bites by a cluster of
infected sandflies. It is worthy to
say that those workers were
mostly Egyptian immigrating or
temporary farmers and builders
with no educational background
about leishmaniasis. In accord-
ance, El-Buni et al. (1993) re-
ported that multiple sores over
the limbs and even on the trunk
represent the typical clinical
presentation of ZCL, mostly ob-
served in foreign workers of de-
velopmental land projects. In the
present study most cases present-
ed at 1-2 month duration from
the onset of lesion. This duration
almost coincide with the period
after start of overt ulceration and
before the start of spontaneous
healing, which provide sufficient
motivation for consultation.

Likewise, Harman (1992) report-


ed that ulceration of L.major le-
sions can occur as early as 2
weeks and healing starts at about
3 month duration. Most observed
lesions had a moderate size from
1-3 cm attained within duration
of 1-2 months. Although lesion
size depends on its duration,
some observed lesions however
(13.5%), reached a large size
from 5 to 12 cm in diameter. Un-
less this was due to secondary
bacterial infection which delays
healing (Potter et al., 1983),
large lesion size can be attributed
to increased strain virulence or
low or altered host immune sta-
tus (Bell, 1995). Since the pre-
vailing L.major strain in Libya is
known to be (Mon-25)- (Ashford
et al.,1976 and El-Buni et
al.,2000-b), the observed large
lesion size can be explained by a
diminished cell-mediated im-
mune response where the ulcer
is superficial with minimal mar-
ginal infiltration and induration
(fig.13) or alternatively, due to
hypersensitivity reaction with an
exaggerated CMI response where
in this case the lesion margin is
over-infiltrated and lesion itself
is swollen and crusted
(Bell,1995); this was liable to
occur in children and allergic pa-
tients (fig.15,16). As regards the
presently observed lesion type,
the ulcerative form constituted
the majority of lesions (77.3%).
A reasonable explanation for this
is that most patients could
choose to present shortly after
ulceration took place when the
condition became more serious.
Ulceration is explained by anoxia
due to pressure ischemia by the
cellular infiltrate underneath or
by an immuno-pathologic reac-
tion (Neva and Brown, 1994).
Likewise, Talari et al. (2006) in
Iran, detected ulcerative lesion
type in 60.7%, papulo-nodular in
27.4%, sporotrichoid in 6% and
impetiginous in 2.5% of cases. In
addition, Hepburn (2003) report-
ed that infection begins by a
papule which enlarges and then
ulcerates; the typical lesion is a
painless ulcer with raised indu-
rated margin and necrotic base
often covered with an adherent
crust of dried exudate, located on
exposed site and measuring from
0.5 to 3 cm in diameter. The
presently observed form se-
quence is almost typical of moist
oriental sore caused by L. major
and was also described by other
authors (Harman, 1992;
Moschella and Hurley, 1992;
Garcia, 2001). The characteristic
rapid development and early ul-
ceration with exudation is at-
tributed to the fact that L.major
is basically a zoonotic parasite of

small animals like rodents and is


not adapted to humans, where the
reaction is more severe and ex-
aggerated relative to that in ro-
dents which is frequently asymp-
tomatic (Bell, 1995). Only two
rare lesion types were observed
in special cases. Sporotrichoid
type was observed in a diabetic
patient, where subcutaneous
nodules were observed proximal
to the primary lesion present on
the forearm. In confirmation,
Moschella and Hurley (1992)
reported that local lymphatic
spread may occur in L.major in-
fection with solitary or multiple
subcutaneous nodules occurring
proximal to the initial lesion be-
tween it and the regional lymph
nodes. Empetiginous type was
observed in an allergic child
where vesicles and pustules sur-
rounded by erythema existed
masking the primary lesions.
Masmoudi et al. (2005) in Tuni-
sia examined facial lesions and
reported that empetiginous form
occurred frequently in children
and that such infected lesions
should treated first by antibiotics.
Diagnosis in established en-
demic areas is usually made on
basis of a clinically typical lesion
in conjunction with appropriate
history of exposure, however
apart from a relatively toxic and
costly chemotherapy; there are a
number of mimics which should
be excluded before starting spe-
cific treatment, such as bacterial
and fungal infections, eczema,
tuberculosis, leprosy and basal
cell carcinoma (Harman, 1992).
Therefore, parasitological con-
firmation should be the first step
for management of CL particu-
larly in a recent endemic rural
area like Sirte where dermatolo-
gists are not always available. In
the present study, the majority of
the examined clinical cases were
confirmed parasitological by
Giemsa -stained skin slit smear.
Leishmania amastigotes could be
detected in smears taken from
ulcer margin in (79.5%) out of
151 examined cases. This satis-
factory result could be explained
by the observed finding that most
lesions were still active and non-
healing with a relatively short
duration of 1-2 months .In addi-
tion, we believe that there were
some important key steps in the
technique which were done
properly and raised the positivity
of amastigote detection, includ-
ing: (1) Samples were deliberate-
ly taken from advanced and in-
durated purple-colored part of
the ulcer margin, which is ex-
pected to be most infiltrated by
parasitized macrophages. This
was achieved by meticulous ex-
amination of the lesion with the

aid of a hand lens. (2) Complete


haemostasis was achieved to
avoid contamination by hemo-
globin debris, making interpreta-
tion difficult. (3) Four smears
were taken from the marginal slit
in each case and examined for an
adequate time. (4) Proper Giem-
sa staining improves the mor-
phology and results in clearly
differentiated kinetoplast, nucle-
us and cytoplasm. In confirma-
tion, Navin et al.(1990) compar-
ing different diagnostic tests,
achieved 84% slit smear sensitiv-
ity, he reported that smears made
from lesion border with scalpel
were more likely to yield diagno-
sis than those from aspirates, im-
pression smears or histologic
sections. He mentioned that a
major advantage of simple scrap-
ing over biopsies is that more
samples can be taken with little
increase of trauma to the patient.
This author could obtain a sub-
stantial increase in sensitivity
from 70% to 97% - of the finally
confirmed cases -when the num-
ber of smears taken from each
lesion was increased from one to
four. In addition, Ramirez et al.
(2000) reported 78.3% sensitivity
of dermal scrapings mostly taken
from well-defined indurated ul-
cer margins, in a total of 115 pa-
tients. In comparison, they re-
ported that although aspirate cul-
ture technique by needle is easy
and non-traumatic method of
parasitological diagnosis, it re-
quires special equipment is time
consuming and contamination is
often in culture under field work
conditions. He found that 25 out
of 70 patients, who were also di-
agnosed by microscopy, had
negative results by aspirate cul-
ture. Furthermore, he reported
that the sensitive PCR technique
which is costly and require a
special laboratory infrastructure
is more routinely used in epide-
miological study rather than for
clinical diagnosis particularly in
old world CL as the treatment is
not species-oriented. He con-
cluded that direct microscopic
examination of lesion scraping,
still continues to be the most
widely applied diagnostic meth-
od due to the ease of perfor-
mance, low cost and speed of
technique. On the other hand,
(20.5%) of the examined cases
were negative for amastigotes;
probably because they mostly
had less active lesions either very
early or possibly healing lesions
older than 3 months duration,
with reduced margin infiltration
as indicated by reduced color and
induration. The presence of non-
observable secondary bacterial
infection could also be a cause of
negativity (Edrissian et al.,

1990). In agreement, Garcia


(2001) reported that the ability to
detect parasite in skin scrapings
depends on the number of
amastigotes which is related to
the level of the host immune re-
sponse, the presence of bacterial
or fungal contamination and
whether the specimen is taken
from an active or healing lesion.
In the present study, the skin slit
technique could not be done in
12 patients (7.3%) due to non-
consent of the patients; they were
diagnosed on both clinical and
positive therapeutic response ba-
sis. Therefore, the present results
prove that Giemsa slit smear is a
reliable direct diagnostic tech-
nique, available in rural areas
with limited laboratory equip-
ment and is quite suitable for ac-
tive non-healing lesions with a
short duration.
In the present study, although
spontaneous healing is usually
completed in an average period
of six months (Harman, 1992),
all presenting patients preferred
to receive treatment to speed up
healing and minimize scar for-
mation. The pentavalent antimo-
ny compound, sodium stiboglu-
conate (Pentostam) has been
used since decades for specific
treatment of all forms of leish-
maniasis and remains to be the
mainstay of disease treatment.
The biochemical basis of its ef-
fectiveness is through inhibition
of ATP synthesis by Leishmania
amastigotes (Blum et al., 2004).
Therefore, the drug was chosen
for treatment of the cases; how-
ever the route of administration
was determined according to the
site and size of lesions and the
patient condition in each separate
case. Intralesional Pentostam
proved to be quite suitable for
treatment of all medium sized
active lesions which formed the
majority in our cases. It was used
successfully with good response
in (60.1%) of patients. This can
be explained by the fact that this
route provides the highest local
concentration of this specific
drug, if complete blanching of
the lesion is achieved after injec-
tion. The local pain experienced
by the patient during the neces-
sary strict intra-dermal injection
of the drug, was generally tolera-
ble. The infrequent weekly dos-
age with minimal systemic side
effects and needless hospitaliza-
tion has made the local injection
a relatively suitable choice for
most patients. Likewise, El-Buni
et al.(1997-b), treating 199 ZCL
patients referred to Tripoli cen-
tral hospital from different loca-
tions of the endemic North-West
region, reported that all patients
responded well to sodium

stibogluconate. Sharquie et al.


(1988), treating 130 lesions of
ZCL, 94.6% showed complete
healing; he recommended in-
tralesional route as safe and ef-
fective method of treating acute
CL. Local infiltration was stud-
ied in Saudi Arabia (Tallab et
al.,1996), 2-15 blanching infiltra-
tions were needed to achieve
cure rates of 72-99%. Uzun et al.
(1999) in Turkey treated 76% out
of 3074 CL cases by intralesional
injection with pentavalent anti-
mony. WHO (1990) documented
that the most common treatment
in most parts of the world for CL
is the intralesional application of
pentavalent antimony. These
drugs were proved to be superior
to local Paromomycin in treat-
ment of CL (Faghihi and
Tavakolikia, 2003). Systemic
Pentostam was the second
choice; it required hospitalization
for prior investigation and con-
trol of any possible side effects
particularly in older patients. It
was also given in repeated daily
dosage; therefore it was restrict-
ed to certain types of lesions for
which local injection was rela-
tively less suitable. It was indi-
cated in lesions at sensitive sites
like facial lesions (particularly in
females and children) and over-
joint lesions, multiple lesions
(more than 2), large lesions (5
cm or more), slow-healing le-
sions , in immuno-suppressed or
diabetic patients and in rare spo-
rotrichoid and empetigoid le-
sions. The drug was used suc-
cessfully to treat (30.9%) of cas-
es by slow intravenous or intra-
muscular routes depending on
the stage and progress of the le-
sion with minimal subjective
gastro-intestinal and musculo-
skeletal side effects. Blum et al.
(2004) reported that clinical ex-
perience has shown that systemic
pentavalent antimonies were ef-
fective in severe infections with
big or multiple lesions and those
over the face or joints. Likewise,
they suggested that the dose was
increased from 10-20mg/kg/day
to achieve complete treatment
preventing relapse or develop-
ment of resistant, as reduced ef-
ficacy was associated with lower
dosage and no higher toxicity
was found with the higher doses.
Herwaldt and Berman (1992)
reported that despite the side ef-
fects sometimes associated with
systemic treatment including
myalgia, arthralgia, bone marrow
depression, liver enzyme eleva-
tion or cardiac arrhythmia, this
route remains the alternate effec-
tive therapy for all species caus-
ing CL. The oral anti-fungal Flu-
conazole was tried with satisfac-
tory results in 4 cases with mul-

tiple lesions unfit for systemic


Pentostam .Although the drug
can be hepato-toxic; it was well
tolerated at a dose of 200 mg dai-
ly for 6 weeks. Alrajhi et al.
(2002) in Iran used the drug in
similar dose achieving cure rate
of 79%, with 3 month follow up.
They reported that Fluconazole
was well tolerated with promis-
ing results in L.major infections.
Local hypertonic saline was used
as complementary therapy in
chronic slow-healing or deep le-
sions to stimulate formation of
granulation tissue and promote
the healing process. Sharquie
(1994) in India achieved 96%
local therapy with intralesional
sodium chloride (7%) solution
which acted on the parasite via
osmotic pressure mechanism.
Cryotherapy with liquid nitrogen,
although a non-specific modality
of treatment was successful in 9
cases with small papulo-nodular
lesions.(less than 1cm); such ear-
ly lesions are usually less active
with a smaller number of para-
sites and less chance of relapse.
Khan and Muneeb (2005) in Pa-
kistan reported that cryotherapy
was useful for small lesions with
diameter less than 1 cm. In Tur-
key (Gurei et al., 2000) the cure
rate after 2 sessions of cryother-
apy; followed for 3 months was
77% compared to 85% following
intralesional Pentostam. This
modality is labor-intensive and is
applied basically to the margin to
diminish the parasite without
causing scar. It is not suitable for
multiple, infected or complicated
lesions. In the present study, sec-
ondary bacterial infection of le-
sion was in 14.7% of cases; man-
ifested by purulent exudate, local
pain and tenderness and some-
times enlarged draining lymph
nodes. It occurred frequently on
the legs and feet and could be
attributed to lack of cleaning and
non-covering of the lesion (Gar-
cia, 2001). Secondary infected
lesions are difficult to diagnose
or treatment, therefore bacteria
must be eliminated first. They
usually delay healing and pro-
duce large ulcers (Potter et al.,
1983) possibly by partially kill-
ing the parasite reducing the nec-
essary antigenic stimulus for
production of an effective local
cell-mediated response or by
changing the type of cellular re-
action; diagnosis could also be
difficult for the same reasons. In
addition, slit smear or intrale-
sional Pentostam are contraindi-
cated as they can spread the in-
fection to deeper planes. These
lesions should be completely
treated by drying agents, local
and systemic antibiotics for ten
days at least as this will help

healing by improving the im-


mune response as well as the
therapeutic response. Edrissian et
al. (1990) in Iran reported that
secondary bacterial infection was
common in CL was 26.5% in le-
sions confirmed positive by skin
scraping and 45% in clinically
suspected but parasite negative
lesions. They recommended the
use antibiotic before slit smear or
specific chemotherapy. In the
present study the majority of
cases were followed up for 3
months after completion of
treatment without evidence of
relapse proving that the local
parasite strain is sensitive to the
chosen drugs. Criteria of treat-
ment were based on WHO defi-
nition of clinical healing which
complete re-epithelialization is
observed up to 3 months after
treatment (Mayrink et al., 2006).
Blum et al. (2004) stressed on
the importance of follow up, as
lesions may demonstrate only
partial response after 20 days of
treatment and may not heal until
several weeks later. Follow up
was made on clinical basis as
direct slit smear which proved to
be sensitive in active lesions was
not expected to be so in healing
ones; the test rapidly converted
negative even with spontaneous
healing (Zakraoui et al.,1995).
The sensitive PCR was not a test
of cure; it can remain positive
several years after clinical cure
(Schnabach et al., 1998).
Fear of deep scar formation
expressed by all patients was a
significant stimulus for seeking
medical treatment. Depression
with anxiety, however, due to
possible non-cosmetic appear-
ance particularly in lesions on
face or hand or because of inca-
pacitating joint affection, -
depending on the education level
led to reduced body image satis-
faction and self-esteem with con-
sequent withdrawal and non-
participation in work or social
activity. The lack of basic
knowledge among patients
(58.3%) about the disease trans-
mission, prevention, healing pro-
cess and conventional medical
treatment, certainly contributed
to increased exposure, delay in
notification and treatment. The
authors emphasize the utmost
importance of health education
as a potential effective tool for
avoiding disease circumstances
and encouraging early consulta-
tion and treatment, thus prevent-
ing or minimizing a possibly
deep scar. It should be offered in
advance to the population at risk,
particularly land-workers for
whom the disease should be re-
garded as an occupational hazard
with all its implications. Applied

health education can be im-


proved by utilizing home health
nurses as source of information
beside physicians in health cen-
ters. In none or less endemic
countries like Egypt, where im-
ported cases -by far- exceed in-
digenous cases, there is a real
need for increased awareness of
the disease as many workers are
ignorant of the risks, take no per-
sonal prophylactic measures and
experience delay in diagnosis
followed by inappropriate treat-
ment upon their return. It is a
must that such workers should be
informed better by broadcasting
media about prevention before
departure and properly diagnosed
and treated after arrival at home.
The study stressed on the im-
portance of applying integrated
personal prophylactic measures
particularly at infection season as
being the most practical means
available for disease prevention,
including complete protective
clothing and repellants especially
outdoors, insecticide-treated bed
nets indoors, fine window
screens, filling wall crevices,
avoiding exposure from dusk to
dawn or sleeping at or near
ground level outside or inside
house. This research draws phy-
sician attention for the im-
portance of the early diagnostic
clinical criteria inferred from this
study, where any painless small
red brown nodule, in the endemic
area (or in home-country of tem-
porary land-workers), on ex-
posed skin, progressive for
weeks, unresponsive to local an-
tibiotics, tending to form fine
white scales on surface or yellow
exudate, should be managed as a
moist oriental sore until proved
otherwise. Health personnel in
the rural areas should be ade-
quately trained to practice the
Giemsa slit smear technique to
be done routinely as the first step
of management, once the above
criteria are fulfilled. Pentostam
should be made available at the
rural health centers, where close
follow up and complete treat-
ment can be more guaranteed.
Conclusion
Farmers and construction workers
including Egyptian immigrants were
proved to be particularly prone to
infection from close sand rat bur-
rows.ZCL should be regarded as an
occupational disease; workers must
receive health education and
prophylactic measures at the proper
time. Animal shelters should be
ventilated and separated from hous-
es to reduce domiciliary infection.
Physicians in rural areas in Sirte and
Egypt should receive refreshment
course to be able to recognize early
the described local lesion features
and their development. They should

be trained to perform the reliable


Giemsa slit smear as first step of
management; being available, rapid,
specific and sensitive in all active
non-healing lesions. Intralesional
pentostam proved to be effective
and safe; it should be resorted to
unless systemic route is indicated.
The drug must be available at rural
health centers.
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Explanation of Figures
Fig.1: Psammomys obesus-large, yellow with whitish legs.
Fig.2: Sand rat burrows-in salty soil with salty plant food.
Fig.3: Meriones libycus-small, yellowish, hairy tail end.
Fig.4: Female sandfly on biting-erect wings,hairy body.
Fig.5: Animal shelter-near houses,densely covered.
Fig.6: Early red papule, 5mm,persistent,painless.
Fig.7: Nodule and papule (arrow) on back of forearm.
Fig.8: Close view of typical early nodule covered with fine white scales, just starting
exudation and ulceration (arrow).
Fig.9: Isophoric facial lesions covered with fine white scales.
Fig.10: Active ulcer,covered by loose brown crust.
Fig.11: Deep crater ulcer with raised rolled edge.
Fig.12: Mature facial ulcer with thick rolled edge.
Fig.13: Large exudative ulcer on forearm.

Fig.14: Large active ulcer with indurated margin.


Fig.15: Over-wrist swollen crusted lesion.
Fig.16: Isophoric multiple swollen lesions on child hand.
Fig.17: Sporotrichoid form,with subcutaneous nodules(arrow).
Fig.18: Impetigenous form in an allergic child.
Fig.19: Large purulent lesion on foot dorsum.
Fig.20: Painful secondary infected nasal skin lesion.
Fig.21: Depressed facial scar deformity of untreated lesion.
Fig.22: Skin slit smear made radial on active margin.
Fig.23: Giemsa-stained smear, showing Leishmania amastigotes inside macrophage
with rod-shaped kinetoplast (arrow).
Fig.24: Untreated active facial ulcers in a child.
Fig.25: After treatment with systemic pentostam
Fig.26: Intralesional pentostam injected under ulcer base&margin.
Fig.27: Cryotherapy applications,with liquid nitrogen.
Fig.28: Map of ZCL - endemic Northwest region of Libya :
1-Sirte town (free).
2- Hisha (examined).
3- Al-Gadaheya (examined.).
4-Taurgha (epidemic 2005).
5- Ben-Walid (old endemic focus).
6- Gharian (old focus).
7-Yafran (old focus).
8- Nalut (oldest focus).
9-Tripoli (free).

Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 907 - 917
RELATIONSHIP BETWEEN SERUM CYTOKINES PROFILES AND
HEPATIC FIBROSIS IN SCHISTOSOMIASIS MANSONI:
AN EXPERIMENTAL STUDY
By
REDA R. EL-GAMAL, SOAD M. NADA, NAHED E. MOUSTAFA,
HANY A. AFIFY, GHADA M. FATHY, ASHRAF, S. METWALLY
AND RANIA S. HAMZA
Department of Parasitology, Faculty of Medicine, Zagazig University,
Zagazig, Sharkia Governorate, Egypt
Abstract
.
Forty of eighty mice (10 each group) were infected with S. mansoni cercariae
and sacrificed at 3 weeks (G-A), 6 weeks (G-B), 12 weeks (G-C) and 16 weeks
(G-D) post infection (P.I). The other forty mice were used as control groups of
ten mice each. There were highly significant difference between egg counts
after 12 weeks & 16 weeks of infection compared to 6 weeks P.I. The maxi-
mum egg count and mature eggs were in 6
th
week P.I while dead eggs reached
the peak at 16
th
weeks P.I. Liver egg counts showed maximum followed by in-
testinal and then, stool egg counts. A highly significant differences in hydroxy-
proline, TGF-B1and IL-4 of infected than in controls and their peak at 16
weeks P.I. A significant difference in the IFN- in the infected than in controls
with peak occurred at 6 weeks P.I. and declined after that reaching a low level
at 16 weeks P.I. A highly significant positive correlation was between TGF-
B1and IL4 and significant negative correlation between IFN- and both IL4 &
TGF-B1. A highly significant and significant negative correlation between
TGF-B1 and egg count at 12 & 16 weeks P.I respectively. Negative correlation
was between IL-4 and egg count at 16 weeks P.I. But, significant positive cor-
relation was between IFN- with the egg count at 16 weeks P.I. A significant
negative correlation was between TGF-B1 and oogram at 6 & 16 weeks P.I, but
highly significant positivity was between IFN- and oogram at 16 weeks P.I. A
significant negative correlation was between IL-4 and oogram at 16 weeks P.I.
A significant positive correlation was between levels of hydroxyproline and
TGF-B1 at 12 & 16 weeks P.I. Highly significant negative correlation between
hydroxyproline and IFN- was at 12 weeks P.I with significant and highly sig-
nificant positive correlation between hydroxyproline and IL4 at 12 & 16 weeks
P.I.
Key words: Cytokines, Hepatic fibrosis, Schistosoma mansoni, mice.

Introduction
Schistosomiasis is the second most
important parasitic disease world-
wide after malaria which continues
to be a significant cause of parasitic
morbidity and mortality (Burke et
al., 2009). Schistosomiasis mansoni,
a major cause of hepatic fibrosis in
many developing countries, triggers
a granulomatous inflammatory reac-
tion in response to its eggs that
lodge in the liver. The egg antigens
are eliminated slowly, and the per-
sistent granulomatous response
leads to prolonged matrix synthesis
and hepatic fibrosis (Wahl et al.,
1997). The liver fibrosis gained a
greater attention since 1980. This
attention was firstly directed to-
wards the components of extra-
cellular matrix (ECM) in the liver
with gradual shift towards exploring
the cellular and molecular basis of
fibrosis. The cellular mediators
mainly cytokines, chemokines, cell
receptors and ECM degradation
mechanisms that drive fibrosis pro-
gression and regression were exten-
sively investigated (Bataller and
Brenner, 2005).
This work aimed to study the as-
sociative relationship between cyto-
kine profiles and hepatic fibrosis in
experimental schistosomiasis as ev-
idenced histopathologically and bio-
chemically.
Material and Methods
Eighty Swiss albino mice
weighting 20-25 gm each were ob-
tained from Theodor Bilharz Re-
search Institute. Forty (ten mice,
each group) were infected with S.
mansoni cercariae (Liang et al.,
1987) and sacrificed (Smithers and
Terry, 1965) at 3 weeks (GA), 6
weeks (GB), 12 weeks (GC) and16
weeks (GD) post infection. The oth-
er forty mice were used as control
groups of ten mice each. Parasito-
logical, histopathological, biochem-
ical and immunological studies were
performed.
Egg count per gram stool was
done by Kato thick smears (Martin
and Beaver, 1968), egg count in liv-
er and intestine was after Cheever
(1968), and oogram was after Pelle-
grino et al. (1962)
Determination of number and size
of liver egg granuloma (Attallah et
al., 2006) and detection of collagen
in liver egg granuloma (Masson,
1929) were done
Determination of 4- hydroxypro-
line level was after Bergman and
Loxley (1963).
The levels of IFN-, IL-4 and
TGF-B1 in serum were measured by
ELISA (Montenegro et al., 1999).
Statistical analysis: Data was ana-
lyzed by version 11 software (SPSS,
1999).
Results
The results were shown in tables (1 to 8) and figures (1 to 6).

Table 1: Relation between mean values of egg counts (egg/ gram) in stool, tis-
sues (intestinal &liver) and Oogram different weeks after infection.
Method
Weeks
Kato-smears Liver eggs Intestinal eggs Oogram
Mean SD Mean SD Mean SD
Immature Mature Dead
Mean SD Mean SD Mean SD
6 weeks 1151.8 1.99 4785.3 2.3 2565.9 1.7 371.3 1.8 573.2 1.4 56.7 1.6
12weeks 891.5 2.5 2953.8 3.5 1564.6 3.6 583.2 3.2 340.4 2.0 76.4 1.6
16 weeks 317.1 2.3 1570.5 2.2 1120.2 1.87 780.9 2.1 236.7 2.3 163.2 1.0
*P <0.001 <0.001 <0.001 <0.001 <0.001
Table 2: Hepatic hydroxyproline (ug/gm) in tissues different weeks post infec-
tion.
Hyp level
Weeks
Infected Control
T P
Mean SD Mean SD
3 weeks 49.6 5.3 26 1.0 13.8 <0.001
6 weeks *298 1.1 33.6 0.5 712.3 <0.001
12 weeks *500.7 31.5 37. 3 0.5 46.5 <0.001
16 weeks *599.8 11.0 32.3 1.3 162.3 <0.001
Table 3: Level of TGF-B1 different weeks post infection.
TGF-B1
Weeks
Infected Control T P
Mean SD Mean SD
3 weeks 31.2 1.5 28.4 2.3 3.2 *<0.05
6 weeks *57.7 6.3 21.5 4.0 15.3 **<0.001
12 weeks *536.9 218.8 23.5 6.2 7.4 **<0.001
16 weeks *697.9 219.3 20.0 5.7 9.77 **<0.001
Table 4: Level of IFN- different weeks post infection.
IFN-
Weeks
Infected Control T P
Mean SD Mean SD
3 weeks 61.2 1.0 6.6 0.4 158.2 **<0.001
6 weeks *118.8 5.8 6.58 0.4 60.9 **<0.001
12 weeks *29.4 6.6 6.4 0.4 10.95 **<0.001
16 weeks *9.3 1.1 6.6 0.4 7.59 **<0.001

Table 5: Level of IL4 in different weeks post infection.


IL4
Weeks
Infected Control T P
Mean SD Mean SD
3 weeks 15.0 0.6 7.3 0.4 33.96 **<0.001
6 weeks *31.75 0.4 7.23 0.14 168.49 **<0.001
12 weeks *282 20.4 7.5 0.36 42.4 **<0.001
16 weeks *290 11.5 7.5 0.4 77.3 **<0.001
A
B
C
r = -0.87, P<0.05
0
50
100
150
200
250
300
350
0 20 40 60 80 100 120 140
IFN-
I
L
-
4
Fig. 1: Correlation between cytokines levels in mice infected with S. mansoni
(A= IFN-&TGF-B1, B= IL4&TGF-B1, C= IL4& - IFN-).
r = 0.92, P<0.001
0
50
100
150
200
250
300
350
400
450
0 200 400 600 800 1000
TGF-BI
I
L
-
4
r = -0.78, P<0.05
0
20
40
60
80
100
120
140
0 200 400 600 800 1000
TGF-BI
I
F
N
-

Table 6: Correlation between cytokines levels and liver egg counts different
weeks post infection.
Correlation
Weeks
Egg counts and Cytokines
TGF-B1 IFN- IL4
r p R p r p
6 weeks 0.57 >0.05 0.03 >0.05 0.2 >0.05
12 weeks -0.75 **<0.001 0.03 >0.05 0.38 >0.05
16 weeks -0.61 *<0.05 0.84 **<0.001 -0.71 *<0.05
Table 7: Correlation between cytokines levels and oogram different weeks post
infection.
Correlation
Weeks
Oogram and Cytokines
TGF-B1 IFN- IL4
r P r P r P
6 weeks -0.63 *<0.05 0.037 >0.05 0.3 >0.05
12 weeks 0.51 >0.05 0.02 >0.05 0.33 >0.05
16 weeks -0.64 *<0.05 0. 63 **<0.001 -0.59 *<0.05
Table 8: Correlation between levels of hydroxyproline and cytokines levels in
mice infected with S. mansoni different weeks post infection.
Correlation
Weeks
Hydroxyproline and Cytokines
TGF-B1 IFN- IL4
r p r p r p
3 weeks 0.29 >0.05 0.16 >0.05 -0.16 >0.05
6weeks 0.03 >0.05 -0.58 >0.05 0.12 >0.05
12 weeks 0.66 *<0.05 -0.98 **<0.001 0.79 *<0.05
16 weeks 0.68 *<0.05 -0.12 >0.05 0.71 **<0.001
Discussion
In the murine model of schistoso-
miais, type 1 cytokines such as IFN-
and activated macrophages have
been correlated with immunity. The
type 2- associated cytokines such as
IL-4, IL-13 and IL-10 inhibited
classical macrophage activation and
implicated in granuloma formation
and fibrogenesis around tissue-
deposited eggs (Morais et al., 2002).
In the present results, all groups
showed that the maximum egg
count in the 6
th
week P.I including
egg counts (egg/gram) in stool and
tissues (intestinal &liver) and ma-
ture ova in oogram.
The liver egg counts was the max-
imum followed by the intestinal
then the stool egg counts, that went
with El shafei et al. (2002) who re-
ported highly significant difference
between egg counts after 12 weeks
and 16 weeks of infection compared
to 6 weeks. Silva et al. (2000) found

that the number of eggs per gram of


tissue was greater in the liver than in
the intestines, this difference being
statistically significant (p<0.05). In
addition to the oogram in different
weeks after infection the maximum
number of mature eggs appears in
the 6
th
week of infection, while, the
peak of dead eggs at the 16
th
week
post infection that is supported by
El shafei et al. (2002). All this may
be due to the beginning of matura-
tion of the worm and deposition of
the eggs in the tissues at 6 weeks.
We found a highly significant dif-
ferences in the levels of hydroxy-
proline of infected than in control
groups and its high peak occurred at
16 weeks post infection. It means
that the levels of hydroxyproline
increase with the duration of infec-
tion, that agreed with Olds et al.
(1985) who reported that hepatic
hydroxyproline of infected animals
was higher between 16 & 20 weeks
P.I.. Adewusi et al. (1996) and El
shafei et al. (2002) showed a signif-
icant increase in overall hydroxy-
proline level of the liver 8 weeks
after infection of mice with S. man-
soni and reached its high peak be-
tween 16 and 20 weeks. The peak of
TGF-B1 occurred at 16 weeks P.I.
which becomes statistically signifi-
cantly higher when we compare the
infected groups with the control
one. The results were in accordance
with Zhu et al. (2000) who reported
that The level of serum TGF-beta 1
in experimental group was signifi-
cantly higher than that in controls
(P<0.05) 8, 10, 12 & 16 weeks after
infection by schistosomiasis. Sever-
al reports indicate that TGF- 1 is a
regulatory cytokine that is mainly
produced by regulatory T cells
which provides an effective mecha-
nism of control of the progression of
fibrosis in association with IL-10
(Kitani et al., 2003; Hesse et al.,
2004). A significant difference is
higher in the IFN- in the infected
than in the control groups and its
high peak occurred at 6 weeks post
infection then declined after that
reaching a low level at 16 weeks
post infection. Boros (1994) found
that IFN- was produced very early
at the inception of the liver granu-
lomatous response by the time
granulomas reached maximal size
where its production declined. Also
these results were correlated with
Wynn et al. (1995) who said that the
production of IFN- was highly
suppressed, especially after 6 weeks
of infection. Caldas et al. (2008)
conclude that in the acute phase
there is a mixed expression of
Th1and Th2 cytokines with predom-
inance of Th1 in the early infection.
The higher and lower productions of
IFN- and IL-10, respectively, in
acute as compared to chronic schis-
tosomiasis, partially explain the lack
of modulation of the immune re-
sponse in acute patients. The results
may be due to profound negative
effect of parasitic eggs on schisto-
some antigen driven IFN- produc-
tion. A significant difference is
higher in the IL4 in the infected than
the controls with its high peak oc-
curred at 16 weeks P.I.. Caldas et al.

(2008) supported these results as


they found that IL4 increased after 6
weeks of infection to reach high
level at 16 weeks post infection. The
present study showed a highly sig-
nificant positive correlation between
TGF-B1and IL4 and significant
negative correlation between IFN-
and both IL4 & TGF- B1. This
agreed with by studies in murine
schistosomiasis showing the devel-
opment of fibrosis required for the
production of profibrotic cytokines
IL-2, IL-4 (Kaplanp et al., 1998),
correlated with TGF-B1 synthesis
(Jacobs et al., 1998), and IL-12 and
IFN- (Wynn et al., 1995).
A highly significant and signifi-
cant negative correlation between
TGF-B1 and egg count at 12 & 16
weeks P.I. respectively. Negative
correlation was between IL-4 & egg
count at 16 weeks P.I. Highly sig-
nificant positive was between IFN-
with egg count at 16 weeks P.I.
Silva et al. (2004) found an associa-
tion between Th2 cytokine produc-
tion and intensity of infection and
that the intensity of S. mansoni in
patients was positively correlated
with IL-4 levels, but negative with
IFN- production. A negative signif-
icant was between TGF-B1 and
oogram at 6 & 16 weeks P.I. while
highly significant positive was be-
tween IFN- & and oogram at 16
weeks P.I. Also a negative signifi-
cant was between IL-4 and oogram
at 16 weeks P.I. These results were
in contrary to Pearce et al. (1991)
who reported that Thl responses in
mice with eggs were profoundly
down-regulated. Besides, the injec-
tion of eggs into vaccinated mice
resulted in a reduction of antigen
and mitogen-stimulated Thl func-
tion accompanied by a coincident
expression of Th2 responses, that
coincident with induction of Th2
responses. The down-regulation of
Thl cytokine production may be an
important immunological conse-
quence of helminthes related to host
adaptation. A positive significant
was between levels of hydroxyl-
proline and TGF-B1 at 12 & 16
weeks P.I. Also, highly negative
significance was between hydroxyl-
proline and IFN- at 12 weeks P.I.,
and significant and highly positive
significance was between hydroxyl-
proline and IL4 at 12 &16 weeks
P.I.. Mola et al. (1999) noted that
fibrosis development was correlated
with increased egg antigen-driven
TGF-B1 and IL-4 production, sug-
gesting an etiologic role of these
cytokines in the development of
fibrosis. Regulation of immuno-
pathology in schistosomiasis may be
more complex in man than in ani-
mal models, with multiple media-
tors capable of regulating disease
progression (Wilson et al., 2007).
Major down-regulator of fibrosis
was IFN-, but IL-4 played the role
of pro-inflammatory cytokine in
animal models of schistosomiasis
(Wynn et al., 1993; Chensue et al.,
1997). Severe portal fibrosis cases
were associated with low levels of
IFN- & high levels of TNF- (Hen-
ri et al., 2002; Booth et al., 2004).

In the present study, acute stage


granulomas appeared as predomi-
nantly exudative (cellular), fre-
quently exhibiting a central halo of
necrosis and a sprinkling of poly-
morphonuclear eosinophils. During
the chronic phase hepatic granulo-
mas differed from those of the acute
phase in showing a tendency to con-
fluence and in being smaller and
with the cells and collagen fibers
becoming more packed. Eosinophils
were less numerous while macro-
phages and fibroblasts were promi-
nent as reported by Silva et al.
(2000). In the present work the col-
lagen fibers of hepatic granulomas
in each sample were noticed by us-
ing Masson trichrome stain as it ap-
pears as a green color where the
fibroblast deposited it at the periph-
ery of the granuloma which changed
to fibrocellular and fibrotic granu-
loma in the intermediate and the
chronic stage after infection respec-
tively. This agreed with Silva et al.
(2000) who found that the acute
phase differed from intermediate
and chronic phase of the infection as
there were tendency of the granulo-
ma to confluence and being smaller
and the cells and collagen fibers
became more packed.
Conclusion
A good correlation between hepatic
fibrosis as measured biochemically
by hydroxyproline levels & semi-
quantitatively by histopathological
sections and cytokines levels was
demonstrated. There were positive
correlation between levels of hy-
droxyproline and TGF-B1. Negative
correlation was between hydroxyl-
proline & IFN- with positive cone
between hydroxyproline and IL4.
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venting fibrosis induced by schisto-
some infection. Nature, 376:594-6.
Wynn, T.A.; Eltoum, I.; Cheever,
A.W.; Lewis, F.A.; Gause, W.C.;
Sher, A., 1993: Analysis of cyto-
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induced by eggs of S. mansoni. J.
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917
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 917 - 932
AN EXPERIMENTAL STUDY FOR EVALUATING THE EFFICACY OF
CERCARIAL VACCINE AGAINST SCHISTOSOMA MANSONI
By
SIRRIA M. EL-MARHOUMY, KHOLOUD A. EL-NOUBY, MOHAMED
A. EMARA AND DINA M. ABOU RAYIA
Department of Parasitology, Faculty of Medicine, Tanta University,
Egypt
Abstract
This study assessed the effectiveness of autoclaved cercarial vaccine (ACV)
in protection against Schistosoma mansoni infection in 125 Swiss albino mice
classified into two main groups: GI: a control group. GII: a test vaccinated
with ACV, in a single dose of 0.1ml of 10
4
ml ACV (G.IIa), double dose; 0.2ml
(G.IIb) and two single doses 2 weeks apart (G.IIc). Four weeks later, all mice
were challenged with S. mansoni cercariae and sacrificed 10 weeks post infec-
tion (P.I.). The results revealed that the vaccine in a single dose (G.IIa) induced
a high level of protection against S. mansoni infection. There was a significant
reduction in the mean number of adult worm (91.12%), ova/gram liver
(91.87%), ova/gram intestine (89.09%) and number & size of granulomas in
liver (92.92% & 43.53% respectively). Besides, ACV induced a significant
increase in the level of IL-10 mRNA expression as compared to the control
group.
Key words: autoclaved cercarial vaccine, Schistosoma mansoni, Massons tri-
chrome, fibronectin and IL-10
_________________________________________________________________________________________________________________
Introduction
Schistosomiasis remains one of the
most important and widespread par-
asitic diseases and constitutes an
important public health problem
globally (Krautz-Peterson et al.,
2007). It severely threatens the
health of about 5-6 hundred million
people in the world (Soliman and
Aly, 2005). In Egypt it causes a
great socioeconomic problem and
has deleterious effects on various
tissues and organs of human body
(Feachen et al., 1989). The brunt of
this parasitic disease mainly falls on
the liver which is the main organ of
intermediary metabolism. The dis-
ease is associated with daily produc-
tion of eggs by intravascular worms.
When trapped in liver sinusoids,
these eggs cause an inflammatory
response, leading to cell-mediated
granuloma formation and ultimately
918
to hepatic fibrosis (Elliott, 1996).
The granuloma cells secrete factors
that can stimulate collagen and fi-
bronectin synthesis in fibroblasts.
Fibronectin is a major interstitial
matrix protein with binding sites for
a variety of molecules and with
multiple biological activities. It may
serve to recruit additional fibro-
blasts and also may aid in maintain-
ing the structural integrity of granu-
lomas (Moreno and Bataller, 2008).
Public health measures such as snail
control and drug treatment have
achieved some success in few areas,
but the overall prevalence of the
disease continues to rise. Therefore,
alternative measures of control such
as vaccine development remain a
high priority (Bergquist et al., 2005;
Wilson and Coulson, 2006; McMa-
nus and Loukas, 2008).
Understanding the cytokine re-
sponse is important in terms of both
development of a vaccine and con-
trol of pathology. Of particular in-
terest are cytokines secreted by
CD4+ and CD8+ T cells. CD4+ cell
responses are polarized into T help-
er (Th1& Th2 types). Th1 cells se-
crete interleukin (IL)-2 and IFN-s
which are required for host immune
responses. Th2 cells produce IL-4,
IL-5, IL-13, and IL-10, which facili-
tate antibody production and limit
Th1 response. The interplay be-
tween Th1 and Th2 cytokines may
be important in regulating hepato-
cellular damage infection. Of par-
ticular importance is the capacity of
IL-10 to down-regulate production
of proinflammatory cytokines, such
as TNF-, IL-1, and IFN- & IL-2
from T cells. Endogenous IL-10
reduced intrahepatic inflammatory
response and limits hepatotoxicity
in several models of liver injury
(Nelson et al., 2000; Jenkins et al.,
2005).
Since mice manifest a schistoso-
mal disease state similar to that seen
in humans, several candidate puri-
fied antigens (Ag) for a defined
vaccine have been described in the-
se murine models by James and
Pearce (1988) who recommended
further testing. However, none of
these purified or recombinant Ag
confers the level of protection after
vaccination with radiation-attenuat-
ed cercariae (Olsen et al., 1998 and
Gryseels, 2000). Studies using op-
timally irradiated cercariae of Schis-
tosoma mansoni to vaccinate mice
declared that worm burdens from
challenge infections can be reduced
by 90% as compared with non vac-
cinated control mice (Richter et al.,
1993 and Chan et al., 1997). More-
over, laboratory and field studies
using attenuated vaccines have
shown that immunization is an ef-
fective method for controlling this
helmithic infection, by limiting the
intensity of the disease and the
transmission of infection. However,
the use of attenuated parasite vac-
cine for human immunization is
considered impractical and poten-
tially dangerous (Yole et al., 1996).
Autoclaved vaccine containing the
whole killed parasite was tried
919
against leprosy (Krotoski et al.,
1993) and cutaneous leishmaniasis
(Bahar et al., 1996; Hashemi et al.,
1997) with good promising results.
Therefore, it would be interesting in
this study to make a trial using au-
toclaved cercarial vaccine (ACV)
against schistosomiasis mansoni to
assess the immunogenicity of this
vaccine and to detect its role in pro-
tection against challenge infection
in experimentally infected mice.
Material and Methods
Biomphalaria alexandrina were
purchased from Biological Unit
Theodor-Bilharz Research Institute,
Imbaba, Egypt. Schistosoma man-
soni cercariae were shed from the
snail after exposure to direct light at
28
o
C for 4 hours and parasite free,
male Swiss albino mice (125) 4 to 5
weeks old and weighted 20-25 gm
were housed and infected in accord-
ance with institutional guidelines.
Vaccine preparation (Hashemi et
al. 1997): autoclaved cercarial vac-
cine (ACV) was prepared from
freshly librated cercariae of S. man-
soni from infected B. alexandrina,
adjusted to the required concentra-
tion of (10
4
/ml balanced salt solu-
tion 0.9%), collected in glass con-
tainers and autoclaved under pres-
sure of 15 Ib at 121
o
C for 15
minutes.
Mice were classified into two
main groups: GI: 50 mice served as
a control which further subdivided
into: GIa: infected control and GIb:
infected control with Bacilli Clamett
-Guerin (BCG) by intradermal (i.d.)
inoculation of 5x10
6
colonies form-
ing units (CFU) over the sternum as
an adjuvant (James et al., 1985).
GII: 75 mice served as a vaccinated
group and subdivided into: G IIa: 25
mice received 0.1ml ACV as a sin-
gle dose containing 1000 cercariae/
mouse. GIIb: 25 mice received 0.2
ml ACV as a single dose containing
2000 cercariae /mouse. GIIc: 25
mice received two doses each of
0.1ml ACV containing 1000 cercar-
iae /mouse 2 weeks apart.
Immunization was done by intra-
dermal injection of ACV over the
sternum (Eissa et al. 1998). BCG
was included in the inoculum at
5x10
6
CFU as an adjuvant. Vac-
cinated mice were kept in separate
labeled insect-proof cages.
Four weeks after the beginning of
vaccination, animals of control and
test groups were infected in a dose
of 10010 S. mansoni cercariae/
mouse. Each one was exposed sepa-
rately to cercariae by tail immersion
in 4.5ml dechlorinated water for an
hour (Radke et al., 1971). Mice of
each group were kept separately in
their cages and maintained on the
same laboratory diet under similar
conditions. Ten weeks post infec-
tion (P.I), all were sacrificed and
blood samples were taken for level
of IL-10 gene expression in lym-
phocytic cells.
The mice were subjected to adult
worm count by animal perfusion
(Duvall and De Witt, 1967): A
combined intraperitoneal injection
of an anaesthetic and heparin pre-
920
vented clotting while the mice were
perfused into conical sedimentation
glasses for collecting and counting
the adult worms. The reduction %
(R%) induced by vaccine was calcu-
lated by comparing mean worm
burden in vaccinated (V) and con-
trols (C) where %R=(C-V)/Cx100
(Yole et al., 1996).
The liver and intestine of each
mouse were immediately removed
and subjected to:
Tissue egg count: Parts of liver
and caecum were used for tissue-
egg count by KOH digestion
(Cheever, 1968). One gram from
liver and caecum were weighed, and
put in a test tube containing 2ml of
5% KOH at room temperature for
overnight. Next day, all tubes were
incubated at 37
o
C for 6hr. Each tube
was shaken, and then 0.1ml of the
digest was examined for S. mansoni
eggs. Total number of eggs in 1
gram liver & 1 gram caecum were
calculated.
Formalin-fixed, paraffin embed-
ded liver tissues were cut into sec-
tions 5m in thickness and stained
in haematoxylin and eosin (H&E)
for histopathological examination.
The number of the detected granu-
lomas was calculated and the com-
position was assessed. The granu-
lomas were classified into three
types (cellular, fibrocellular & fi-
brous) according to the predominant
component (Costa-Silva et al.,
2002). Granuloma size was meas-
ured by the use of ocular microme-
ter lens fitted on a light microscope.
The mean diameter of each granu-
loma was measured in two perpen-
dicular diameters. For each section,
ten granulomas were measured and
the mean diameter of all granulomas
was calculated (Jacobs et al., 1997).
Masson's trichrome stain to high-
light fibrotic changes and to confirm
granuloma type (Bancrofti and
Gamble, 2002).
Immunohistochemical study was
performed as a fibrotic marker to
assess the degree of fibrosis in the
granulomas in 10 randomly selected
specimens from each group. Tissue
sections (each of 5m thickness) cut
on positively charged slides (Bi-
ogenex) were immersed in xylene
for 1.5 hours, then into descending
grades of alcohols and lastly into
distilled water. Epitope retrieval for
fibronectin was done using protein-
ase K solution in humid chamber for
20 minutes at 37C. Serum blocking
for antibodies was done by normal
goat serum blocking solution for 30
minutes. Primary monoclonal anti-
mouse antibody for fibronectin
(clone FBN11- LAB VISION) was
incubated with tissue sections over-
night in humid chamber (refrigera-
tor). Endogenous peroxidase was
blocked by peroxidase blocking so-
lution for 10 minutes, then applica-
tion of biotinylated secondary anti-
body for 30 minutes. The tissue sec-
tions were incubated with streptavi-
din-HRP detection system solution
(Vector laboratories). Each step was
followed by washing with phos-
phate buffered saline (PBS). Color
development was done using 33
diaminobenzedene (DAB) as a sub-
921
strate with Mayers hematoxylin as
a counter stain. A semiquantitative
evaluation of the intensity of stain-
ing was performed using scores
from 0-3, with 0 indicating the ab-
sence of positive staining; 1 indi-
cating the presence of mild staining
(+), 2 indicating the presence of
moderate staining(++) and 3 indicat-
ing the presence of marked staining
(+++) after Torbenson et al. ( 2002)
Determination of the level of IL-
10 gene expression: lymphocytes
were separated from the collected
blood samples {10 randomly select-
ed from each studies group} and
processed to measure level of IL-10
gene expression in lymphocytes.
MagNA Pure Compact Nucleic Ac-
id Isolation Kit I (Roche, Germany)
was used for isolation of total RNA
from lymphocytes. The messenger
RNA levels of IL-10 were deter-
mined using RT-PCR technique.
The primers used generated bands
of 296 base pair (bp.) for IL-10; also
- globulin gene with 661bp. was
amplified and used as a reference
control. Clonal DNA was synthe-
sized at 42
o
C for 30min., then
RTase was inactivated at 94
o
C for
10min. Lastly DNA was amplified
with final extension time of 5min. at
72
0
C. The primer used for IL-10 by
PCR was 3-GCAG GA CTT TAA
GGG TTA CT-5, 5-TTC ATG
GCC TTG TAGAC A CC-3. PCR
products were separated by electro-
phoresis in 20% agarose gel, visual-
ized by U.V. trans-illuminator and
photographed. The intensity of the
bands was quantified by gel pro-
analyzer and the results were ana-
lyzed by computer soft ware. The
level of IL-10 was determined as the
percentage of gene expression of the
target gene and control gene -
globulin in optical density. The
amount of RT-PCR product was
normalized as a ratio to the values
obtained for - globulin as internal
standard for each sample.
Statistical analysis: Data were
analyzed using mean, standard devi-
ation, analysis of variance
(ANOVA) t-test, P value & reduc-
tion %. Analyses were processed
according to the conventional pro-
cedures using Statistical Program of
Social Sciences (SPSS) software for
window, version 10.0.
Results
Regarding the effect of autoclaved
cercarial vaccine (ACV) in combi-
nation with Bacilli Clamett-Guerin
(BCG) on the number of adult worm
recovered by perfusion technique
(Tab. 1) showed a statistical signifi-
cant reduction in the mean number
of adults S. mansoni in vaccinated
Gs compared to control (GIa)
(P<0.001). The R% was 91.12%,
78.97%&82.77% in GIIa, GIIb &
GIIc respectively. The highest sig-
nificant reduction was in GIIa.
As regards the effect ACV on the
tissue egg count, Table (1) showed a
statistical significant decrease in the
mean number of S. mansoni ova/
gram liver (%R was 91.87%,
83.86% & 85.97%) and in the mean
number of S. mansoni ova/gram in-
922
testine (%R was 89.09%,83.52%
&86.35%) in vaccinated GIIa, GIIb
& GIIc respectively compared to
control GIa (P < 0.001). The highest
significant reduction was reported in
GIIa than GIIb & GIIc. There was
no statistical significant difference
in parasitological findings between
GIa and GIb (Tab. 2).
Regarding the effect of ACV on
the mean number and size of the
granulomas detected in liver sec-
tions. There was a statistical signifi-
cant reduction (Tab. 3) in number
(92.92%,87.36%&89.40%) and size
of granulomas/liver (43.53%,
28.95% & 30.83%) in GIIa, GIIb &
GIIc respectively as compared to the
control ones (P< 0.001). The highest
significant decrease was detected in
vaccinated GIIa. There was no sta-
tistical difference regarding number
and size of granulomas between GIa
and GIb (P>0.05) (Tab. 4).
The results of the liver sections
and control infected group revealed
the presence of large sized granulo-
mas of fibrocellular and fibrotic
types. The fibrocellular granulomas
showed collection of inflammatory
cells with predominant eosinophils,
macrophages and lymphocytes.
Also, few neutrophils with scarce
active fibroblasts and plasma cells
were present (Figs.1&2). In respect
to the vaccinated GIIa the number
of granulomas decreased signifi-
cantly (Fig.3). All granulomas were
of fibrocellular type (Fig4.). As re-
gards the vaccinated GIIb and GIIc,
no gross histopathological differ-
ence was detected between them.
The number of granulomas in both
groups was greatly reduced. Most
granulomas became of fibrocellular
nature while few granulomas were
of fibrotic type with diminished cel-
lularity (Figs. 5&6).
The immunohistochemical assay
of fibronectin used as a fibrotic
marker was moderate (++) to
marked (+++) brownish staining of
fibronectin-Ab in granulomas in
controls where macrophages were
the main cell type (Fig.7) localized
along reticulin positive fibers; spin-
dle cells, (presumptive fibroblasts).
While in vaccinated groupIIa, the
granuloma detected showed mild
(+) to moderate staining (++) of fi-
bronectin-Ab (Fig.8) indicating less
extensive fibrotic process in vac-
cinated compared to controls. No
definite difference was in the vac-
cinated Gs IIb&IIc. The distribution
of groups according to fibronectin
Ab expression was given (Tab. 5).
Vaccinated Gs showed mostly grade
1 & 2 staining, but controls showed
mostly grade 2 & 3. Difference be-
tween both groups was statistically
significant (P<0.05).
The immunogenicity of ACV was
assessed by measuring the level of
IL-10 gene expression in mice lym-
phocytic blood cells. Level was sig-
nificantly increased (Fig. 9, 10) in
vaccinated Gs to 1.8540.464,
0.5300.191 & 0.7170.122 for Gs
IIa, IIb & IIc respectively Vs 0.185
0.077&0.4340.064 for GIa & GIb
respectively (P<0.001). Statistically
significant difference was between
GIIa and GIIb & IIc (P < 0.001). No
923
statistical difference was between
GIIb & GIIc and non difference was
between GIa &GIb (P >0.05).
Table 1: Mean number of adults recovered and tissue eggs in infected control
and vaccinated groups.
Group
Mean adult worm Mean egg count/ gram tissue
Liver Intestine
Mean SD + % R Mean SD + % R Mean SD + % R
GIa 32.10 1.56 - 3012.5 459.25 - 3187.33 872.82 -
GIIa 2.85 0.66 91.12 244.73 3.61 91.87 347.79 23.56 89.09
GIIb 6.65 0.921 78.97 486.11 52.09 83.86 525.04 72.75 83.52
GIIc 5.53 0.931 82.77 422.65 114.81 85.97 434.79 70.75 86.35
P1 t. test (12.31) P. <0.001* t. test (16.78) P. <0.001* t. test (10.11) P. <0.001*
P2 t. test (10.61) P. <0.001* t. test (6.12) P. <0.001* t. test (6.73) P. <0.001*
P3 t. test (1.65) P. >0.05 t. test (1.55) P. v>0.05 t. test (3.18) P. >0.05
%R= comparing mean worm burden in vaccinated (V) and control (C) where %R=(C-V)/ Cx
100, P1 comparison between GIIa & GIIb, P2 comparison between GIIa & GIIc, P3 comparison
between GIIc & GIIb >0.05 non significant , <0.001 highly significant*
Table 2: Adults and tissue eggs in infected and BCG-infected controls.
Group Mean adult worm
Mean egg count/ gram tissue
Liver Intestine
Mean SD + t-test Mean SD + t-test Mean SD + t-test
GIa 32.10 1.56 - 3012.5 459.25 - 3187.33 872.82 -
GIb 30.8 1.38 1.7 2620 118.77 1.33 2990 356.89 1.9
P>0.05
P between GIa & GIb= >0.05 non significant
Table 3: Number and size of granulomas/liver section in infected and vac-
cinated groups.
Group Number of granulomas Size of granulomas
Mean SD + % R Mean SD + % R
GIa 64.73 3.48 - 342.89 58.4 -
GIIa 4.58 0.78 92.92 193.58 19.15 43.53
GIIb 8.18 1.14 87.36 243.6 43.57 28.95
GIIc 6.86 0.95 89.40 237.16 12.64 30.83
P1 t. test (10.50) P. <0.001* t. test (2.59) P. <0.05*
P2 t. test (9.26) P. <0.001* t. test (3.66) P. <0.05*
P3 t. test (3.15) P. >0.05 t. test (0.98) P. >0.05
%R= comparing mean worm burden in vaccinated (V) and control (C) where %R=(C-V)/Cx100
P1 between GIIa & GIIb =0.05 non significant, P2 between GIIa & GIIc=<0.001 highly signifi-
cant **
P3 between GIIc & GIIb =<0.05 significant*
Table 4: Number and size of granulomas/liver section in infected and BCG-
infected controls.
924
Group Number of granulomas Size of granulomas
Mean SD + t-test Mean SD + t-test
GIa 64.73 3.48 - 342.89 58.4 -
GIb 59.83 2.28 7.5 296.18 39.15 2.59
P >0.05
P between GIa & GIb =>0.05 non significant
Table 5: Distribution of fibronectin expression in 10 sections in all groups.
Group Grade (0) staining Grade (1) staining Grade (2) staining Grade (3) staining
N. % N. % N. % N. %
GIa 0 0 1 10 2 20 7 70
GIb) 0 0 2 20 3 30 5 50
GIIa 0 0 3 30 6 60 1 10
GIIb 0 0 4 40 4 40 2 20
GIIc 0 0 3 30 5 50 2 20
Distribution of significant difference between Gs Ia&Ib and Gs IIa,IIb &IIc, P<0.05.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
IL-10
GroupIa
GroupIb
GroupIIa
GroupIIb
GroupIIc
Fig. (9) Fig. (10) )
Fig. 9: IL-10 gene expression in all groups.
Fig. 10: Gel electrophoresis for detection of IL-10 mRNA by RT-PCR stained with ethidium bromide show-
ing Lane1: molecular weight size marker. Lanes 2-8: show IL-10 expression with different densities at 296
base pair (bp). - globulin used as an internal reference control at 661bp.
Discussion
The dream of a vaccine against
schistosomiasis remains a powerful
driving force in research. In this
respect, a trial was done in the pre-
sent study to assess the immunogen-
icity of a special type of S. mansoni
vaccine which is the autoclaved cer-
carial vaccine (ACV). The level of
protection obtained by a vaccine
according to Williams et al. (2002)
is the mean percentage reduction of
adult worm burden in an immunized
group of experimental animals in
comparison to an untreated group
925
using uniform challenge. In this
context, the results of the current
work showed that ACV confers a
high level of protection especially if
given as a single dose as in GIIa
that showed a significant reduction
in worm burden (91.12%) with
marked reduction in egg deposition
in the liver (91.87%) and in the
large intestine (89.09%). The histo-
pathological examination of the liv-
er sections revealed marked reduc-
tion in number (92.92%) and size
(43.53%) of hepatic granulomas.
This was probably due to the higher
level of activation of the immune
system as a direct consequence of
vaccination. These findings agreed
with Eissa et al. (1998), who report-
ed a high level of resistance by
ACV (96.9%) against S. mansoni
infected mice. However, the protec-
tive level obtained by ACV in the
present study (91.12 %) was higher
than that of Schimidt and Roberts
(1996), who recorded 63% & 70%
by using UV-irradiated cercariae
and gamma irradiated cercariae re-
spectively. They added gamma-
irradiated cercariae in immunization
against schistosomiasis was imprac-
tical because the gamma irradiation
equipments were only available in
central institutes and the transport of
these irradiated larvae to distant ru-
ral areas requires continued deep
freezing proved impractical. El-Ridi
et al. (1997) reported that highly
effective immunization of mice with
the UV-attenuated cercarial vaccine
led to a significant impairment in
challenge worm egg production.
Zhu et al. (2005) found that vac-
cination with UV-irradiated cercari-
ae of S. japonicum induced high
protective level against challenge
infection.
The reduction of worm load and
egg deposition in tissues using ACV
may be interpreted by lymphocytic
activation and/or antibody produc-
tion. This prevented migration of
schistosomula within vasculature
preventing them from reaching the
hepatic portal system, thus dimin-
ishing the number of worms, ova
and granulomas (Eissa et al., 1998).
The results revealed that, intrader-
mal inoculation of BCG alone 4
weeks before infection with S. man-
soni (GIb) did not cause a statisti-
cally significant difference in worm
load, tissue egg count, number and
size of hepatic granulomas as well
as IL-10 gene expression as com-
pared to infected controls. This
agreed with James et al. (1985) and
Eissa et al. (1998) who reported that
BCG acting as a potent adjuvant in
association with larval antigens un-
der conditions which was not pro-
tective when administered alone.
BCG was included as an adjuvant.
Using double dose (0.2ml) of
ACV (GIIb), or two single doses
(each of 0.1ml) two weeks apart
(GIIc), resulted also in a significant
reduction in worm burden (78.97%)
and (82.77%) respectively. This re-
duction is manifested in egg deposi-
tion in the liver (%R was 83.86% in
GIIb and 85.97 % in GIIc, and egg
deposition in the caecum (%R was
83.52% in GIIb & 86.35% in GIIc).
926
The histopathological examination
of liver showed a marked reduction
in size (28.95% in GIIb & 30.83%
in GIIc) and number of granulomas
(87.36% in GIIb & 89.40% in GIIc).
In the current work, single vac-
cination (GIIa) resulted in a signifi-
cant increase in protective level than
in GIIb & GIIc. So, multiple vac-
cinations with ACV were not re-
quired to induce a greater protec-
tion. This agreed with Eissa et al.
(1998) who found that, a single dose
of ACV (10
5
/ml) gave more protec-
tion than with subsequent vaccina-
tions. Also, Donald et al. (2004)
found that vaccination with radia-
tion-attenuated cercariae against S.
japonicum in mice, resulted in re-
duction of worm burden (40%-
50%), but with subsequent vaccina-
tion, no additional protection oc-
curred. On the contrary, Hs et al.
(1981) showed that one, three &
five vaccinations with X ray-
attenuated cercariae induced pro-
gressively higher levels of immuni-
ty. Mangold and Dean (1986) and
Delgado and Mclaren (1990) found
that the first exposure of mice to
irradiated attenuated cercariae gave
a large increment in protection but
with subsequent vaccination a mar-
ginal increase was produced. The
discrepancy may be related to the
method of vaccine preparation or
the immunizing dose, which may
produce immune tolerance with
subsequent vaccination in some cas-
es or potentiate the immune re-
sponse in other cases. Yole et al.
(1996) who investigated the effect
of different doses of radiation-
attenuated cercariae in monkeys
found that increasing number of
vaccinations beyond a certain point
instead of boosting protection di-
minished it, and attributed that to
development of a tolerant state in-
duced by frequent vaccinations.
In the current work, the effect of
ACV on histopathological and im-
munohistochemical changes in liver
sections showed that most of the
granulomas in the vaccinated group
were of fibrocellular nature as evi-
denced by mild to moderate staining
of fibronectin-Ab{grade 1&2}. This
may refer as ACV induced high lev-
el of activation of immune system
shown by increased IL-10 mRNA
expression. The increased produc-
tion of IL-10 limits the action of the
pro-inflammatory cytokines respon-
sible for granuloma formation thus
resulted in marked reduction in
granuloma size and cellularity. This
led to subsequent amelioration of
liver pathology and fibrosis (Hu et
al., 2004). Similar histopathological
changes were reported by Eissa et
al. (1998). Zhou et al., (2000) on
investigating the role of fibronectin
(FN) on hepatic stellate cells in liver
fibrosis induced by CCl4 reported
that the expression of fibronectin
(FN) receptor positive cells detected
by immunohistochemistry reached
its peak at the 10
th
week which
coincided with the higher expres-
sion of fibronectin reported in the
present control group study.
Interleukin 10, the chief among
soluble mediators with known im-
927
munoregulatory function, has wide-
ranging regulatory effects upon an-
tigen presentation, co-stimulation
and development of acquired T cell
responses (Hogg et al., 2003). This
immunoregulatory effect was highly
beneficial to the host concerning
regulation of the inflammatory re-
sponse against egg deposition led to
reduction of both granuloma size
(Moore et al., 2001) and influx of
inflammatory cells and has a protec-
tive role against fibrogenesis (Hu et
al., 2004).
In the current work, ACV had a
high protective level against schis-
tosomiasis shown by significant in-
crease in IL-10mRNA expression in
vaccinated group. No doubt, the use
of ACV stimulated activation of
local Th2 cells in liver tissues and
increased the production of IL-10
that led to subsequent amelioration
of liver pathology and fibrosis. Je-
sus et al. (2004) reported that IL-10
was important to reduce the pathol-
ogy of acute schistosomiasis and
that the level of IL-10 decreased in
hepatosplenomegaly patients. Zhang
et al. (2007) reported that not only
the antifibrotic effects of IL-10 on
experimental hepatic schistosomia-
sis but also the fibrotic changes
were partially reversed by simulta-
neous administration of IL-10. They
explained that as IL-10 could inhibit
the activation of hepatic stellate
cells (HSCs) and made an antifibro-
genic process come into effect.
In the present study, increased IL-
10 mRNA expression agreed with
many authors. Cook et al. (1993)
found that the spleen cells from
mice infected with S. mansoni, re-
leased no detectable cytokines and
expressed only IL-10 mRNA prom-
inently and Wynn et al. (1993)
found increased expression of IL-10
in lung tissue containing schistoso-
mal granulomas. Betts and Wilson
(1998) reported that IL-10 mRNA
was highly identified in total RNA
extracted from granuloma of S.
mansoni infected mice. El-Malky et
al. (2001) found that IL-10 mRNA
was prominently represented in liver
cells of mice immunized with gam-
ma-irradiated cercariae. Further-
more, Donald et al. (2004) reported
that peripheral blood mononuclear
cells (from resistant individuals to S.
japonicum in China) were found to
produce significantly greater
amount of IL-10 in response to par-
asite extracts and recombinant anti-
gens in vitro. Pacififica et al. (2006)
observed a high level of IL-10 in
splenocytes of vaccinated mice with
Sm22.6 antigen against schistoso-
miasis. But, Jenkins et al. (2005)
found that using radiation-
attenuated cercariae (induced high
level of protective immunity in
mice) failed to induce IL-10. This
discrepancy may be related to the
type of the used vaccine and the
probable different immune response
it elicited.
A logic question: is it possible to
develop vaccine against a complex
metazone parasite such as Schisto-
soma? There are a lot of evidences
that offer grounds for optimism in
this concern; one of them is that
928
human populations in endemic areas
invariably develop some degree of
protection naturally (Hagan et al.,
1991). Another fact is that a number
of successful anti- parasite veteri-
nary vaccines have been developed,
some of which were used as re-
combinant vaccines against Echino-
coccus granulosus (Dalton and
Mulcany, 2001). The most hopeful
finding is that a vaccine against S.
haematobium named Bilhvax, using
one vaccine candidate S. haemato-
bium glutathione-s-transferase
(GST) successfully passed the stage
of industrial scale-up and safety
testing and underwent phase II hu-
man clinical trials (Donald et al.,
2004).
Conclusion
ACV proved to be a promising
method in controlling S. mansoni as
it confered a high level of resistance
reaching 91.12% which is high
among other types of vaccines test-
ed by others. It causes marked re-
duction in the size and number of
granulomas with subsequent ame-
lioration of liver pathology and fi-
brosis. ACV does not require ex-
pensive equipment, just an auto-
clave, which is present in any insti-
tute. Furthermore, safety is thought
to be present in using this vaccine as
it is a type of heat- killed vaccine
and not live-attenuated one that may
have the risk of back mutation to a
wild form. Verification of the safety
of ACV was actually investigated
by Eissa et al. (2003) who found
that liver and renal function tests
done to mice vaccinated with ACV
were not affected. Further research-
es should be done focusing on pre-
cise purification of antigens in this
vaccine and evaluation of the actual
immune response elicited in humans
as experimental animal researches
despite providing crucial insights
into host/parasite relations, no ani-
mal model reflects the human situa-
tion well enough to permit transla-
tion of animal data directly to hu-
man host (Bergquist et al., 2002).
So, taking researches to the human
clinical trials is not only a hope but
also a must.
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Legend of figures
Fig. 1: Liver section of a control mouse (Ia) showing multiple typical schistosomal granulomas.
Some of fibrocellular type, others are fibrotic (arrows). Some showed S. mansoni ova (H&E, X
160).
Fig. 2: Liver section of a control mouse showing multiple schistosomal granulomas. Majority of
fibrotic type with greenishly-stained fibrous tissue. Others of fibrocellular type (Masson Tri-
chrome X 160).
Fig. 3: Liver section of a vaccinated mouse (IIa) showing a schistosomal granuloma, with a sig-
nificantly smaller size (arrow) than control group. Note some dilated central vein (H&E, X 160).
Fig. 4: Liver section of a vaccinated mouse (IIa) showing a fibrocellular granuloma. Note some
dilated veins (Masson Tirchrome, X 400).
Fig. 5: Liver section of a vaccinated mouse (IIc) showing 2 schistosomal granulomas of fibocel-
lular type around S. mansoni ova (arrow). Note dilated congested central veins (H&E, X 160).
Fig. 6: Liver section of a vaccinated mouse (IIc) showing fibrocellular schistosomal granuloma
(arrow). Note dilated congested central veins (Masson Tirchrome, X 160).
Fig. 7: A liver section of a control mouse showing dense fibrosis . Note marked staining of gran-
uloma for fibronectin-Ab (arrow) (grade 3) (Streptavidin-Biotin DAB, X 200)
Fig. 8: A liver section of a vaccinated mouse (IIa) showing little fibrotic change in granuloma.
Note moderate staining for fibronectin-Ab (grade 2) (Streptavidin-Biotin DAB, X 200).
918

Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 943 - 95
SEROLOGICAL DETECTION OF TOXOPLASMA GONDII IN
CHRONIC RENAL FAILURE PATIENTS AND RENAL TRANSPLANT
RECIPIENTS
By
SAYEDA M. AUFY
1
, ABEER M. A. MAHGOUB
1
*, MOHAMED GAMAL
EL-DIN SAADI
2
and MARWA ADEL ELMALLAWANY
1
Departments of Parasitology
1
, and Internal Medicine
2
, Faculty of Medi-
cine, Cairo University, Egypt
Abstract
Toxoplasma gondii antibodies were detected in 78 patients with renal disease
by ELISA. Patients were classified according to the renal status; chronic renal
failure patients not on haemodialysis (G1=19), chronic renal failure patients on
regular haemodialysis (G2=30), renal transplant recipient (G3=29) and 13
normal controls. Anti-Toxoplasma IgG & IgM antibodies were 36.8% & 10.5%
in renal failure patients not on haemodialysis, 56.7% &16.7% in patients on
regular haemodialysis and 69% &24.1% in renal transplant recipients versus
23.1% & 0% in controls with statistical significant difference for Toxoplasma
IgG antibodies only. Anti-Toxoplasma IgG antibodies levels of G3 were lower
than that of G1. It was observed that the more the exposure to dialysis, the
more the risk of toxoplasmosis. It was found that 85.71% of renal transplant
recipient seropositive cases for anti-Toxoplasma IgM antibodies were detected
in one year post-transplantation and 14.28% of cases after the first year of
transplantation.
Key words: Toxoplasma gondii, renal, haemodialysis, transplant, ELISA, IgG
& IgM antibodies.
Correspondence: Abeer Mohamed Aly Mahgoub (goubya@hotmail.com)
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Toxoplasma gondii is a ubiquitous
protozoan parasite that infect one-
third of world population. Within
immunocompetent humans, toxo-
plasmosis is benign and self limiting
(Barbosa et al., 2009), whereas,
toxoplasmosis is a major opportun-
istic infection that may lead to mor-
bidity and mortality in immuno-
compromised individuals (Meeka et
al., 2001). Chronic renal failure pa-
tients suffer from impairment of cell
mediated immunity either due to
uremia or due to interventions used
in their therapy, including dialysis
and transplantation with subsequent
immunosuppressive therapy and
multiple blood transfusions (Weiss

and Dubey, 2009). Toxoplasmosis


in these patients usually occurs as a
consequence of the recrudescence
of a latent infection acquired before
the onset of immune suppression.
However, it may occur also due to
recently acquired acute infection
with the parasite (Montoya et al.,
2001). Clinical signs are non-
specific and insufficient for a defi-
nite diagnosis because toxoplasmo-
sis mimics several other infectious
diseases (Remington et al., 2001).
Diagnosis of toxoplasmosis is by
serological, histological, and mo-
lecular methods, or by some combi-
nation of them (Montoya et al.,
2002). The direct recovery of T.
gondii from biological samples is
often impractical. Consequently,
serological diagnosis represents the
most widely used approach for de-
fining the stage of infection (Sen-
sini, 2006). Serological tests were
routinely applied to detect IgM &
IgG specific antibodies, including
IFT and ELISA, with the latter
showing higher sensitivity and spec-
ificity (Remington et al., 2004).
The work aimed to study the
prevalence of positivity for anti-
Toxoplasma antibodies (IgG &
IgM) in patients with chronic renal
failure and renal allograft recipient
by using ELISA.
Subjects, Materials and Methods
The study compromised 91 pa-
tients, 51 male and 40 female with
age ranges between 8.5 & 70 year,
selected from Internal Medicine
Department, Outpatients Clinics,
and King Fahd Haemodialysis Unit,
Kasr El Aini Teaching Hospital in
2008. They were classified into four
groups according to their renal sta-
tus. G1: 19 chronic renal failure
patients who were not subjected to
haemodialysis. G2: 30 chronic renal
failure patients who were on regular
haemodialysis. G3: 29 renal trans-
plant recipients. G4: 13 control
cases with no history of renal trou-
bles and serum urea and creatinine
within normal range.
All cases were subjected to com-
plete history taking using a structur-
al questionnaire, clinical examina-
tion has been carried out for every
case for signs suggestive of Toxo-
plasma infection, serum blood sam-
ples were collected from all cases
for renal function test (serum urea
and creatinine) and for serological
analysis for anti-Toxoplasma gondii
IgG and IgM antibodies.
Detection of anti-Toxoplasma
IgM antibodies in sera was done
using IgM capture immunoen-
zymatic assay (Herbrink et al.,
1987). Bioelisa TOXO IgM (Im-
munocapture) kit (Biokit, S.A. Bar-
celona, Spain) used for semi-
quantitative detection of IgM anti-
bodies against T. gondii in human
sera.
Detection of anti-Toxoplasma
IgG antibodies was by using ELISA
(Derouin et al., 1987). NovaLisa
Toxoplasma IgG-ELISA (NovaTec
Immuno-diagnostica GmbH (D-
63128 Dietzenbach, product number
TOXG0460, Germany) was used for

qualitative and quantitative meas-


urement of T. gondii antibodies in
human serum.
Statistical evaluation: All data
were processed using SPSS (1 0.0)
for Windows 98. Quantitative vari-
ables were expressed using mean
and standard deviation; they were
compared using t-student's test
(paired and unpaired). Pearson cor-
relation is used to correlate two
quantitative variables. Qualitative
variables were compared using Chi-
square test for comparisons between
proportions when appropriate. Val-
ues of P < 0.05 were considered
significant and P < 0.01highly sig-
nificant.
Results
Table 1: Specific anti-Toxoplasma IgM & IgG antibodies by ELISA.
Group Anti-Toxoplasma IgM* Anti-Toxoplasma IgG**
G1 (n=19)
-ve +ve -ve +ve
17 (89.5%) 2 (10.5%) 12 (63.2%) 7 (36.80%)
G2 (n=30)
25 (83.3%) 5 (16.7%) 13 (43.3%) 17 (56.7%)
G3 (n=29)
22 (75.9%) 7 (24.1%) 9 (31%) 20 (69%)
G4 (n=13)
13(100%) 0 (0.00%) 10 (76.9%) 3 (23.1%)
Total (n=91)
77 (84.6%) 14 (15.4%) 44 (48.4%) 47 (51.6%)
*Significant difference between all groups (P =0.3).
**Significant difference between all groups (P= 0.021).
Table 2: Specific anti-Toxoplasma IgM& IgG antibodies in all groups.
G3
(n=29)
G2
(n=30)
G1
(n=19)
Control
(n=13)
Anti-Toxoplasma
7.6924
5.42612
7.4877
5.80811
4.9703
6.58520
4.7291
2.2767
Mean SD
IgM*
175.8772
174.94709
189.5237
223.36718
95.1405
115.68056
56.3662
86.33521
IgG**
*Significant difference between control and G1versus G2 & 3 (P =0.013).
**Significant difference between G2 versus control (P = 0.03). **Significant difference between G3 versus control
(P=0.033). **No significant statistical difference in G1 versus control (P= 0.084).
**Anti-Toxoplasma IgG antibodies levels of G3 lower than G1.
Comparing all groups regarding
kidney function tests (serum urea
and creatinine), there was a highly
significant difference between all
groups (P= 0.00 & 0.00 respective-
ly). According to Pearson correla-
tion, there was a significant correla-
tion between creatinine level and
anti-Toxoplasma IgG antibodies
level in G1. A high creatinine level
corresponded to a high anti-
Toxoplasma IgG antibodies level
and vice versa (r=0.507, p=0.05).
No correlation between creatinine
level and anti-Toxoplasma IgG an-
tibodies level in all other groups.

Table 3: Haemodialysis and anti-Toxoplasma IgG & IgM antibodies in renal


patients.
Anti-
Toxoplasma
+ve history of
haemodialysis
-ve history of
haemodialysis Total
N % N % N %
IgG*
+ve
33 75.00 11 25.00 44 100.0
-ve
15 44.12 19 55.88 34 100.0
IgM**
+ve
11 78.57 3 21.43 14 100.0
-ve
37 57.81 27 42.19 64 100.0
*Significance difference (P= 0.02), **No significance difference (P= 0.131).
Table 4: Anti-Toxoplasma IgM and IgG antibodies and duration of dialysis in
G2 and duration of transplantation in G3.
Anti-
Toxoplasma
MeanSD haemodialysis
duration in G2 in months
MeanSD transplantation
duration in G3 in months
IgM -ve 47.446.94 57.00950.98554
+ve 76.815.85 8.0 6.1101
IgG -ve 40.237.51 33.466750.45
+ve 61.599.67 50.4554.84761
More the exposure to dialysis led
to toxoplasmosis more risks. A sig-
nificant correlation was found be-
tween duration of haemodialysis
and anti-Toxoplasma IgG antibodies
in G2. The increase in duration of
haemodialysis corresponded to a
high level of anti-Toxoplasma IgG
antibodies and vice versa (r=0.384,
p=0.05). No significant correlation
was between duration of dialysis
and anti-Toxoplasma IgG antibodies
level in renal transplant recipient
patients. 85.71% of renal transplant
recipient positive cases for anti-
Toxoplasma IgM antibodies were
detected within one year post-
transplantation and 14.28% of cases
after the first year of transplanta-
tion, with a highly significance dif-
ference (P =0.004). But, there was
no significance difference between
positivity for anti-Toxoplasma IgG
antibodies and duration of trans-
plantation (P= 0.671).
Discussion
In the present study, anti-
Toxoplasma IgG antibodies were
detected by ELISA which proved to
be highly specific and sensitive as
compared with other tests (Johnson
et al., 2005; Alvarado-Esquivel et
al., 2006).
In the present study, IgM capture
immunoenzymatic assay was used
to overcome false-positive reactions
caused by rheumatoid factor and
false-negative reactions caused by
blockage by anti-Toxoplasma IgG
antibodies and eliminate potential
interference by IgG and other iso-
types by binding only IgM antibod-
ies and unbound antibodies are re-

moved by washing (Meeka et al.,


2001; Gras et al., 2004).
In the present study, positivity for
anti-Toxoplasma IgG antibodies
was 23.1% in control G, 36.8% in
G1, 56.7% in G2 & 69% in G3 with
significant difference between them.
None of control G was positive for
anti-Toxoplasma IgM antibodies
(0%) while 10.5% of G1, 16.7% of
G2 and 24.1% of G3 were positive
for anti-Toxoplasma IgM antibodies
without significant difference be-
tween groups. The detected differ-
ence between seroprevalence be-
tween groups could be attributed to
the difference in patients renal sta-
tus with its subsequences as dialy-
sis, blood transfusion and intake of
immunosuppressive drugs. These
risk factors were the only significant
difference existed between Gs. The
results agreed with Yazar et al.
(2003), Ocak et al. (2005), and Ros-
tami et al. (2006). They reported
wide variations in toxoplasmosis
prevalence due to variation in geo-
graphical areas. Prevalence of toxo-
plasmosis varied greatly among dif-
ferent geographical areas, cultural
behavior and hygienic standards
that led to marked differences even
within the same country (Barbosa et
al., 2009).
In the present study, on comparing
anti-Toxoplasma IgM antibodies
level among groups; there was sig-
nificant difference between control
and renal failure not on haemodialy-
sis versus renal failure patients on
regular haemodialysis and renal
transplant recipient ones. Also, the
present results showed that anti-
Toxoplasma IgG antibodies level of
renal failure not on haemodialysis
was higher than control G but with-
out significant difference. The anti-
Toxoplasma IgG antibodies level of
renal failure patients on regular
haemodialysis was higher than con-
trol G with a significant difference.
Anti-Toxoplasma IgG antibodies
level of renal transplant recipient
patients was higher than control G
with a significant difference.
Whereas anti-Toxoplasma IgG anti-
bodies level of renal transplant re-
cipient patients were less than renal
failure patients on regular haemodi-
alysis.
These results may be attributed to
renal failure patients not on haemo-
dialysis group and control group
were not exposed to the risk of dial-
ysis and renal transplantation. How-
ever, the former had more immunity
impairment than the latter. Renal
failure patients on regular haemodi-
alysis were at more risk than control
group being exposed to dialysis and
blood transfusion. Whereas renal
transplant recipient group is the
most risky group being exposed to
dialysis, blood transfusion, trans-
plantation and life-long immuno-
suppressive drugs. Anti-Toxoplasma
IgG antibodies level of renal trans-
plant recipient group was less than
that of renal failure patients on
regular haemodialysis group might
be due to effect of immunosuppres-
sive drugs. Thus, reactivation of
toxoplasmosis had occurred in some
of the present cases as serological

profile compatible with reactivation


including increased IgG antibody
titers, or less frequently, increased
titers of acute phase antibodies
(Yazar et al., 2003; Montoya and
Liesenfeld, 2004).
The present study showed a sig-
nificant correlation between anti-
Toxoplasma IgG antibodies level
and creatinine level in renal failure
not on haemodialysis group. A high
creatinine level corresponded to a
high anti-Toxoplasma IgG antibod-
ies level and vice versa. But, there
was no significant correlation be-
tween creatinine level and anti-
Toxoplasma IgG antibodies in other
groups. This might be explained as
creatinine level reflected the renal
function that affected the immune
status controlling toxoplasmosis.
But, creatinine level was within
normal range among positive and
negative control G. In haemodialy-
sis group, creatinine level was vari-
able according to haemodialysis
sessions. In renal transplant creati-
nine level was controlled to the
normal range.
Regarding dialysis and its relation
with toxoplasmosis, 78.57% of
positive IgM renal cases versus
57.81% of negative IgM renal cases
exposed to dialysis with high with-
out statistical significant difference.
Moreover, 75% of seropositive IgG
renal cases versus 44.12% of nega-
tive IgG renal cases exposed to di-
alysis with a statistical significant
difference. Thus, haemodialysis pa-
tients were at more risk of toxo-
plasmosis (Abbas et al., 1996;
Yazar et al., 2003; Ocak et al.,
2005) as in the present study.
On studying the effect of duration
of dialysis in renal failure patients
on regular haemodialysis group and
its relation with toxoplasmosis, it
was found that the more the expo-
sure to dialysis, the more the risk of
toxoplasmosis. The results agreed
with Yazar et al. (2003) and Ocak et
al. (2005). There was a significant
correlation between duration of di-
alysis and anti-Toxoplasma IgG an-
tibodies level in renal failure on
regular haemodialysis patients. In-
crease in duration of dialysis corre-
sponded to a higher anti-
Toxoplasma IgG antibodies level
and vice versa. This agreed with
Abbas et al. (1996). Whereas there
was no significant correlation be-
tween duration of dialysis and anti-
Toxoplasma IgG antibodies level in
renal transplant recipient group.
In the present study, the mean du-
ration of transplantation for positive
IgG was higher than negative IgG.
On comparing positivity for IgG in
relation to duration of transplanta-
tion, there was no significant differ-
ence. But duration of transplantation
in relation to positivity of IgM was
statistically significance. The mean
duration of transplantation for posi-
tive IgM was lower than mean level
of negative IgM. This may be due to
high dose of immunosuppressive
drugs during early post-
transplantation period. Thus, renal
transplant recipient becomes more
susceptible for toxoplasmosis in
early post-transplantation period.

Then, the doses of immunosuppres-


sive drugs decrease with increase of
post-transplantation period. This
also could explain that 85.71% of
positive IgM renal transplant recipi-
ent in the present study were detect-
ed in the first post-transplan-tation
year and 14.28% were later. T.
gondii infection was reported be-
tween one day and 13 month after
transplantation and reactivations
were described up to 7 years after
transplantation (Renoult et al.,
1997; Giordano et al., 2002; Wulf et
al., 2005; Macpherson, 2005).
Conclusion
Toxoplasmosis is a worldwide
zoonotic parasite. Besides, the hae-
modialysis and/or renal transplanta-
tion is a true health problem. No
doubt, the awareness of transplant
recipients about the potential risks
of acquisition of infectious diseases
due to regular administration of
suppressive drugs may lead to low
prevalence rate of infection.
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 951 - 962
PREVALENCE OF ASYMPTOMATIC BACTERIURIA IN EGYPTIAN
CHILDREN AND ADOLESCENTS WITH TYPE 1
DIABETES MELLITUS
By
MONA A. SALEM
1
, RANDA M. MATTER
1
, ABEER A. AB-
DELMAKSOUD
1
AND SHERIN A.EL MASRY
2
Departments of Pediatrics
1
and Clinical Pathology
2
, Faculty of Medicine,
Ain Shams University, Cairo 11566, Egypt
Abstract
The prevalence of asymptomatic bacteriuria (ASB) and associated risk factors
were investigated in 100 Egyptian children and adolescents with type1 diabetes
mellitus and 100 age and sex matched healthy controls. All were subjected to
clinical evaluation and assessment of mean random blood glucose, mean glyco-
sylated hemoglobin (HbA1c); microalbuminuria and midstream urinary sam-
ples were collected for complete urine analysis and two consecutive urine cul-
tures and sensitivity tests. The prevalence of ASB was higher among diabetics
than controls (30% versus 14%, p<0.01) and was more among older age
(p=0.033) and female patients (p<0.001); especially postpubertal. Microalbu-
minuria (36.7%) and microvascular complications (50%) were significant risk
factors for ASB in patients while metabolic control and disease duration were
not relevant to ASB (p>0.05). Pyuria was a strong predictor of bacteriuria in
patients (80%) and controls (100%). The most common isolates were E. coli in
patients (30%) and Pseudomonas in controls (57.1%). Gram positive isolates
were detected in 46.7% of diabetic patients but not in controls. ASB is more
prevalent among type 1 diabetic patients in the pediatric age group. Screening
for ASB is warranted in diabetic patients with risk factors especially if pyuria is
detected in their urine analysis.
Key words: Type 1 diabetic children, adolescents, complications, bacteriuria,
E. coli.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Asymptomatic bacteriuria (ASB)
was defined as the presence of 10
5
colony-forming units (CFUs)/mL of
one and the same bacterial species
on two consecutive days without
symptoms of urinary tract infections
(UTIs) (Nicolle et al., 2005).
Knowledge of risk factors for UTIs
in diabetic patients is important to

identify patients in need for therapy


to prevent serious complications.
ASB increases risk for UTI among
patients with diabetes (Geerlings et
al., 2000a). Evidence from various
epidemiological studies suggests
that ASB and symptomatic UTI are
more common in patients with dia-
betes mellitus (DM) than among
those without DM (Ribera et al,
2006). Diabetic patients have a two
to three fold higher prevalence of
ASB and are at risk for developing
more serious consequences (Geer-
lings et al., 2001). The presence of
ASB was the major risk factor for
developing symptomatic urinary
tract infection (Ribera et al, 2006).
The prevalence of ASB in diabetes
mellitus has not been clearly estab-
lished (Matteucci et al., 2007). Few
studies addressed the prevalence of
ASB in diabetics in the pediatric age
group (Rzsai et al., 2003, 2006).
This study was designed to assess
the prevalence of the asymptomatic
bacteriuria among Egyptian children
and adolescents with type 1 diabetes
mellitus and to recognize the possi-
ble associated risk factors.
Patients, Subjects and Methods
A case control study was conduct-
ed on 100 children and adolescents
with type 1 diabetes mellitus. Pa-
tients were randomly selected from
those regularly attending the Diabe-
tes Clinic, Children's Hospital, from
May 2008 to June 2009. They were
33 males (33%) and 67 females
(67%) and their ages ranged
from 2 to 18 years old with mean of
11.344.32 years. Patients with his-
tory of urologic disease (such as
stricture urethra, posterior urethral
valve and meatal stenosis), patients
with definite urinary tract infection
at time of the study and uncoopera-
tive patients were excluded from the
study. Informed consent was ob-
tained from all subjects after ap-
proval by the Local Ethical Com-
mittee.
Patients were subdivided into two
groups to study the effect of glyce-
mic control on the development of
ASB. Group I: included 50 patients
with type 1 diabetes with good gly-
cemic control (HbA1c < 7.5%); this
group aged 2 to 18 years old with
mean of 10.224.79 years. They
were 21 males and 29 females with
mean disease duration 4.633.62
years. Group II: included 50 pa-
tients with type 1 diabetes with poor
glycemic control (HbA1c > 7.5%);
this group aged 2 to 18 years old
with mean of 11.344.23 years.
They were 12 males and 38 females
with mean disease duration
4.353.14 years. One hundred age
and sex matched healthy children
and adolescents were included as a
control group. They were 63 fe-
males (63%) and 37 males (37%),
their ages ranged from 2 to 18 years
old with mean of 10.224.76 years.
Data recorded from patients in-
cluded; demographic data, diabetes
duration, glycemic control, therapy
and any history suggestive of com-
plications or UTIs. Clinical assess-
ment included; general anthropo-

metric measures of weight, height


and body mass index (BMI) accord-
ing to Egyptian percentiles as well
as systemic examination for any
evidence of complications.
Laboratory investigations includ-
ed mean random blood glucose
(MRBG) over the last year follow
up was recorded. Mean glycosylated
hemoglobin (HbA1c) was calculat-
ed by using HPLC (high purified
liquid chromatography) after Gold-
stein et al. (1986). HbA1c target
range of <7.5% was recommended
for all age groups (ISPAD, 2009).
Urinary microalbumin was per-
formed using immune turbidimetric
methods prior to the study. Persis-
tent microalbuminuria was defined
when two of three samples showed
an excretion rate of 30-300 g/mg
creatinine (Viberti et al., 1982).
Mid-stream clean catched urine
samples were collected for urine
analysis for RBC, pus cells, crystals
or casts and two urine cultures and
sensitivity samples were collected on
two consecutive days. Urine speci-
mens were placed on blood agar and
MacConkey by a standard loop and
incubated for 48 hours to recognize
the type, colony count of the causa-
tive organisms and their sensitivity to
antibiotics. If the plate yielded 3 or
more different microorganisms, the
urine was regarded as contaminated
(Geerlings et al., 2001). Leukocyturia
(pyuria) was diagnosed by two meth-
ods. The semiquantitative method by
microscope defined leukocyturia as
>5 cells/high power field (HPF) at
400 magnification. The second was
the dip-stick method (Boehringer,
Combur
10
-Test M, Roche Diagnostics
GmbH, Mannheim, Germany). Leu-
kocyturia was diagnosed if either the
microscopic finding or the dip-stick
test was positive (Rzsai et al., 2006).
The presence in a culture of at least
100,000 bacteria per milliliter of
urine usually provides conclusive
evidence of infection in patients with
symptoms, a count of 100,000 bacte-
ria per milliliter in patients without
symptoms indicates asymptomatic
bacteriuria, a count of < 10,000 bacte-
ria per milliliter of urine usually pro-
vides no evidence of infection; a
count between 10,000 and 100,000
bacteria per milliliter of urine usually
provides query result that must be
repeated to confirm the evidence of
infection (Nicolle, 2001). Microscop-
ic examination of urinary sediment
was done to detect any bilharzial ova
or Trichomonas spp.
Statistical analysis: data were sta-
tistically analyzed using SPSS pro-
gram (Statistical Package for Social
Sciences) software version 17. De-
scriptive statistics were done for
numerical data by mean, standard
deviation and minimum& maximum
of the range, while they were done
for categorical data by number and
percentage. The analyses were done
for quantitative variables using t-test
in two groups with parametric data
and Mann Whitney U in cases of
two groups with non parametric.
Analytical analyses were done for
qualitative data using Chi square
test for parametric variables and
Fishers Exact test for non paramet-

ric ones. P value < 0.05 was consid-


ered significant.
Results
Diabetic patients and control group
were comparable as regard their age
and gender. However; patients had
significant microalbuminuria and py-
uria (15% & 38% respectively) when
compared to control group (p<0.001).
ASB was significantly detected in
patients (30%) more than the controls
(14%) [OR=2.63, CI=1.29-5.34
(p<0.01)]. No Schistosoma haemato-
bium ova or Trichomonas vaginalis
trophozoites were detected in urine
analysis of patients or controls. There
was no significant difference between
the two subgroups of patients as re-
gards age, sex, presence of microal-
buminuria or ASB. Furthermore;
metabolic control level did not influ-
ence the spectrum or type of organ-
isms detected in positive cultures
(tab. 1).
Diabetic patients with ASB were
older with mean age of 12.873.33
years (p=0.033), female patients
were frequently affected than males
(OR =0.148, CI= 0.04-0.54), 36.7%
of them had significant microalbu-
minuria (OR= 9.55, CI=2.73-33.44)
and 50% of them had significant
history of microvascular complica-
tions (OR=2.89, CI=1.18-7). Meta-
bolic control (either mean random
blood glucose or HbA1c) and dis-
ease duration were not related to the
development of ASB (p>0.05) (tab.
2). Twenty seven (90%) of diabetic
patients with ASB were females and
3(10%) were males (p<0.001),
while in control group, 71.4% of
ASB was detected in females com-
pared to 28.6% in males; but this
difference did not reach statistical
significance (p>0.05).There was no
significant difference between pre-
pubertal and postpubertal female
patients regarding development of
microalbuminuria, pyuria or diabet-
ic complications (p>0.05). ASB was
more prevalent in postpubertal fe-
male patients (tab. 3) compared to
prepubertal female patients but did
not reach statistical significance
[OR=2.86, CI=0.95-8.6 (p=0.056)].
Growth of different microorganisms
did not vary significantly between
prepubertal and postpubertal female
patients (p>0.05).
Gram positive organisms were sig-
nificantly more prevalent in patients
(46.7%) than in controls (0%). E.
coli was the most common organ-
ism; detected in 30% of positive
cultures in patients and Pseudomo-
nas sp. was detected in 26.7% with
variable growth of Enterococci
(16.7%), S. aureus (6.7%), S. co-
agulase negative (23.3%), Acineto-
bacter (3.3%) and Candida (3.3%).
Controls showed growth of gram
negative organisms with Pseudo-
monas in 57.1% of positive cultures
and E. coli in 42.9% (fig. 1). 80% of
patients with ASB showed pyuria in
urine analysis and positive urine
cultures were in 63.2% of all pyuria
cases (tab. 2). Pyuria was a predic-
tor for positive urine cultures in pa-
tients with 80% sensitivity (CI=
0.66-0.89), 80% specificity (CI=

0.74-0.84), 63% positive predictive


value (CI = 0.52-0.70) and 90%
negative predictive value (CI=0.83-
0.95). In controls sensitivity and
specificity was 100% (fig. 2).
Antibiotic sensitivity in patients'
positive cultures showed sensitivity
in 81.8% to morepenam, 70.6% to
4
th
generation cephalosporins and
68.4% to aminoglycosides followed
by sensitivity to 3
rd
generation ce
phalosporins, amoxicillin-clavula-
nate (60% and 35% respectively).
There was no significant differ-
ence between diabetic patients and
controls regarding sensitivity to var-
ious antibiotics (p>0.05). Nitrofu-
rantoin showed sensitivity in up to
94.1% of patients (p<0.05) (fig. 3).
Antibiotics sensitivity did not vary
significantly with changes in HbA1c
or between prepubertal and postpu-
bertal females (p<0.05).
Table 1: Clinical and laboratory characteristics of diabetic patients and con-
trols, and the two subgroups of diabetic patients.
Variant
Diabetic
n=100
Control
n=100
Test P value
Group I
n=50
Group II
n=50
Test P value
Age (years)
11.34
4.32
10.22
4.76
-1.68
#
0.09
10.22
4.79
11.34
4.23
-0.7
#
0.49
Male/female 33/67 37/63 0.35** 0.55 21/29 12/38 3.66 ** 0.056
Microalbuminuria
(ug/mg creat) n(%)
15(15%) 0(0%) 16.21** <0.001 5 (10%) 10(20%) 1.96** 0.16
Pyuria n (%) 38(38%) 14(14%) 14.96** <0.001 24 (48%) 14 (28%) 4.24** 0.04
ASB n (%) 30(30%) 14(14%) 7.46** 0.006 15(30%)
15
(30%)
0** 1
Gram positive
n (%)
14
(46.7%)
0(0%)
P = 0.007*
8
(46.2%)
6 (40%)
0.30** 0.58
Gram negative
n (%)
17
(56.7%)
14
(100%)
8
(53.8%)
9 (60%)
*Fishers Exact test, **Chi square test, One case with gram +ve & -ve organisms.
Table 2: Clinical and laboratory characteristics of diabetic patients with asymp-
tomatic bacteriuria and patients with negative urine culture and sensitivity.
Variant +ve C&S (ASB) n= 30 -ve C&S n= 70 Significance P value
Age (years) 12.873.33 10.694.42 -2.13
#
0.033
Male/female 3/27 30/40 10.25** <0.001
Disease duration (years) 5.083.29 4.243.41 -1.34
#
0.18
Microvascular complication n (%) 15 (50%) 18 (25.7%) 5.6 ** 0.018
RBG (mg/dl) 166.2352.89 158.7760.88 -0.72
#
0.48
HbA1c (%) 7.952.09 7.61.57 0.82
##
0.41
Microalbuminuria (ug/mg creat) n (%) 11 (36.7%) 4 (5.7%) 15.78** <0.001
C&S= culture and sensitivity,
##
Student t test **Chi Square test
#
Z value of Mann-Whitney U test
Table 3: Comparison between prepubertal and postpubertal diabetic females.
Variant Prepubertal n=24 Postpubertal n=43 significance P value
Microvascular complications n (%) 6(25%) 19(44.2%) 0.41** 0.52
Microalbuminuria (ug/mg Creat) n (%) 4 (16.7%) 9 (20.9%) P= 0.76*
Pyuria n (%) 10 (41.7%) 22 (51.2%) P= 0.46*
ASB n (%) 6(25%) 21(48.8%) 3.63** 0.056
Gram positive n (%)
Gram negative n (%)
3(50%)
3(50%)
11(52.4%)
11(52.4%) P= 1*
**Chi Square test, *Fishers Exact test, One postpubertal diabetic with gram positive & negative organisms.

Fig. 1: Distribution of different cultured organisms among diabetic patients and controls with ASB (Staph
coagul ve=Staph coagulase negative).
Fig. 2: ROC curve showing that pyuria had 80% sensitivity & specificity to predict ASB in patients (AUC=
0.8, CI =0.7-0.9)(A) and 100% sensitivity & specificity in controls (AUC =1,CI= 1)(B).
A B
0
20
40
60
80
100
E
-
C
o
l
i
E
n
t
e
r
o
c
o
c
c
i
P
s
e
u
d
o
m
o
n
a
s
S
t
a
p
h

a
u
r
e
u
s
S
t
a
p
h

c
o
a
g
u
l

-
v
e
A
c
i
n
e
t
o
b
a
c
t
e
r
C
a
n
d
i
d
a
Patients
Control

Fig. 3: Sensitivity of different cultured organisms to antibiotics in patients and controls with positive
cultures(Amox-clav=amoxicillin-clavulanate, Aminoglyc.=aminoglycosides, 3
rd
gen. ceph.=3
rd
generation
cephalosporins,4
th
gen. ceph.=4
th
generation cephalosporins).
Discussion
Diabetes mellitus as a group of
metabolic diseases is characterized
by chronic hyperglycemia resulting
from defects in insulin secretion,
insulin action, or both (ADA, 2008).
Diabetics are more likely to have
asymptomatic and symptomatic
bacteriuria (Patterson and Andriole,
1997; Geerlings et al., 2000b) with
increased risk for pyelonephritis and
subsequent impairment of renal
function (Geerlings et al., 2001).
In this study, the prevalence of
ASB was significantly higher in
Egyptian type 1 diabetic children
and adolescents (30%) than in the
control group (14%). Also, Ma-
kuyana et al. (2002) reported that
the prevalence of ASB was 32% in
diabetics and 11% in non diabetics
in urban black population. Rzsai et
al. (2003) reported that the preva-
lence of ASB was 10.1% of 178
type 1 diabetic children and adoles-
cents compared to 2.6% of 194
school aged controls. Kayima et al.
(1996) investigated one hundred and
thirty five diabetic patients and fif-
teen patients had positive cultures
showing11.1% incidence of ASB.
Possible reasons for the higher
prevalence of ASB in diabetes may
include increased residual urine
volume or impairment of several
aspects of host defense mechanism
(Rzsai et al., 2003). Urinary IL-8
was elevated in children with type1
DM with significant bacteriuria
compared with the non diabetics
(Rzsai et al., 2006).
In the present study, patients with
ASB were significantly older
(P=0.033) than those without. This
result agreed with Geerlings et al.
(2000b), Meiland et al. (2004) and
Odetoyin et al. (2008) who con-
0
2 0
4 0
6 0
8 0
10 0
A
m
o
x
-
c
l
a
v
.
A
m
i
n
o
g
l
y
c
.
M
o
r
e
p
e
n
a
m
3
r
d

g
e
n
.

c
e
p
h
.
4
t
h

g
e
n
.

c
e
p
h
.
Patients
Control

cluded that age was a risk factor for


ASB in diabetic patients.
Female sex was a significant risk
factor for ASB in diabetic patients.
Frippiat (2005) reported that most
of the research about ASB in diabet-
ic females showed a prevalence of
about 7-13%, which was 3-4 times
higher than non diabetic ones.
Matteucci et al. (2007) concluded
that the main risk factor for ASB in
diabetic patients was female sex and
Yayli et al. (2003) observed low
prevalence of ASB in boys. It was
explained by the high circumcision
frequency in Turkey which was also
the situation in Egypt. El-Gamal and
Saleh (1991) screened 450 urine
samples collected from rural school
children in Egypt and revealed that
the incidence of asymptomatic bac-
teriuria was 7% with predominance
in females (11%) than in males
(3.6%).
ASB was borderline significantly
higher among postpubertal diabetic
females (P= 0.056). This is mostly
due to the onset of menstruation at
puberty and its relation to sexual
activity. A gradual increase in the
incidence of asymptomatic bacteriu-
ria was noted in girls from prepu-
bertal age to postpubertal age (Ku-
mar et al., 2002). Geerlings et al.
(2000b) suggested that age, dura-
tion, metabolic control, and compli-
cations of diabetes were the most
common risk factors for ASB in
diabetic females.
Lack of correlation between ASB
in diabetic patients and metabolic
control of diabetes is in agreement
with Nicolle et al. (2005) who
found that bacteriuria was usually
correlated with the presence of
long-term complications rather than
with metabolic parameters of dia-
betic control. However, the present
results disagreed with Meiland et al.
(2004) and Bonadio et al. (2004) in
their studies on diabetic women.
There was no significant relation-
ship between the duration of diabe-
tes and the increased prevalence of
ASB. Also, Zhanel et al. (1991) re-
ported that the prevalence of asymp-
tomatic bacteriuria was not influ-
enced by the type or duration of di-
abetes. However, Meiland et al.
(2004) found that the duration of
diabetes was powerful predictor of
ASB in diabetic women.
In the present study, pyuria was
more prevalent in diabetic patients
than controls (P <0.001). This result
agreed with Rzsai et al. (2003)
who found that the leukocyturia
tended to be more frequent in dia-
betic patients than in controls (14.4
versus 7.6%; P= 0.052). Nicolle et
al. (2005) reported that pyuria was
present with asymptomatic bacteriu-
ria in ~32% of young females and
70% of diabetic patients. In the pre-
sent study, pyuria was a strong pre-
dictor for positive urinary cultures
in both patients and controls being
detected in 80% of positive cultures
in patients and in 100% in controls.
Fihn (2003) reported that sensitiv-
ity of pyuria was 95% with 71%
specificity. The prevalence of ASB
was higher in patients with microal-
buminuria (P <0.001). Matteucci et

al. (2007) stated that the main risk


factor for ASB in diabetic patients
was UAE (urinary albumin excre-
tion). The likelihood of ASB in-
creased with higher UAE levels.
The current study revealed that the
presence of microvascular compli-
cations was an important risk factor
for ASB in young diabetics (P=
0.018). This agreed with results of
Ribera et al. (2006).
Urine cultures of patients showed
growth of 56.7% of gram negative
organisms with E. coli in 30% and
Pseudomonas in 26.7% with varia-
ble growth of gram positive organ-
isms, while controls showed only
gram negative organisms with E.
coli in 42.9% and Pseudomonas in
57.1%. Rzsai et al. (2006) found
that 36.4% of the bacteriuria was
gram-negative and 63.6% gram-
positive in diabetic patients. Nicolle
(2001) found that E. coli was the
most common organism isolated in
80-90% of positive cultures in pa-
tients and controls. Also, Rzsai et
al. (2003) reported that E. coli was
in only about a quarter of patients
with Uropathogenic E. coli (UPEC)
being the predominant etiologic
agent of UTI in diabetic individuals.
Makuyana et al. (2002) reported
that the commonest bacterial organ-
ism isolated in diabetics were E.
coli (26%) followed by Staphylo-
coccus aureus (21%), Streptococcus
group B (14%), Streptococcus
group D and non-lactose fermenting
(7%), Pseudomonas (7%), Klebsiel-
la and Proteus (2%). Kayima et al.
(1996) found that E. coli was isolat-
ed in 40% of diabetic patients and
gram negative bacilli made up
66.7% of the isolates. The low rates
of E. coli were found in type 1 dia-
betic women with ASB (Geerlings
et al., 2000b).
The spectrum of pathogenic bac-
teria causing ASB and UTI is dif-
ferent. The reason for this may be
that the virulence factors are differ-
ent in ASB and UTI (Rzsai et al.,
2003).
In the current study, there was no
significant difference between dia-
betic patients and controls as re-
gards sensitivity of the isolates to
commonly used antibiotics. Sensi-
tivity to nitrofurantoin reached up to
94.1% in patients.
Kayima et al. (1996) found that
isolates were poorly sensitive to the
regularly available antibiotic ampi-
cillin (33% sensitive). Nitrofuranto-
in inhibited growth in 93% of the
isolates. Other antimicrobials with
over 80% sensitivity level included:
gentamicin, ceftazidime, amoxicil-
lin-clavulanate and cefuroxime.
They are expensive or require par-
enteral administration. Although
the isolated organisms were those
usually isolated in UTIs, they were
not sensitive to the commonly
available antibacterial agents.
Odetoyin et al. (2008) found that
the isolated organisms were largely
resistant to common antibiotics test-
ed such as cotrimoxazole and gen-
tamicin but susceptible to nitrofu-
rantoin. Makuyana et al. (2002)
concluded that gentamicin, nitrofu-
rantoin and ampicillin were the

most effective antimicrobials in the


majority of isolates. Certain isolates
exhibited some bacterial resistance
to conventional antibiotics. Most of
the isolates were resistant to one or
more antibiotics in the study on
school going children (Kumar et al.,
2002).
The eradication of microorgan-
isms that cause UTIs has been re-
ported to be more difficult in diabet-
ic patients than in non diabetic pa-
tients because of an increased fre-
quency of multidrug resistance
(Geerlings et al., 2000b). Treatment
may reduce the overall proportion
of time infected in the long term and
carriage of a unique strain, but most
treatment regimens were followed
by subsequent recolonization (Dalal
et al., 2009). The treatment of ASB
in type 1 diabetic females did not
appear to prevent UTI (Harding et
al., 2002) and may promote the in-
vasion of more virulent pathogens
(Stein and Fnfstck, 1999). A care-
ful follow-up of pediatric patients
was warranted before anti-biotic
therapy can be considered (Rzsai
et al., 2003) as type 1 diabetic pa-
tients with ASB had a tendency to-
ward complications of UTIs. Anti-
microbial management of bacteriu-
ria in type 1 diabetics must focus on
the prompt identification and effec-
tive treatment of symptomatic epi-
sodes. It is recommended to consid-
er diabetic patients with symptomat-
ic UTI as having a complicated UTI
and therefore to treat them for a pe-
riod of 7-14 days (Geerlings, 2008).
Conclusion
The prevalence of ASB is more
among type 1 diabetic children and
adolescents, especially females in
the postpubertal period. Microalbu-
minuria and history of microvascu-
lar complications are major risk fac-
tors for ASB in young diabetics.
Pyuria is a strong predictor for
positive urinary cultures in both pa-
tients and controls. The most com-
monly isolated organism in urinary
cultures of diabetics was E.coli.
Thus, Screening of ASB in young
diabetics with risk factors is rec-
ommended to avoid development of
complicated UTI.
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dmeyer, H.M.; England, J.D.;
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Harding, G.K.M.; Zhanel, G.G.;
Nicolle, L.E., 2002: Antimicrobial
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tes), 2009: Assessment and moni-
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 963 - 973
RISK FACTORS PREDISPOSING TO TOXOPLASMOSIS IN
CHRONIC RENAL FAILURE PATIENTS AND
RENAL TRANSPLANT RECIPIENTS
By
ABEER M A MAHGOUB
1*
, SAYEDA M. AUFY
1
, MOHAMED GAMAL
EL-DIN SAADI
2
AND MARWA ADEL ELMALLAWANY
1
Departments of Parasitology
1
, and Internal Medicine
2
, Faculty of Medi-
cine, Cairo University, Cairo, Egypt
Abstract
This work evaluated risk factors predisposing to toxoplasmosis in chronic
renal failure patients and renal transplant recipients. The present study included
91 cases classified according to their renal status into four groups; control
group, renal failure patients not on haemodialysis, renal failure patients on reg-
ular haemodialysis and renal transplant recipients group. The age groups (<20)
and (30- ) had the highest positivity for anti-Toxoplasma IgG & IgMantibodies
in comparison to the other age groups. The results showed no sex difference in
positivity rate for anti-Toxoplasma IgG & IgMin groups. There was no signifi-
cant difference between groups regarding risk factors for contracting toxoplas-
mosis, clinical presentation suggestive of toxoplasmosis and diabetes mellitus.
There was significant difference between all groups as regarding intake of im-
munosuppressive drugs and blood transfusion.
Key words: toxoplasmosis, IgG, IgM, renal failure, haemodialysis, transplant,
immunosuppressive drugs, diabetes.
Correspondence: *Dr. Abeer Mohamed Aly Mahgoub, Email: goubya@
hotmail.com, Phone: 00227925500/ 0020105315500).
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Toxoplasma gondii is the most
common human parasite world wide
and one that infects a wide range of
hosts. Nearly one-third of humanity
has been exposed to this parasite
(Dubey, 2005). Infection rate in
human and other animals differ
from one country to another. The
reasons have been attributed to
many variations; human behavior
such as cooking habits, number of
cats living outdoors and other varia-
tions which are still not fully under-
stood (Gilbert et al., 2000). In
Egypt, the overall prevalence in
1992 was 31.4% (Zawawy et al.,
2000). The prevalence of the Toxo-
plasma infection varies depending

on the age of population and geo-


graphical area. The incidence of
infection increases with age (Parija,
2004). In immunocompetent pa-
tients, toxoplasmosis was benign
and self limiting (Barbosa et al.,
2009). But, toxoplasmosis proved to
be a major opportunistic infection
that may lead to morbidity and mor-
tality in immunocompromised indi-
viduals (Meeka et al., 2001). Chron-
ic failure patients suffered impair-
ment of cell mediated immunity
either due to uremia or interventions
used therapy, as dialysis and trans-
plantation with subsequent immu-
nosuppressive therapy and multiple
blood transfusions. The immune-
compromised hosts with T cell de-
fects include AIDS patients, hema-
tological malignancies patients (es-
pecially Hodgkins disease and oth-
er lymphomas), organ transplant
recipients, and patients on immuno-
suppressive therapy with cortico-
steroids and cytotoxic drugs (Weiss
and Dubey, 2009). In these patients
infection usually occurs as a conse-
quence of the recrudescence of a
latent infection acquired before the
onset of immune suppression. How-
ever, it may occur also due to re-
cently acquired acute infection
(Montoya et al., 2001). The role of
reactivation of latent infections in
the production of disease in immu-
nosuppressed adult was recognized
at outset of solid organ transplantta-
tion (Ruskin and Remington, 1976).
This study aimed to clarify risk
factors predisposing to toxoplasmo-
sis among chronic renal failure pa-
tients and renal transplant recipi-
ents.
Subjects, Materials and Methods
The study included 91 patients, 51
male and 40 female with age ranged
between 8.5 & 70 year. They were
classified into four groups according
to their renal status. G1: 19 chronic
renal failure patients who were not
subjected to haemodialysis. They
were 12 male & 7 female selected
from Internal Medicine wards and
outpatients clinics requesting follow
up for renal status and/or treatment
of other medical problems as hyper-
tension, diabetes mellitus and ane-
mia. G2: 30 patients with chronic
renal failure on regular haemodialy-
sis. They were 16 male and 14 fe-
male with haemodialysis period be-
tween 7 & 132 month selected
from King Fahd Haemodialysis
Unit. G3: 29 renal trans-plant recip-
ients. They were 17 male & 12 fe-
male with post renal transplanta-tion
period ranging from 1 to 180
months selected from renal trans-
plantation outpatient clinic request-
ing follow up. G4: 13 control cases
with no history of renal troubles
with normal serum urea and creati-
nine. They were 6 male & 7 female
selected from trauma causality of
orthopedic department.
All cases were subjected to a
complete history taking using a
structural questionnaire with special
emphasis on risk factors to toxo-
plasmosis as blood transfusion, im-
mune suppression as diabetes and/or

taking immunosuppressive drugs.


Clinical examination was carried
out for signs and symptoms sugges-
tive of toxoplasmosis such as fever,
enlarged lymph nodes, hepatosple-
nomegaly, and/or skin rash. Serum
blood samples were collected for
renal function test (urea and creati-
nine) and for anti-Toxoplasma
gondii IgG and IgM antibodies.
Anti-Toxoplasma IgM was done
by IgM-capture immunoenzymatic
assay (Herbrink et al., 1987), using
TOXO-IgM (Immunocapture) kit
(Biokit, S.A. Barcelona, Spain).
Anti-Toxoplasma-IgG antibodies
was done by ELISA (Derouin et al.,
1987) by using NovaLisaToxo-
plasma IgG-ELISA (NovaTec Im-
mundiagnostica GmbH (D-63128
Dietzenbach, product, Germany) .
Statistical evaluation: All data
were processed using SPSS (1 0.0)
for Windows 98. Quantitative vari-
ables were expressed using mean
and standard deviation; they were
compared using t-student's test
(paired and unpaired). Pearson cor-
relation is used to correlate two
quantitative variables. Qualitative
variables were compared using Chi-
square test for comparisons between
proportions when appropriate. P
<0.05 was considered significant &
P < 0.01 highly significant.
Results
Comparing sex distribution in the
groups showed no statistical signifi-
cant difference (P=0.689). The ages
ranged between 8.5 and 70 year.
The mean age of control group was
39.94.16. The mean age of renal
failure not on haemodialysis group,
renal failure on regular haemodialy-
sis group and renal transplant recip-
ient group was 49.893.09,
42.172.39, 27.091.98 respective-
ly. Age distribution in groups
showed highly statistical significant
difference between them (P=0.00).
Clinically, of 91 patients, 3 gave
history of fever with lymphadenopa-
thy, 2 gave history of lymphade-
nopathy, 2 gave history suggestive
of retinochoriditis and 15 had histo-
ry of pregnancy miscarriage. There
was no statistical significant differ-
ence among the four study groups
(P=0.52, 0.51, 0.57 and 0.16 respec-
tively).
The distribution of risk factors for
contracting toxoplasmosis among
the four study groups was elicited
(tab. 1). Occupation was investigat-
ed if it requires exposure to any of
the above mentioned risk factors or
not. Occupation that requires expo-
sure to risk factors was considered
occupation with expected risk (e.g.
farmer) and that does not necessitate
exposure to risk factors was consid-
ered as occupation without expected
risk (e.g. employer). Comparing
history of risk factors for contract-
ing toxoplasmosis in the study
groups, there were no statistical sig-
nificant differences between them.
Table 1: Risk factors for contracting toxoplasmosis among all groups.

Variants
Control
N. %
Renal failure not
on haemodialysis
N. %
Renal failure on
haemodialysis
N. %
Renal trans-
plantation
N. %
Total
N. %
P
value
Occupation: risky 4 30.80 8 42.10 12 40.00 11 37.90 35 38.50
not risky 9 69.20 11 57.90 18 60.00 18 62.10 56 61.50
Residence: Urban 9 69.20 12 63.20 18 60.00 15 51.70 54 59.30
0.719
(NS)
Rural 4 30.80 7 36.80 12 40.00 14 48.30 37 40.70
Cat contact: Yes 2 15.40 4 21.10 5 16.70 4 13.80 15 16.50
0.929
(NS)
No 11 84.60 15 78.90 25 83.30 25 86.20 76 83.50
Soil contact: Yes 3 23.10 6 31.60 11 36.70 9 31.00 29 31.90
0.852
(NS)
No 10 76.90 13 68.40 19 63.30 20 69.00 62 68.10
Eat unwashed
vegetables: Yes
2 15.40 2 10.50 4 13.30 4 13.80 12 13.20 0.98
(NS)
No 11 84.60 17 89.50 26 86.70 25 86.20 79 86.80
Eat undercooked
meat: Yes
3 23.10 3 15.80 6 20.00 7 24.10 19 20.90
0.911
(NS)
No 10 76.90 16 84.20 24 80.00 22 75.90 72 79.10
Table 2: History of risk factors for toxoplasmosis among groups.
*No significant difference in all groups regarding history of Diabetes (P =0.166). *Highly significant dif-
ference in all groups regarding history of intake of immunosuppressive drugs (P =0.00). *Highly significant
difference in all groups regarding history of blood transfusion (P =0.00).
Regarding the relation between
age and the prevalence of the anti-
Toxoplasma IgG & IgM antibodies
in the present study, it was found
that the age groups of (<20) had a
high positivity for anti-Toxoplasma
IgG antibodies. As the age in-
creased, positivity was also in-
creased with age but less markedly,
until reach the age group (30-) year
then positivity started to decrease.
In addition, it was also observed
that a high positivity for anti-
Toxoplasma IgM antibodies was
encountered in age (<20) with a less
increase in higher age group until
reach the highest peak (30-). So, it
was observed that the age groups
(<20) and (30-) had the highest
positivity for anti-Toxoplasma IgG
Group Diabetes Immuno-suppressive drugs Blood transfusion
+ve -ve +ve -ve +ve -ve
GI
6 (31.6%) 13 (68.4%) 4 (21.1%) 15 (78.9%) 0 (0.0%) 19 (100%)
GII
4 (13.3%) 26 (86.7%) 5 (16.7%) 25 (83.3%) 25 (83.3%) 5 (16.7%)
GIII
3 (10.3%) 26 (89.7%) 29 (100%) 0 (0.0%) 29 (100%) 0 (0.0%)
GIV
1 (7.7%) 12 (92.3%) 0 (0.0%) 13 (100%) 0 (0.0%) 13 (100%)
Total
14 (15.4%) 77 (84.6%) 38 (41.8%) 53 (58.2%) 54(56.3%) 37 (40.7%)

& IgM antibodies in comparison to


the other age groups.
On studying the relation between
sex and the prevalence of anti-
Toxoplasma IgG & IgM antibodies
in the present study, it was observed
that out of the 47 seropositive cases
for anti-Toxoplasma IgG antibod-
ies, 20 females (42.6%) and 27
males (57.4%) were encountered
without statistical significant differ-
ence (p=0.473).Whereas, of 14 posi-
tive cases for anti-Toxoplasma IgM
antibodies, 6 females (42.9%) and
8 males (57.4%) were encountered
without statistical significant differ-
ence (P=0.928).
After serological test for anti-
Toxoplasma IgG & IgM antibodies
were performed, it was found that
regarding the 3 patients who gave
history of fever with lymphadenopa-
thy; one was positive IgG & IgM
while the other 2 patients were only
positive for IgG. The 2 patients with
history of lymphadenopathy without
fever were positive IgG & IgM, the
2 patients who gave history of reti-
nochoriditis; one was positive IgG
& IgM and second was positive for
IgG and negative IgM . Of 15 fe-
male who had history of bad out-
come of pregnancy, 19.1% were
positive for IgG & 14.3% were
positive for IgM.
There was statistical significance
between lymphadenopathy and posi-
tive IgG and between lymphade-
nopathy and positive IgM (P=0.026
& 0.004 respectively). But, there
was no significance difference be-
tween positive IgG in relation to
fever, retinochoriditis and bad out-
come of pregnancy (P=0.088, 0.026
&0.502 respectively) as well as no
significance difference between pos-
itive IgMin relation to fever, retino-
choriditis and bad outcome of preg-
nancy (P=0.381, 0.17, 0.81 respec-
tively).
Table 3: IgG & IgM positivity and toxoplasmosis risk factors.
*Significant difference (P=0.028) between Diabetes & IgG. *No significance (P=0.901) between Diabetes &
IgM. *Significant difference (P=0.011) between immunosuppressive drugs & IgG.. *Significant difference
(P=0.014) between immunosuppressive drugs & IgM. *Significant difference (P=0.025) between blood
transfusion & IgG. *No significant difference (P=0.14) between blood transfusion & IgM.
Discussion The present study included 91
cases classified according to their
Anti-
Toxoplasma
Diabetes Immuno-Suppressive drugs Blood Transfusion
+ve -ve Total +ve -ve Total +ve -ve Total
11
(23.4%)
36
(76.6%) 47
27
(54%)
23
(46%)
50
35
(79.5%)
9
(20.5%)
44
IgG +ve
-ve
3
(6.8%)
41
(93.2%)
44
11
(39.3%)
17
(60.7%)
28
19
(55.9%)
15
(44.1%)
34
IgM +ve
2
(14.3%)
12
(85.7%)
14
11
(78.6%)
3
(21.4%)
14
12
(85.7%)
2
(14.3%)
14
-ve
12
(15.6%)
65
(84.4%)
77
27
(42.2%)
37
(57.8%)
64
42
(65.6%)
22
(34.4%)
64

renal status into four groups; control


group, renal failure patients not un-
dergo haemodialysis, renal failure
patients undergo haemodialysis and
renal transplant recipients group.
Multiple factors were associated
with the occurrence of toxoplasmo-
sis, including route of transmission,
climate, cultural behavior and hy-
gienic standards. This combination
led to marked differences even with-
in the same country (Amendoeira et
al., 2003; Rorman et al., 2006).
In the present study, the risk
factors included living in rural are-
as, eating raw or unwashed vegeta-
bles and fruits, contact with cats,
handling and consumption of raw or
undercooked meat and occupation.
This was to evaluate the socioeco-
nomic and environmental pattern
which might predispose to T. gondii
infection. Living in rural areas had a
higher risk of toxoplasmosis than
those in urban areas (Etheredge and
Frenkel, 1995; Garcia et al., 1999).
This may be due to more expo-
sure, in rural areas, to the sources of
infection in unfavorable envi-
ronment contaminated by the oo-
cysts (Spalding et al., 2005). The
vegetables and water contaminated
with oocysts were an important
mechanism of T. gondii transmis-
sion (Cook et al., 2000). A signifi-
cant association between eating raw
or unwashed vegetables and fruits
and T. gondii seroconversion was
reported (Baril et al., 1999). So, the
toxoplasmosis infected the vegetari-
ans and the herbivores (Rifaat et
al., 1977; Amin and Morsy, 1997).
Baril et al. (1999) and Kravetz
and Federman (2005) emphasized
the relevance of cats in toxoplasmo-
sis as the most likely method for
transmission, particularly in school
children (El Shazly et al., 1996).
Also, Spalding et al. (2005) cleared
that contact with contaminated soil
was among factors that contribute
to Toxoplasma infection. Epidemio-
logical studies and several outbreaks
have identified the handling and
consumption of raw or undercooked
meat as a source of the toxoplasmo-
sis (Carme et al., 2002; Dubey and
Jones, 2008). Montaya (2002)
demonstrated that clinical presenta-
tions suggestive of the toxoplasmo-
sis include fever, lymphadenopathy
and retinochoriditis. In the present
study, clinical presentations sugges-
tive of toxoplasmosis were infre-
quent.
There was no statistical significant
difference between study groups
regarding risk factors for contract-
ing toxoplasmosis, clinical presenta-
tion suggestive of toxoplasmosis
and diabetes mellitus. Whereas there
was statistical significant difference
between the four study groups as
regarding dialysis, intake of immu-
nosuppressive drugs and blood
transfusion. These differences could
be attributed to the difference in the
renal status with its subsequences in
interventions and therapy between
the study groups.

Concerning the relation between


age and seropositivity in the present
study, there was increase in positivi-
ty with increase in age and the high
IgG positivity and IgM positivity
were among groups with age be-
tween 20 and 30 years old and be-
low 20 years. This agreed with
Wong et al. (2000). Also, Petersen
et al. (2001) reported that increasing
positivity was marked in children up
to 9 years, and in higher age groups,
the increase was less marked up to
50 years when peak values were
reached, the positivity declined.
Spalding et al. (2005) deduced that
increasing positivity with increasing
in age might correlate with more
exposure to several environmental
risk factors.
In the present study, no sex differ-
ence in positivity of IgG & IgM.
This agreed with Assmar et al.
(1997) and Sobral et al. (2005). On
the other hand, the results disagreed
with Ghorbani et al. (1981) who
found the prevalence of seroposi-
tivity for anti-Toxoplasma antibod-
ies higher in females than in males
and disagreed with Excler et al.
(1988) who found higher prevalence
in males.
The present study showed signifi-
cance difference between lymphad-
enopathy and positive IgG & IgM.
But, significance difference was
neither between positive IgG nor
between IgM in relation to fever,
retinochoriditis and/or bad pregnan-
cy outcome. In the present study,
clinical presentations suggestive of
infection were not frequent. This
agreed with reports that acute Toxo-
plasma infection was often clinical-
ly silent (Thulliez 1992; Remington
et al., 2001). Also, Kravetz and
Federman (2005) found that T.
gondii infections might progress to
sub-clinical form in 90% of cases or
might present non-specific signs.
In the present study, 23.4% of IgG
positive cases were diabetic versus
6.8% of negative ones with a statis-
tical significant difference. Howev-
er, no significant difference was
between positivity and negativity
for IgM in them. Kasper and Booth-
royd (1993) found that diabetic pa-
tients were more susceptible to tox-
oplasmosis as diabetes affected the
immune status.
In the present study, there were
54% of positive IgG renal cases ver-
sus 39.29 % negative IgG ones re-
ceived immunosuppressive drugs
with a statistical significant differ-
ence. Besides, 78.57% of renal posi-
tive IgM cases versus 42.19 % of
seronegative IgM renal ones re-
ceived immunosuppressive drugs
with a significant difference.
Liesenfeld and Remington (2000)
stated that intake of immunosup-
pressive drugs was considered one
of the toxoplasmosis risk factors.
The present study showed that
79.55% of positive IgG renal cases
versus 55.88% of negative IgG renal
cases received blood transfusion
with a significant difference. Be-
sides, 85.71% of positive IgM renal
cases versus 65.63% of negative
IgM renal ones received blood
transfusion without significant dif-

ference. Toxoplasma antigen was


detected in blood from chronically
infected individuals (Sarwat et al.,
1993; Dupon et al. 1995; Elsheikha
and Morsy, 2009). However, blood
transfusion rarely transmits the tox-
oplasmosis and greater risk occurs
after granulocyte transfusion. This
positivity in patients with a history
of blood transfusions might not be
related only to exposure to blood
transfusion but might be attributed
also to high number of repeated
blood transfusions among renal pa-
tients.
Generally speaking, in Arab coun-
tries, toxoplasmosis prevalence
ranged between 37% in Jordan
(Morsy and Michael, 1980), 37.4%
in Saudi Arabia (Abbas et al.,
1986), 37.5% in Libya (Kassem and
Morsy, 1991) and up to 95.5% in
Kuwait (Behbehani and Al-Karmi,
1980).
Conclusion
No doubt, improving the level of
knowledge about toxoplasmosis and
relevant risk factors will have obvi-
ous influence on the withdrawing
the infection rate amongst immuno-
suppressed patients. Health system
managers should continue to offer
education that help prevention of
infectious disease as toxoplasmosis
in immunosuppressed patients.
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Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 975 - 990
PATHOLOGICAL STUDIES OF DIFFERENT GENOTYPES OF
HUMAN CRYPTOSPORIDIUM EGYPTIAN ISOLATES IN
EXPERIMENTALLY MICE
By
AMANY M. EIDA, MOHAMED M. EIDA
2
AND AMINA EL-DESOKY
3
Departments of Parasitology Tropical Medicine
2
, and Pathology
3
, Fac-
ulties of Medicine
1,2
and Veterinary Medicine
3
, Suez Canal University,
Ismailia, Egypt.
Abstract
The genotyping of cryptosporidium clinical isolates obtained from 36 gas-
trointestinal symptomatic patients were identified by nested PCR for amplifica-
tion of 18S rRNA followed by RFLP analysis using Ssp1 and Vsp1, and then
pathological changes between different cryptosporidium genotypes were eval-
uated in experimentally infected mice. Cryptosporidium genotypes (C. parvum,
C. hominis & C. melegridies) were detected (66.7%, 27.7% & 5.6%) respec-
tively in human isolates. Different degrees of pathological changes were found
among infected mice by different Cryptosporidium genotypes. Moderate and
severe degrees of pathological changes with infection score ranging from 2 to
4 were found in all infected mice with C. parvum (except one isolate), while
mild degree of pathological changes with infection score of 2 was found in all
mice with C. melegridies. The results showed statistically significant relation
between genotype and pathological degrees. There were no differences in the
average number of oocysts per smear in Group and Group I while in Group
I, there was no oocysts shedding.
Keyword: Cryptosporidium, genotypes, PCR-REFLP analysis, experimental
study.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Cryptosporidium parvum is one of
the most important intestinal para-
sites infecting man. It has a world-
wide distribution (Xiao et al., 2000)
and it is responsible for a major di-
arrheal diseases as well as water-
borne outbreaks (Hlavsa et al.,
2005). August and September of
2001, the incidence of cryptosporid-
iosis was 55.1 per 100,000 per year
as compared to 3.1 per 100,000 per
year for the remainder of the sur-
veillance period (p < 0.0001) (Kev-
in and Deirdre, 2005). In developing
countries, Cryptosporidium is re-
sponsible for 8-19% of cases of di-

arrheal disease (Fayer et al., 2000;


Tzipori and Ward, 2002). Cryptos-
poridiosis remains a clinically sig-
nificant opportunistic infection in
immunocompromised patients caus-
ing potentially life-threatening diar-
rhea (Pozio and Morales, 2005).
Also, the parasites not only infect
humans, but also cause morbidity in
farm animals, leading to economic
losses (Sunnotel et al., 2006). In
Egypt, zoonotic cryptosporidiosis
proved a health problem (Massoud
et al., 2008) with a prevalence rate
of 17% (Abdel-Messih, et al.,
2005). There were at least 11 Cryp-
tosporidium sp., and new species
and genotypes are regularly de-
scribed (Fayer et al., 2001; Morse et
al., 2007). Cryptosporidium is rec-
ognized as containing assemblages
of species which are genotypically
heterogeneous but morphologically
largely identical (Xiao and Rayan,
2004). The main causative agents of
human cryptosporidiosis are geno-
type1 (human) and genotype2 (bo-
vine) of the C. parvum (Llorente et
al., 2007). Human infections with
other zoonotic species as C. canis
and C. felis, C. meleagridis were
reported (Xiao, et al., 2001; Gatei,
et al., 2002; Leoni et al., 2006). Dif-
ferences between isolates of C. par-
vum have been shown at the molec-
ular level by several techniques.
The existence of four different al-
leles in Cryptosporidium isolates of
human and bovine origin was
demonstrated (Enemark et al.,
2002). The severity of infection in
man and animals varied depending
on the specific C. parvum isolate
(Pozio et al., 1992; Tzipori et al.,
1994; Okhuysen et al., 1999). Pre-
vious studies of the 18S rRNA gene
have shown that the locus can be
useful for the specific identification
of most Cryptosporidium sp. (Mor-
gan et al., 2000; Tiangtip and
Jongwutiwes, 2002; Xiao et al.,
2001). A limited number of isolates
was typed from the developing
countries (Tiangtip and Jong-
wutiwes, 2002). The significance of
the different Egyptian species,
genotypes and histopathological
changes in the gastrointestinal tract
are not yet clearly identified.
The present study aimed to iden-
tify genotypes of Cryptosporidium
clinical isolates from patients target-
ing hyper variable region of 18S
rRNA gene and evaluate the histo-
pathologic changes in gastrointesti-
nal tract and oocyst shedding in ex-
perimentally infected mice by dif-
ferent genotypes.
Subjects, Material and Methods
Stool samples were collected
from 211 patients with gastrointes-
tinal symptoms. The samples were
examined immediately by wet
mount smear stained with
Lugol

iodine and formalin ethyl


acetate concentration method to ex-
clude the presence of other para-
sites. Every sample was subjected to
Sheather

s floatation method and


examined by kinyoun
`
s stain to de-
tect Cryptosporidium oocyst and to
exclude the presence of Cyclospora

and Isospora. Diagnosis was con-


firmed by immunochromatografic
lateral flow immunoassay (R-
Biopharm, Germany). Briefly, one
ml extraction buffer was added to
100l of stool samples and homog-
onised by vortex mixer, allowed to
sediment for 3 minutes. The super-
natant (200-500l) was transferred
into another clean tube and strip test
immersed in the prepared sample to
the appropriate line then read after 5
min. In +ve sample, red and blue
band appear. In ve sample, blue
band appear only. In non valid test,
no visible band or combination of
bands different from mentioned
lines.
A total 32 positive samples were
diagnosed by kinyoun
`
s stain and
another 6 samples were diagnosed
by immunochromatografic test.
Each positive sample for Cryptos-
poridium oocysts was divided into
two parts: first was preserved in
potassium dichromate and stored at
4C as a source of live oocysts for
experimental infection of mice and
second part was stored at -20C un-
til required for DNA extraction and
genotyping by PCR. For extraction
of DNA: Pea-sized of frozen stool
was scraped in 1.5 ml eppendorf
tube and suspended in 500l lysis
buffer. Rupture of oocyst were done
by heating tubes for 15 minutes at
80C, freezing for 30 min at -80C
and repeated 7 times (Kim et al.,
1992). Sample DNA extraction was
performed according to the manu-
facturer's protocol (Qiagen Kit Ltd,
UK). Further genomic analysis was
done by nested PCR protocol for
amplification of 18S rRNA fol-
lowed by RFLP analysis (Xiao et
al., 1999). A PCR product of 1,325
bp was amplified using external
primers: forward. 5 TTCTAGAGC-
TAATACATGCG3and reverse 5
CCCTAATCCTTCGAAACAGGA
3. Primers for the secondary nested
PCR step, a PCR product that was
826 to 864 bp long (depending on
isolates) was amplified by using a
primary PCR product and internal
primers: 5 GGAAGGGTTGTA TI-
TATTAGATAAAG3` and reverse
5 AAGGAGTAAGGAACAACC-
TCC A3. Reagent concentration
and cycling conditions was used in
both run, 10 x PCR buffer, 25 mM
Mg C
l2
, 2.5 Mm deoxynucleoside
triphosphate mixture, 100nM pri-
mers, and 1U of Taq polymerase
(Perkin Elmer, Biosystems, UK).
Cycling conditions for each run was
as following: Tubes were subjected
to 30 cycles of 94 min followed
by 94 for 45s, 55 for 45s, 72

for a min. and then by a final exten-


sion at 72 for 7 min. The different
species were identified by use of
restriction digestion of the second-
ary product. For restriction fragment
length analysis 20 L of secondary
PCR product was digested in a
50L reaction mixture containing
10 units of Ssp1 or Vsp1 (Fermen-
tas, Life Science) for genotyping
and 5L of appropriate restriction
buffer at 37
o
C overnight. The di-
gested products were fractionated
on a 2% agarose gel containing eth-

idium bromide at a concentration of


0.5g/mL.
Cryptosporidium oocysts were
isolated from patients stool using
the cold sucrose flotation method.
Fecal material was placed in a 50ml
centrifuge tube and 30 ml of sucrose
solution (specific gravity 1.2) were
added, shaken vigorously, laid-over
with 5ml of DW, and centrifuged at
4C at 411xg for 30 min. Ten ml of
top layer of sucrose solution were
transferred to new 50ml centrifuge
tubes plus 40ml of DW. After cen-
trifugation at 1,643g for 15 min.,
25ml of supernatant was decanted
and 25ml of DW were added. After
another centrifugation at 1,643xg
for 15 min., supernatant was de-
canted and 1,000l of DW were
added. Finally, the suspension was
mixed well and transferred to 1.5 ml
tube (Yananto and Veeraseatakul,
2002) and stored in 2.5% potassium
dichromate at 4 until used. A
volume of stool was mixed with
four volumes of saturated solution
of sodium chloride in distilled wa-
ter, centrifuged 300 rpm at 4
o
C for 5
min., 5ml of supernatant containing
oocysts were mixed with 45 ml. of
distilled water and re-centrifuged
300 rpm at 4
o
C for 5 minutes then
oocysts in pellet were suspended in
PBS (Ungar et al., 1986).
The number of oocysts present in
suspension was counted in a modi-
fied Neubauer hemacytometer after
mixing 0.2ml with 0.8ml of mala-
chite green (malachite green, 0.16 g;
distilled water, 100ml) (Castro-
hermida et al., 2004).
Eight week old mice were orally
challenged with oocysts. The infect-
ing inoculum 10
4
oocysts/mouse)
inoculated intraoesophagealy in
0.1ml phosphate buffered saline
(PBS, pH 7.4) (Pavlasek, 1982;
Yang and Healey, 1994). Each
mouse was kept in an isolated la-
beled cage. Feces were collected in
2.5% potassium dichromate every
two days starting on day 2 post in-
fections (P.I.). Isolation of oocysts
was by the cold sucrose flotation
method (Yananto and
Veeraseatakul, 2002.) Oocysts
shedding were recorded for 30 days
in three mice groups (GrI, GrII &
GrIII). Each group contained three
mice/ genotype was done as follow-
ing: the numbers of oocysts on
slides stained by Kinyouns acid
fast stain were counted, and means
for 20 fields were calculated at
400 magnification. If no oocysts
were seen in 20 fields, the entire
slide was examined. The number of
oocysts was categorized as follow-
ing; no oocysts; 1< 5 oocysts per
smear ; 2, 5 to 50 oocysts per
smear; 3, 50 to 100 oocysts per
smear and 4 >100 oocysts per
smear (Rasmussen and Healey,
1992). Three mice/each clinical
isolates were euthanized with ether
and killed on day 9 P.I. Entire di-
gestive tract of each mouse was
fixed in 10% buffered formalin and
used for histopathology study. Two
separate hematoxylin and eosin
stained, paraffin-embedded cross
sections of each intestinal segment
were assessed to cause histopatho-

logic changes ranked (scores) after


Appleyard and Wallace (1995).
They were: 0 =no parasite, 1= a sin-
gle parasite or a cluster of parasites
limited to one villus/crypt unit or
one colonic crypt per section; 2 =
two or three villi/crypt unit or two
or three colonic crypts per section
with various numbers of parasites;
3= parasites in many crypts with
some villus or surface epithelium
involvement; and 4= extensive in-
fection of crypts and villi or surface
epithelium, usually associated with
epithelial damage (Leitch and He,
1999).
Results
Positive cases were 32 diag-
nosed by kinyoun
`
s stain and anoth-
er 6 samples were diagnosed by
immunochromatografic test. Posi-
tive PCR was 36/38 (94.8%). PCR
reactions resulted in 837, 831, 833
bp amplified bands according spe-
cies (fig.1). RFLP analysis of nested
PCR products showed that only 3
cryptosporidium genotypes were
identified among all the human clin-
ical isolates. C. parvum, C. hominis,
C. melegridies were in 66.7%,
27.7% & 5.6% respectively. Diges-
tion of secondary products by Ssp1
showed that C. parvum and C. hom-
inis generated three bands of 449,
270 &111 bp and C. melegridies
generated three bands of 449, 254
&111 bp (Fig.2). C. parvum and C.
hominis were differentiated by Vsp1
digestion pattern. C. parvum pro-
duced two visible bands of 627 &
104 bp, C. hominis generated two
visible bands at 556 & 104 bp and
C. melegridies generated visible
bands of 104, 171, 456 (Fig.3).
Different pathological changes
were detected among mice infected
by different genotypes. Moderate
and severe pathological changes
with infection score ranged from 2
to 4 were in all C. parvum mice
with except in one isolate while
mild pathological changes with in-
fection score of 2 found in all in-
fected mice with C. melegridies.
Fisher-exact test showed highly sta-
tistical significa-nce between geno-
type and pathological changes
(Tab.3). Regarding to oocyst shed-
ding, in GrI no oocysts discharged,
in GrII oocysts discharged in day 5
post-infection (PI) with gradual in-
crease in number with a peak on day
9 PI. One cleared infection by day
19 P.I., two mice cleared infection
by day 13 P.I. In GrIII oocysts dis-
charged in day 5 PI in two and in
day 7 P.I. in one with a peak on day
11 P.I. and cleared by day 17 (Tab.
4).
Table 1: Prevalence of cryptosporidium genotype among human isolates.
VspI
digestion
SspI
digestion
PCR amplicon
size
Total cryptosporidium
Isolates (n=36) N %
627-104
556-104
104-171-456
111-270-449
111-270-449
111-254-449
831
837
833
C. parvum 24 66.7 .
C. hominis 10 27.7
C. melegridies 2 5.6

Table 2: Cryptosporidiosis clinical pictures and genotype in 36 patients.


Symptoms No. Diarrhea Abdominal pain Fever Vomiting Mixed
Genotype No. % No. % No. % No. % No. %
C. parvum 24 10 42 6 25 2 8 3 12.5 3 12.5
C. hominis 10 4 40 2 20 2 20 1 10 1 10
C. melegridies 2 1 50 1 50 - - -
P-value = 0.983 (not significance)
Table 3: Cryptosporidium genotypes and pathologic degree in mice.
Pathological degree C. hominis (10) C. parvum (24) C. melegridies (2)
No 10 (0)
a
0 0
Mild 0 1 (2)
a
2 (2)
a
Moderate 0 5 (3)
a
0
Severe 0 18(4)
a
0
P-value = 0.0001 (highly significance)
Table 4: Summary of oocysts shedding by mice.
Species(mouse no) Day 5 Day7 Day 9 Day 11 Day 13 Day15 Day 17 Day 19 Day21 Day23 Day25 Day 27
Group
C. hominis (1)
C. hominis (2)
C. hominis (3)
-----------------------
Group
C. parvum (4)
C. parvum (5)
C. parvum (6)
-----------------------
Group
C. melegridies (7)
C. melegridies (8)
C. melegridies (9)
- - - - - - - - - - - -
- - - - - - - - - - - -
- - - - - - - - - - - -
---------- -----------------------------------------------------------------------------------------------------
2 2 4 2 2 1 1 - - - - -
1 2 3 1 - - - - - - - -
1 3 4 1 - - - - - - - -
----------------------------------------------------------------------------------------------------------------
1 2 2 4 3 2 - - - - - -
1 1 1 3 2 1 - - - - - -
- 1 2 4 3 1 - - - - - -
*Numbers of oocysts categorized as following: - , no oocysts; 1+ < 5 oocysts per smear ; 2+, 5 to
50 oocysts per smear; 3+, 50 to 100 oocysts per smear and 4+ >100 oocysts per smear.
Fig.1: Nested PCR for Cryptosporidium spp for 18SrRNA. Lane 1 and lane11 showed positive and negative
control and lane 2,4,5,6,8,9,10 showed positive samples
M 1 2 3 4 5 6 7 8 9 10 11
800bp

Fig.2: REFLP analysis for 18SrRNA with Ssp1. lane 1-4 showed C.parvium and C.hominis and lane5-6
showed C.melegridies
Fig.3: REFLP analysis for 18SrRNA with Vsp1. Lane1-4 showed C.parvium and lane 5,6 and 8 showed
C.hominis and lane 7 showed C.melegridies.
Discussion
Cryptosporidium infect a wide
range of vertebrates, and represent a
significant cause of morbidity and
mortality. Cryptosporidiosis is a
common cause of diarrhoeal disease
with a global distribution (Caccio,
2005). Major outbreaks of cryptos-
poridiosis had been occurred in Eu-
rope and United States (Semenza
and Nichols, 2007).
In the present study, only two
samples out of 38 positive samples
were negative by PCR. Samples
were diagnosed as positive by both
kinyoun
`
s stain and immunochroma-
tografic test. This may be due to
400bp
200bp
100bp
M 1 2 3 4 5 6 M
600bp
400bp
200bp
100bp
M 1 2 3 4 5 6 7 8 M

incomplete lysis of oocysts, insuffi-


cient amount of cryptosporidial
DNA in sample or may be due to
degradation of DNA during storage
(Verweij et al., 2004). Also, pres-
ence of PCR inhibitors in stool
samples like complex polysaccha-
rides and biliribin (Menterio et al.,
1997) may play role in negative re-
sults. The possible existence of
many Cryptosporidium sp. fostered
the development of DNA tech-
niques suitable for typing isolates.
The commonly used techniques are
PCR-restriction fragment length
polymorphism analysis (Guyot et
al., 2002; Gatei et al., 2003) and
sequence analysis of taxonomically
relevant loci (Guyot et al., 2001;
Dalle et al., 2003; Soba et al., 2006;
Jex et al., 2008). However, some
PCR methods cannot differentiate
among C. meleagridis and various
genotypes of C. parvum-like para-
sites (Champliaud et al., 1998).
In present study, PCR targeting
hyper variable region of 18S rRNA
gene as 18S rRNA genes occurred
as five copies in entire cryptosporid-
ial genome (Le Blancq et al., 1997)
that reflected in the increased sensi-
tivity of the 18S rRNA-specific
PCR procedure compared to other
procedures. Also, PCR-REFLP of
18S rRNA gene proved useful in
identification of various Cryptos-
poridium genotypes correctly (Ong
et al., 2002; Chalmers et al., 2005;
Leoni et al., 2006).
Many types of Cryptosporidium
sp. were detected in feces of hu-
mans and animals (Leoni et al.,
2006; Morse et al., 2007). In the
present study, only three genotypes
of Cryptosporidium were identified
among all human clinical isolates.
C. parvum, C. hominis and C.
melegridies were detected among
24 (66.7%), 10 (27.7%) and 2
(5.6%) respectively. The present
data were similar to that reported in
Egypt (El-Hamshary et al., 2008);
Saudi Arabia (Al-Brikan et al.,
2008); Kuwait (Sulaiman et al.,
2005) and England (Goh et al.,
2004). In study of El-Hamshary et
al., (2008), both C. parvum and C.
hominis were 68.7% and 26.3% re-
spectively. Al-Brikan et al., (2008)
found that among four genotypes,
C. parvum was the commonest
(42.9%) followed by C. hominis
(37%) while C. melegridies and C.
muris were 2.9% for each one.
Sulaiman et al., (2005) and Goh et
al., (2004) reported that C. parvum
was found to be the most common
species reported in 94% and 84%
cases respectively. Caccio (2005)
and Coupe et al. (2005) reported
that the vast majority of both im-
munocompetent and immunocom-
promised patients were caused by
C. hominis and C. parvum. The
latter confirmed that infection with
other species was found in only
18.5% (13/70) of cases of PCR-
positive cryptosporidiosis. Howev-
er, Nagamani et al. (2007) found
that 29 (69.0%) were C. hominis,
eight (19.0%) were C. parvum.
Mixed infection with C. parvum and
C. hominis was in five (11.9%).
Among 85 patients, five types were

identified: C. parvum human (67),


bovine (8), and dog (2) genotypes,
C. meleagridis (7), and C. felis (1)
by Xiao et al. (2001). Palmer et al.
(2003) suggested that C. muris
might be a global emerging zoono-
tic pathogen of particular im-
portance to humans living in rodents
infested regions as well as poor san-
itation. So, absence of infected cas-
es by C. muris in present study may
be due to the fact that most of the
studied populations had no history
of direct contact with rodent.
In present study, there are no sig-
nificant differences between differ-
ent genotypes and clinical presenta-
tion in patients. This agreed with
Akiyoshi et al., (2003) who reported
that the diarrheal disease in infected
children with C. meleagridis was
indistinguishable from that in chil-
dren with acute illness due to C.
parvum. Also, Xiao et al. (2001)
stated that there was no significant
difference in age, percentage with
diarrhea, or duration of diarrhea for
episodes with human genotype,
compared with those of zoonotic
(bovine, dog, C. meleagridis & C.
felis) genotype. HIV-patients with
C. hominis, C. parvum, C. melea-
gridis showed a range of clinical
pictures of infection, including tran-
sient diarrhea, chronic diarrhea, de-
hydration, and cachexia for each
one (Xiao and Rayan, 2004).
In the present study, C. parvum
and C. meleagridis isolates were
infective to mice while C. hominis
weren't. These results were coordi-
nated with the results of Peng et al.
(1997), Widmer et al. (2000), Srter
et al. (2003) and Darabus et al.
(1997). Peng et al. (1997) reported
that genotype 2 isolates were infec-
tive to mice or calves, whereas gen-
otype 1 isolates were not. These
results support the occurrence of 2
distinct transmission cycles of
Cryptosporidium in humans. Wid-
mer et al. (2000) added that only in
vivo model that exists for this geno-
type is a gnotobiotic piglet model.
Others reported that animals infect-
ed with genotype 1 of C. parvum
are monkeys in the United States
(Spano et al., 1998) and in dugong
dugon in Australia (Morgan et al.,
2000). But, Srter et al. (2000) re-
ported that C. meleagridis was suc-
cessfully passaged in immuno-
suppressed mice. Akiyoshi et al.
(2003) proved it was infectious for
mice, rats, rabbits, and cattle and
that the ability of C. meleagridis to
infect a range of hosts, in contrast to
C. parvum type 1 in particular was
curious and not understood. But,
Srter et al. (2000) reported that
strains of Cryptosporidium differed
with respect to host specificity. So,
in vivo methods require a specific
host for infection (Sangloung et al.,
2006).
In present study, different degrees
of histopathological changes were
found among all infected mice. All
mice infected by C. parvum isolates
(except one) showed highest score
of infection with moderate and se-
vere pathological changes affecting
both small and large intestine while
all mice infected by C. meleagridis

showed score 2 with mild degree of


pathological changes and restriction
to small intestine. The results gave
highly statistical significance be-
tween genotype and pathological
degree. These results were coordi-
nated with Srter et al. (2000) who
reported that the location of C. mel-
eagridis is in small intestine. Aki-
yoshi et al. (2003) reported that mu-
cosal changes due to C. meleagridis
included a slight shortening of villi
and an irregular epithelial surface
layer and was distributed through-
out the small intestine and less dis-
tributed in the large intestine. On
the other hand, histological changes
in the intestinal epithelium due to C.
parvum as villous atrophy and crypt
hyperplasia in the lower small intes-
tine was reported (Heine et al.,
1984) or in the caecum and the co-
lon (McDonald et al., 1992). blunt-
ing of the intestinal villi, crypt hy-
perplasia and inflammation was de-
scribed (Chen et al., 2001 and
Mele et al. ,2004), fusion of villi
and eosinophilia of lamina propria
(Enemark et al., 2003), small and
large intestine mucosa severely
damaged with villous contraction
and little or absent epithelial layer
(Tzipori, et al., 1994) and evident
vacuolation due to complete cell
necrosis (Sanad and Al-Malki,
2007). Most Cryptosporidium spe-
cies infect the epithelium of the gut
but in severe infections, dissemina-
tion can occur to extra-intestinal
sites (Lopez-Velez et al., 1995).
Different genotypes show different
levels of infectivity and this varia-
bility in pathogenicity in different
hosts between Cryptosporidium
species and types may be due to the
existence of specific virulence fac-
tors among species and isolates
(Okhuysen and Chappell, 2002).
In the present study, the pattern of
oocyst shedding showed that onset
of oocyst shedding occurred on the
5
th
in GrII while in GrIII, oocyst
shedding occurred on the 5
th
in two
mice and on the 7
th
day post-
infection in one mouse. It reach a
peak on the day 9 and began to de-
cline gradually until it disappear
completely on 19
th
day P.I in one
mice of GrII and on 13
th
day PI in
other two mice with a peak on the
day 11 and began to decline gradu-
ally and disappeared completely on
17
th
day PI in GrIII. The results
agreed with Tsushima et al. (2003)
and Arafa et al. (2000). The latter
Showed that onset of oocyst shed-
ding occurred on the 3
rd
day with a
sharp rise to reach a peak on the day
9 and began to decline gradually
until it disappear completely on
15
th
day post-infection. Others re-
ported that patent infection in mice
develops in approximately 4 days.
Oocysts are shed for an average of 9
days. They were spontaneously
cleared by the third weeks of age
(Petry et al., 1995). Tsushima et al.
(2003) reported that there were no
significant differences observed in
the average number of oocysts per
gram of feces (OPG) in any cryp-
tosporidium isolates. In infected C.
melegridies and infected C. parvum
mice, the prepatent periods (3 days),

the patencies (death between 10 &


27 days PI), and the number of ex-
creted oocysts was almost identical
(Srter et al., 2000).
Conclusion
No doubt, zoonotic cryptospo-
ridiosis proved to be a serious
health problem worldwide. Further
study is a must to investigate the
application of other animals mod-
els as, pigs to develop monitoring
systems for C. parvum genotype 1.
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Explanation of Figures
Fig. 1 section of ileum showing C.parvium parasite at mucosal surface (arrow head),
intense inflammatory reaction (arrow) and sever edema (E).
Fig. 2 section of ileum showing C.parvium parasite at mucosal surface (arrow head),
inflammatory cells(arrow), moderate atrophy (decrease cryp/villi ratio) (A) and Vacuo-
lation (V).
Fig. 3: section of ileum showing C .melegridies parasite at mucosal surface (arrow
head), vacuolation (v), mild edema (M).
Fig. 4: section of jejunum showing C .melegridies parasite at mucosal surface (arrow
head), mild inflammatory infiltrate (arrow) and mild atrophy (A).

991
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 991 -1013
MOSQUITO FAUNA (DIPTERA: CULICIDAE) AND SEASONAL
ACTIVITY IN MAKKA AL MUKARRAMAH REGION, SAUDI ARABIA
By
A.M. ALAHMED, M.A. AL KURIJI, S.M. KHEIR, S.A. ALAHMEDI
3
,
M. J. AL HATABBI
4
AND M.A.M. AL GASHMARI
5
College of Food and Agricultural Sciences, king Saud University,
Riyadh, King Abdul Aziz City for Science and Technology, Riyadh ,
Ministry of Health, Jeddah, Makka Region
3
, Ministry of Health, Makka Al
Mukarrama, Makka Region
4
, and Ministry of Health,
Al Taif, Makka Region
5
, Saudi Arabia.
Abstract
From March 2004 to February 2006, a mosquito survey was conducted in
Makka Al Mukarrama Region, in the western part of Saudi Arabia, and 19 spe-
cies which belong to 4 genera, were collected: Aedes (2 species), Anopheles (8
species), Culex (8 species) and Culiseta (1 species). The mosquitoes were Ae-
des caspius, Ae. aegypti, Anopheles d'thali, An. gambiae, An. multicolor, An.
rhodesiensis, An. sergenti, An. stephensi, An. subpictus, An. turkhudi, Culex
arbieeni, Cx. laticinctus, Cx. pipiens, Cx. quinquefasciatus, Cx. sinaiticus, Cx.
tigripes, Cx. tritaeniorhynchus, Cx. univittatus and Culiseta longiareolata. Cx.
arbieeni was reported for the first time in Saudi Arabia from Al Taif District.
The physical properties of water of mosquitos larval breeding sites showed
the total dissolved salts (TDS) varied between 70-15552 ppm, pH ranged be-
tween 5.4-11.2 and water temperature varied between 15C in winter to 40.7C
in summer. There was no correlation between these physical properties and the
distribution of mosquito larvae.
Light traps collected 1858 mosquitoes, and adult Culex were the most preva-
lent as 1658 (89.24%) were collected, followed by 121 (6.51%) Aedes, 68
(3.66%) Anopheles and 11 (0.59%) Culiseta. The effects of temperature and
rainfall on seasonal abundance of mosquitoes in the study area are discussed.
Key words: Saudi Arabia, mosquitoes, Makka Al Mukarramah, light traps,
species 2 Aedes, 8 Anopheles, 8Culex and 1Culiseta, larval breeding sites.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
During the past few decades, Saudi
Arabia has witnessed substantial
efforts in social development and
urbanization in all provinces, which
have affected insect fauna, particu-
larly mosquitoes (Diptera: Cu-
991
licidae). Expansion of agricultural
projects and development of water
resources, in addition to the favora-
ble climatic conditions for mosquito
survival and development in West-
ern Region led to creation of more
mosquitoes breeding sites.
Several studies on mosquito fauna
of Saudi Arabia were carried out as
(Mattingly and Knight, 1956; Zaher,
1973; Buttiker, 1981; Will et al.,
1985; Abdullah and Merdan, 1995;
Jupp et al. 2002, Miller et al., 2002;
Abdoon and Al Shahrani, 2003;
Alahmed et al, 2007; Al Ghamdi et
al., 2008), but little on mosquito
fauna of Makka Al Mukarramah
Region.
The present work was undertaken
to study the mosquito fauna and the
seasonal dynamics of adult mosqui-
toes in Makka Al Mukarramah Re-
gion, the western part of Saudi Ara-
bia.
Material and Methods
The western coastal escarpment
can be considered two mountain
ranges separated by a gap at the vi-
cinity of Makka Al Mukarrama. The
northern range seldom exceeds 2100
meters above sea level, and the ele-
vation gradually decreases towards
the south to about 600 meters
around Makka, with some coastal
plains. The climate is hot dry in
summer and warm, slightly humid
in winter. The vegetative cover is
very poor in the study area, except
for some coastal zones. Makka Al
Mukarramah, Jeddah and Al Taif
districts were selected for the study,
because of accessibility, diverse
ecology and abundance of mosqui-
toes.
Fig. 1: Collection sites of mosquitoes in Makkah Al Mukarramah Region, Saudi Arabia.
991
Larval collection: During the peri-
od March 2004 to Feb. 2006, a
mosquito survey was conducted in
Makka Al Mukarramah district. The
study area lies between Lat. 20N-
22N and Long. 39E- 41E (Fig 1).
Weekly field trips were made to
collect mosquito larvae from all po-
tential breeding sites by a standard
mosquito larval dipper with extend-
able handle; and three to five scoops
were taken from each breeding site
(350 ml each). Larvae were extract-
ed, preserved into 80% ethyl alcohol
in glass vials with screw caps, la-
beled and sent to the Entomology
Laboratory, College of Food and
Agricultural Sciences, King Saud
University, Riyadh. Larvae were
mounted as described by R.E. Har-
bach from Natural History Museum
in London (personal Communica-
tion), and identified using standard
identification keys (Hopkins, 1952;
Mattingly and Knight, 1956; Har-
bach, 1988). Representative samples
of identified larvae were sent to the
British Natural History Museum in
London for confirmation.
In each larval breeding site, the fol-
lowing information were recorded:
coordinates of collection site, date
and time of larval collection, current
weather conditions, water tempera-
ture, pH and total dissolved salts
(TDS), degree of water turbidity and
motion, type of breeding site (e.g.
irrigation canals, rain water collec-
tions, ponds or water storage tanks)
and presence or absence of shadow,
algae or aquatic plants in the breed-
ing site.
Adult mosquitoes were collected
from Makka Al Mukarrama, Jeddah
and Al Taif using two CDC and one
standard New Jersey (NJ) light traps
(Bioquip Company, Gardena, CA,
90248-3602, USA) in each collec-
tion site. The CDC and NJ light
traps were attached to a battery that
supplies power, and installed per-
manently near suitable mosquito
breeding sites. The light traps were
operated once every two weeks
from sunset to sunrise the following
day throughout the study period.
The collected mosquitoes were
packed, labeled and transported to
the Entomology Laboratory in Ri-
yadh. Adult mosquitoes were identi-
fied using standard identification
keys of Mattingly and Knight
(1956); Harbach (1988); Glick
(1992) and recorded. Some repre-
sentative samples of identified mos-
quitoes were sent to the British Nat-
ural History museum in London for
confirmation.
Results
A total of 21467 adults and larval
mosquitoes were collected repre-
sented 4 genera and 19 species: Ae-
des (2 species), Anopheles (8 spe-
cies), Culex (8 species) and Culiseta
(1 species). They were Aedes cas-
pius, Ae. aegypti, Anopheles d'thali,
An. gambiae, An. multicolor, An.
rhodesiensis, An. sergenti, An. ste-
phensi, An. subpictus, An. turkhudi,
Culex arbieeni, Cx. laticinctus, Cx.
pipiens, Cx. quinquefasciatus, Cx.
sinaiticus, Cx. tigripes, Cx. tri-
991
taeniorhynchus, Cx. univittatus and
Culiseta longiareolata.
Among 19609 mosquito larvae
(Tab.1) Culex spp. were the most
abundant & 15133 (77.17%) larvae
were collected, followed by 2444
(12.46%) Aedes spp., 1747 (8.91%)
Anopheles spp. and 285 (1.46%)
Culiseta sp. Light traps collected
1858 adult mosquitoes in the study
area (Tab.2), and Culex were most
prevalent as 1658 (89.24%) were
collected, then 121 (6.51%) Aedes,
68 (3.66%) Anopheles, and 11
(0.59%) Culiseta. Cx. arbieeni was
encountered for the first time in this
region from Al Taif District.
Makka Al Mukarrama District: In
this site, 5299 Larvae were collected
(Tab. 4), and Culex larvae were the
most abundant, where 4310
(81.34%) were collected, followed
by 747 (14.1%) Anopheles, 191
(3.6%) Aedes and 51 (0.96%)
Culiseta. Table (5) shows collection
sites of these larvae.
A total of 751 adult mosquitoes
were collected, 231 by CDC light
traps (30.76%) and 520 by NJ light
traps (69.24%). The NJ light trap
was more efficient than CDC light
trap (P< 0.05%). Adult Culex were
the most abundant, and 718 (95.6%)
were collected, followed by 21
(2.8%) Aedes and 12 (1.6%) Anoph-
eles (Tab. 6).
Table 1: Mosquito larvae in different districts.
Total Culiseta Aedes Anopheles Culex District
5299 51 191 747 4310 Makka Al Mukarramah
12973 69 2253 131 10520 Jeddah
1337 165 0 869 303 Al Taif
19609
(100%)
285
(1.46%)
2444
(12.46%)
1747
(8.91%)
15133
(77.17%)
Total (%)
Table 2: Adult mosquito in different districts.
Total Culiseta Aedes Anopheles Culex District
751 0 21 12 718 Makka Al Mukarramah
719 0 50 1 668 Jeddah
388 11 50 55 272 Al Taif
1858
(100%)
11
(0.59%)
121
(6.51%)
68 (3.66%)
1658
(89.24%)
Total
Table 3: Water physical properties of larval sites in different districts.
TDS (ppm) pH Ambient temp (C)
District
Max. Min. Max. Min. Max. Min.
14656 70 11.2 6.4 40.7 15 Makka Al Mukarramah
15552 198 9.5 5.4 33 21.2 Jeddah
11968 365 10.9 7.5 29.5 17.8 Al Taif
991
Table 4: Different mosquito larvae collected from Makka Al Mukarrama Dis-
trict.
Date
Aedes Anopheles Culex Culiset
a
cas
pius
ae-
gypt
i
d'th
ali
gam
biae
multi-
color
rhodesi
ensis
ser-
gen-
ti
sub-
pic-
tus
lati-
cinc-
tus
pipi
ens
quin
que
fas-
cia-
tus
tritae
tae-
nio
rhyn
chus
uni-
vitta-
tus
longi-
areola-
ta
Mar
-04
0 0 19 0 0 0 0 0 0 0 0 0 0 0
Apr-
04
0 0 8 0 0 0 0 0 0 0 0 0 0 0
May
-04
0 0 2 0 0 0 0 0 0 0 0 0 0 0
Jun-
04
0 0 13 10 0 0 0 0 0 0 0 0 15 0
Jul-
04
0 16 3 0 0 13 0 0 0 9 3 0 0 0
Aug
-04
0 0 48 0 0 0 0 0 0 0 0 0 0 0
Sep-
04
0 9 37 0 0 0 0 0 0 3 0 0 5 0
Oct-
04
0 4 11 0 11 0 0 10 0 0 0 0 0 0
Nov
-04
0 2 29 0 0 0 0 6 0 13 0 0 0 0
Dec
-04
0 3 29 0 0 0 0 18 34 19 0 0 0 0
Jan-
05
0 25 53 21 0 0 0 0 44 26 0 0 0 31
Feb-
05
0 39 29 0 0 0 0 0 0 8 0 21 0 0
Mar
-05
0 23 0 0 0 0 0 0 19 11 0 0 0 0
Apr-
05
0 10 37 0 0 0 0 0 0 210 0 0 3 1
May
-05
0 25 0 0 0 0 0 61 0 53 71 0 32 0
Jun-
05
0 0 0 0 0 0 0 79 14 22 14 52 0 0
Jul-
05
0 9 0 0 0 41 0 0 0 23 0 0 14 0
Aug
-05
0 0 37 0 0 0 0 0 0 24 0 0 2 7
Sep-
05
0 0 0 0 0 0 28 0 0 98 0 0 0 0
Oct-
05
0 0 0 0 0 0 0 0 0 48 0 0 0 0
Nov
-05
0 0 0 0 0 0 0 0 13 0 14 0 0 5
Dec
-05
0 1 0 0 4 44 0 0 0 619 0 20 0 0
Jan-
06
1 16 0 0 35 5 0 0 0 663 0 6 0 0
Feb-
06
0 8 0 0 6 0 0 0 0
201
6
0 45 4 7
To-
tal
1 190 355 31 56 103 28 174 124
386
5
102 144 75 51
Table 5: Collection sites of mosquito larvae in Makka Al Mukarrama District.
Site No. Coordinates (N,E) mosquito larvae collected
1 40.12704 21.28898 Anopheles sp.
2 40.11384 21.42901 Anopheles sp.
3 39.52838 21.29252 Anopheles sp.
4 39.55112 21.29638 Anopheles sp.
5 40.12704 21.28898 Anopheles sp.
6 39.58551 21.5831 Anopheles sp.
7 39.49266 21.35609 Anopheles sp. Culex sp.
8 39.43583 21.22553 Anopheles sp
9 39.47678 21.23758 Anopheles sp.
10 39.52521 21.27073 Anopheles sp. Culex sp. Aedes sp.
11 39.5262 21.2707 Aedes sp. Culex sp.
12 39.59303 21.39725 Anopheles sp
13 39.57535 21.4193 Anopheles sp.
14 39.56138 21.34147 Anopheles sp.
991
15 40.0569 21.0745 Anopheles sp.
16 40.0569 21.0745 Anopheles sp.
17 39.42297 21.39775 Anopheles sp. Culex sp.
18 40.09948 21.21869 Anopheles sp.
19 40.09971 21.2187 Aedes sp. Culex sp.
20 39.04362 22.40935 Anopheles sp.
21 39.4312 22.32027 Anopheles sp.
22 39.50762 21.26491 Aedes sp.
23 40.01944 22.18501 Anopheles sp
24 39.47087 21.23758 Anopheles sp
25 39.52521 21.27073 Aedes sp.
26 39.47681 21.23751 Anopheles sp Culex sp.
27 39.40518 21.2559 Anopheles sp.
28 39.58945 21.23874 Culex sp.
29 39.52235 21.24505 Aedes sp.
30 39.52305 21.24709 Anopheles sp.
31 39.52231 21.21729 Culex sp.
32 39.52221 21.24729 Culex sp.
33 40.15195 21.44242 Anopheles sp. Culex sp
34 40.1535 21.4449 Anopheles sp
35 39.58945 21.23874 Culex sp
36 39.52281 21.24729 Culex sp
37 39.52281 21.26729 Aedes sp.
38 39.5262 21.27027 Aedes sp.
39 39.52235 21.24505 Aedes sp.
40 39.52709 21.24709 Culex sp.
41 39.52521 21.27073 Culex sp.
42 39.51534 21.27096 Culex sp.
43 39.52616 21.29329 Culex sp.
44 42.66973 21.49026 Anopheles sp.
45 40.08143 21.36996 Anopheles sp.
46 40.07075 21.34841 Anopheles sp.
47 40.13612 21.37001 Aedes sp.
48 40.47084 21.36833 Anopheles sp.
49 39.51378 21.25766 Aedes sp. Culex sp.
50 39.45834 21.33401 Anopheles sp. Culex sp.
51 39.48935 21.30545 Anopheles sp.
52 39.02202 21.28605 Anopheles sp. Culex sp. Aedes sp.
53 40.01534 22.15864 Culex sp.
54 39.52235 21.24505 Culex sp.
55 40.01512 21.06435 Anopheles sp. Culex sp.
56 40.30722 21.10024 Aedes sp. Culex sp.
57 39.57 21.29401 Aedes sp. Culex sp.
58 39.57055 21.2945 Anopheles sp.
59 39.5705 21.23277 Culex sp.
60 39.15722 22.09157 Culex sp.
61 39.52838 21.29252 Aedes sp.
62 39.56112 21.29638 Aedes sp. Culex sp.
63 39.50424 21.27769 Anopheles sp.
64 39.53917 21.27594 Anopheles sp. Culex sp.
65 39.53202 21.27569 Anopheles sp. Culex sp.
66 39.49232 21.27721 Culex sp.
67 39.50427 21.27662 Anopheles sp. Culex sp.
68 40.13376 21.1076 Culex sp.
69 39.52305 21.24709 Culex sp.
70 39.52235 21.24505 Anopheles sp. Culex sp.
71 39.52521 21.27073 Anopheles sp. Culex sp.
72 39.5262 21.2707 Anopheles sp. Culex sp.
73 39.47087 21.23758 Anopheles sp. Culex sp.
74 40.2141 21.47712 Anopheles sp. Culex sp.
75 40.10025 21.41379 Culex sp.
76 40.10035 21.41279 Culex sp.
991
77 40.2175 21.4776 Anopheles sp. Culex sp.
78 39.48918 21.31517 Anopheles sp. Culex sp.
79 39.48271 21.31146 Aedes sp.
80 39.48206 21.31136 Aedes sp. Culex sp.
81 39.51625 21.25984 Anopheles sp. Culex sp.
82 39.51846 21.25842 Culex sp.
83 39.51019 21.26685 Anopheles sp. Culex sp.
84 39.51711 21.26642 Culex sp.
85 39.51221 21.26732 Culex sp.
87 39.51611 21.252 Anopheles sp.
88 39.55091 21.90689 Anopheles sp. Culex sp.
89 39.55112 21.90638 Culex sp.
90 39.52281 21.24729 Culex sp.
91 39.58945 21.23874 Culex sp.
92 39.40518 21.2559 Culex sp.
Temperature has great effect on
mosquitoes activity. Mosquitoes
were collected in few numbers when
the temperature was between 25 C
-30 C, and disappeared between
July and October when the tempera-
ture was above 35C. A peak of ac-
tivity was attained in January, when
the temperature was 25C (Fig 2).
The effect of rainfall on abun-
dance of mosquitoes was variable
(Fig 3). Very low numbers of mos-
quitoes were collected during and
after rainy season, while a peak of
mosquito activity was attained in
January which is a dry month.
Jeddah District: In this site, 12973
larvae were collected (Table 7), and
Culex spp. were the most abundant
where 10520 larvae (81.09%) were
collected; followed by 2253
(17.37%) Aedes spp., then 131
(1.01%) Anopheles spp. and 69
(0.53%) Culiseta sp. Table (8)
shows different collection sites of
these larvae.
During this study, 719 adult mos-
quitoes were collected from Jeddah
District, while 328 (45.62%) were
collected by CDC light traps and
391 (54.62%) were collected by NJ
light traps. Adult Culex were most
abundant, and 668 (92.91%) were
collected, followed by 50 (6.95%)
Aedes, and only one (0.14%)
Anopheles (Tab. 9).
991
Table 6: Adult Culex collected from Makka Al Mukarrama District.
date
quinquefas-
ciatus
pipiens tritaeniorhyn-
chus
perex-
iguus
torrenti-
um
simpsoni Total To-
tal CDC NJ CD
C
N
J
CDC NJ CD
C
N
J
CD
C
N
J
CD
C
N
J
CD
C
NJ
Mar-
04
0 0 0 1 0 4 0 0 0 0 0 0 0 5 5
Apr-
04
0 1 0 0 2 2 0 0 0 0 0 3 2 6 8
May-
04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Jun-
04
0 0 0 0 0 0 0 1 0 0 0 0 0 1 1
Jul-04 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Aug-
04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Sep-
04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Oct-
04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Nov-
04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Dec-
04
0 0 0 0 0 0 0 2 0 1 0 0 0 3 3
Jan-
05
27 427 0 0 0 0 0 0 130 0 0 0 157 42
7
584
Feb-
05
0 0 0 0 0 0 0 0 22 2
6
0 0 22 26 48
Mar-
05
0 0 0 0 0 0 0 0 13 1
1
0 0 13 11 24
Apr-
05
0 0 0 0 0 0 0 0 11 0 0 0 11 0 11
May-
05
0 0 0 0 0 0 0 0 0 3 0 0 0 3 3
Jun-
05
0 0 0 0 0 0 0 0 0 4 0 0 0 4 4
Jul-05 0 0 0 0 0 0 0 0 0 2 0 0 0 2 2
Aug-
05
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Sep-
05
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Oct-
05
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Nov-
05
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Dec-
05
0 0 6 0 0 0 0 0 2 3 0 0 8 3 11
Jan-
06
6 0 0 0 0 0 0 0 0 6 0 0 6 6 12
Feb-
06
0 0 0 0 0 0 2 0 0 0 0 0 2 0 2
total 33 428 6 1 2 6 2 3 178 5
6
0 3 221 49
7
718
Cont. Table 6: Adult Aedes and Anopheles collected from Makka Al Mukarrama.
Date
Aedes Anopheles
Total
Total aegypti caspius
Ae . Total
d'thali azaniae
Total An .
CDC NJ CDC NJ CDC NJ CDC NJ CDC NJ
Mar-04 0 0 0 6 6 0 6 0 0 6 0 12 12
Apr-04 0 0 0 3 3 0 0 0 0 0 0 3 3
May-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Jun-04 6 4 0 0 10 0 0 0 1 1 6 5 11
Jul-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Aug-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Sep-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Oct-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Nov-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Dec-04 0 0 0 0 0 0 3 0 0 3 0 3 3
Jan-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Feb-05 1 0 0 0 1 0 0 0 0 0 1 0 1
Mar-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Apr-05 0 0 0 0 0 0 0 0 0 0 0 0 0
May-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Jun-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Jul-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Aug-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Sep-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Oct-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Nov-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Dec-05 1 0 0 0 1 0 0 0 0 0 1 0 1
Jan-06 0 0 0 0 0 0 0 0 0 0 0 0 0
Feb-06 0 0 0 0 0 2 0 0 0 2 2 0 2
Total 8 4 0 9 21 2 9 0 1 12 10 23 33
991
Table 7: Aedes and Anopheles larvae collected from Jeddah District.
Date
Aedes Anopheles
caspius aegypti d'thali rhodesiensis stephensi subpictus turkhudi
Mar-04 47 0 11 0 0 0 3
Apr-04 0 333 0 0 0 0 0
May-04 73 111 1 0 0 0 0
Jun-04 21 0 0 0 0 12 0
Jul-04 21 468 0 0 0 9 0
Aug-04 0 9 3 0 0 0 0
Sep-04 13 1 0 0 0 0 0
Oct-04 0 10 2 4 0 0 0
Nov-04 0 33 0 0 0 0 0
Dec-04 0 1 0 0 0 0 0
Jan-05 0 153 0 0 22 0 0
Feb-05 0 0 0 0 0 0 0
Mar-05 9 0 22 0 0 0 0
Apr-05 0 0 0 0 0 0 0
May-05 13 26 0 0 0 0 0
Jun-05 0 36 0 0 0 0 0
Jul-05 0 0 0 0 0 0 0
Aug-05 0 61 0 0 0 0 0
Sep-05 0 127 0 0 0 0 0
Oct-05 8 55 0 0 0 13 0
Nov-05 0 318 0 0 0 0 0
Dec-05 75 37 0 0 0 0 0
Jan-06 0 151 0 0 0 19 0
Feb-06 38 5 10 0 0 0 0
Total 318 1935 49 4 22 53 3
Cont Table 7: Culex and Culiseta larvae collected from Jeddah District
Date
Culex Culiseta
laticinctus pipiens quinquefasciatus sinaiticus tigripes tritaeniorhynchus univittatus longiareolata
Mar-04 11 829 0 0 0 98 11 0
Apr-04 0 890 0 0 0 0 0 0
May-04 60 441 100 0 0 0 0 0
Jun-04 0 439 0 0 0 0 0 0
Jul-04 0 787 0 0 0 0 0 0
Aug-04 0 0 0 0 0 0 0 0
Sep-04 2 0 0 0 0 0 0 0
Oct-04 0 5 0 5 0 0 0 0
Nov-04 0 0 0 0 0 0 0 0
Dec-04 249 48 0 0 0 0 0 0
Jan-05 0 125 0 0 0 0 0 4
Feb-05 0 186 66 0 0 0 0 15
Mar-05 0 126 0 0 0 942 410 12
Apr-05 119 209 0 0 0 179 338 38
May-05 0 249 0 0 4 0 44 0
Jun-05 0 862 0 0 0 0 0 0
Jul-05 161 152 100 360 0 0 0 0
Aug-05 208 40 0 38 0 20 0 0
Sep-05 0 47 0 0 0 0 0 0
Oct-05 73 0 0 0 0 0 38 0
Nov-05 0 83 0 0 0 0 0 0
Dec-05 1 75 0 0 0 165 0 0
Jan-06 12 290 0 0 0 10 0 0
Feb-06 0 686 127 0 0 0 0 0
Total 896 6569 393 403 4 1414 841 69
991
Table 8: Mosquito larvae in Jeddah District.
Mosquito larvae collected Coordinates (N, E)
Cx. sinaiticus Ae. caspius 23.18726 39.21023
Ae. caspius Cx. quinquefasciatus 21.2782 39.15194
Cx. quinquefasciatus 21.2792 39.15329
Cx. pipiens 20.37085 40.90187
Cx. pipiens 21.31898 39.10935
Cx. pipiens 21.31735 39.10133
Cx. pipiens 23.18526 39.21003
Cx. pipiens 21.2782 39.11251
Ae. aegypti Cx. laticinctus 21.2782 39.15311
Ae. aegypti 20.37055 40.90167
An. d'thali 21.31808 39.10535
An. d'thali 21.31235 39.10033
Ae. aegypti 21.31908 39.10735
Ae. aegypti 21.31902 39.1059
Ae. aegypti 21.31235 39.10033
Cx. laticinctus Ae. caspius 21.35627 39.13222
Ae. caspius 21.3275 39.15121
Ae. caspius 21.37252 39.12982
An. d'thali Ae. aegypti Cx. pipiens 21.35736 39.12135
Ae. aegypti An. rhodesiensis 21.37256 39.13222
Ae. aegypti Cx. sinaiticus An. d'thali 21.2375 39.15121
Ae. aegypti Cx. sinaiticus Cx. pipiens 21.31215 39.14141
Ae. aegypti 21.25447 39.19969
Ae. aegypti 21.28107 39.12041
Ae .aegypti 21.28467 39.12127
Ae. aegypti 21.28702 39.12295
Cx. laticinctus 21.33293 39.14024
Cx. pipiens 21.358 39.0943
Cx. pipiens 21.25446 39.19926
Ae. aegypti Cx. laticinctus 21.35873 39.15955
Cx. quinquefasciatus 22.53139 39.1595
Cx. pipiens 23.06603 39.0448
Cx. pipiens 23.07132 39.06823
Cs. longiareolata 23.10138 39.09046
Cx. pipiens 21.2729 39.15009
An. stephensi Cs. longiareolata 23.04266 39.4081
Cx. pipiens 21.3089 39.08183
Ae. aegypti 21.37781 39.09396
Cx. pipiens 21.28062 39.2121
Cx. univittatus 21.24988 39.13006
Cx. pipiens 20.0937 40.18663
Cx. tritaeniorhynchus 21.28064 39.21219
Ae. . aegypti An. d'thali 21.40078 39.4509
Cs. longiareolata 21.27806 39.15073
Cx. pipiens 23.11004 38.51285
Cx. univittatus 22.56994 39.38218
An. d'thali 21.56705 39.0763
Ae. aegypti 21.3286 39.13332
Cx. pipiens 21.33854 39.11943
Ae. aegypti 21.37661 39.10542
Cx. pipiens 21.36761 39.09991
Ae. aegypti 21.28655 39.11846
Ae. aegypti 21.27386 39.11892
Cx. pipiens 21.279 39.14969
Ae. aegypti 21.2975 39.17773
Cx. pipiens 21.30741 39.18928
Ae. aegypti 22.44861 39.0242
Cx. pipiens 21.26943 39.19191
Ae. aegypti 21.26909 39.11745
991
Cx. pipiens 21.356 39.06985
Ae. aegypti 21.31535 39.09318
Cx. pipiens 21.29722 39.10721
Ae. aegypti 21.36392 39.08548
Cx. pipiens 21.37944 39.11677
Ae. aegypti 21.34331 39.13127
Ae. aegypti 21.27593 39.15588
An. d'thali 21.3572 39.12924
Ae. aegypti 20.44956 40.32218
Cx. univittatus 20.21079 40.54446
Ae. aegypti 21.32843 39.11985
Ae. aegypti 21.26996 39.17355
Ae. aegypti Cx. pipiens 21.25451 39.19983
Ae. aegypti 19.58463 43.47791
Ae. aegypti 20.2719 40.43934
Ae. aegypti 21.40421 39.07239
Ae. aegypti Cx. pipiens 21.25503 39.13652
Ae. aegypti Cx. pipiens 21.29047 39.0981
Ae. aegypti 21.32597 39.14451
Ae. caspius 20.27661 40.28201
Cx. laticinctus Ae. caspius 20.29134 40.43066
Cx. tritaeniorhynchus 21.24618 39.12793
Ae. aegypti 21.37167 39.11444
Cx. pipiens 21.27552 39.10593
Ae. aegypti Cx. pipiens 21.36569 39.12556
Ae. aegypti Cx. pipiens 21.47379 39.07878
Ae. aegypti 21.332 39.1388
Cx. laticinctus An. subpictus Cx. tritaeniorhynchus 22.47079 39.20769
Ae. aegypti An. subpictus Cx. pipiens 22.48942 39.28421
Cont. Table (8): Mosquito larvae collected from Jeddah District
Ae. aegypti Cx. quinquefasciatus 21.30841 39.10321
Ae. caspius 21.26919 39.11845
Cx. pipiens 21.3821 39.1413
Ae. caspius An. d'thali Cx. quinquefasciatus 20.20143 39.51371
Ae. caspius Cx. pipiens Cx. quinquefasciatus 20.20153 39.61381
Cx. pipiens 20.20159 39.61389
Cx. pipiens 21 3803 391701
Ae. caspius Cx. pipiens Cx. tritaeniorhynchus 212233 391337
Cx. pipiens 212351 391500
Cx. laticinctus Cx. pipiens Cx. univittatus 213759 391408
An. d'thali Cx. pipiens Cx. tritaeniorhynchus 2135873 3915955
Cx. pipiens 2128583 3915812
Ae. aegypti 2128735 3914616
Cx. pipiens 2126661 3914290
Ae. aegypti Cx. pipiens 21.26919 39.11844
Ae. aegypti Cx. pipiens 2123518 391501
An. d'thali Cx. laticinctus Cx. quinquefasciatus 213433 3913092
Cx. pipiens 2129223 391044
Cx. pipiens 2132543 3909244
Cx. pipiens Ae. caspius Cx. laticinctus 2127117 3918254
Ae. aegypti Cx. pipiens 2137272 3910126
Ae. aegypti Cx. pipiens 2138274 391331
Cx. pipiens 2128249 3915432
An. subpictus Cx. pipiens 2123597 3915543
Cx. pipiens 2126048 3916531
Cx. pipiens 2122270 3913338
Cx. pipiens Ae. caspius 2149223 3927901
Cx. pipiens Ae. caspius 2133587 3914112
Ae. aegypti Cx. pipiens 2139142 3962670
Cx. pipiens 2131132 3911372
Cx. pipiens Ae. aegypti 2135187 3982830
Ae. aegypti Cx. pipiens 2148392 3922190
991
Ae. aegypti Cx. pipiens Ae. caspius 2131548 3910363
Ae. aegypti Cx. pipiens 2148466 3913593
Ae. aegypti Cx. pipiens 2128266 3911572
Cx. pipiens 2148327 3908186
Ae. aegypti Cx. pipiens 2129220 3913205
Cx. pipiens 213075 3914163
Ae. aegypti Cx. pipiens 213529 3908441
Table 9: Adult Aedes and Anopheles mosquitoes from Jeddah District.
Date
Aedes Anopheles
aegypti caspius
Total Aedes
d'thali
Total Anopheles .
CDC NJ CDC NJ CDC NJ
Mar-04 0 0 0 0 0 0 0 0
Apr-04 0 0 0 0 0 0 0 0
May-04 0 0 0 0 0 0 0 0
Jun-04 0 0 0 0 0 0 0 0
Jul-04 0 0 0 0 0 0 0 0
Aug-04 0 0 0 0 0 0 0 0
Sep-04 0 0 0 0 0 0 0 0
Oct-04 0 0 0 0 0 0 0 0
Nov-04 0 0 0 0 0 0 1 1
Dec-04 0 0 0 0 0 0 0 0
Jan-05 0 0 0 0 0 0 0 0
Feb-05 0 0 0 0 0 0 0 0
Mar-05 0 0 0 0 0 0 0 0
Apr-05 0 0 0 0 0 0 0 0
May-05 0 2 0 0 2 0 0 0
Jun-05 3 0 0 0 3 0 0 0
Jul-05 0 0 0 1 1 0 0 0
Aug-05 0 0 0 0 0 0 0 0
Sep-05 0 0 0 0 0 0 0 0
Oct-05 0 0 0 0 0 0 0 0
Nov-05 12 13 1 0 26 0 0 0
Dec-05 11 0 0 0 11 0 0 0
Jan-06 6 0 1 0 7 0 0 0
Feb-06 0 0 0 0 0 0 0 0
Total 32 15 2 1 50 0 1 1
991
Cont Table 9: Adult Culex mosquitoes collected from Jeddah District
Date
Culex
quinquefasciatus tritaeniorhynchus torrentium perexiguus pipiens Total
Total
CDC NJ CDC NJ CDC NJ CDC NJ CDC NJ CDC NJ
Mar-04 0 0 4 0 0 0 0 0 0 0 4 0 4
Apr-04 0 0 4 0 0 0 0 0 0 0 4 0 4
May-04 0 0 0 4 0 0 0 0 0 0 0 4 4
Jun-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Jul-04 0 0 0 3 0 0 0 0 0 0 0 3 3
Aug-04 0 0 2 0 0 0 0 0 0 0 2 0 2
Sep-04 0 0 8 0 0 0 0 0 0 0 8 0 8
Oct-04 0 0 0 0 0 0 0 0 0 0 0 0 0
Nov-04 0 0 27 8 0 9 0 0 0 0 27 17 44
Dec-04 0 13 40 14 30 2 0 0 0 0 70 29 99
Jan-05 0 0 0 11 36 8 0 0 0 0 36 19 55
Feb-05 0 0 0 0 2 3 0 0 0 0 2 3 5
Mar-05 0 0 0 10 17 2 0 0 0 3 17 15 32
Apr-05 0 0 1 0 15 0 0 0 0 0 16 0 16
May-05 1 8 2 2 0 0 0 0 0 0 3 10 13
Jun-05 0 0 17 164 0 0 9 0 0 0 26 164 190
Jul-05 0 0 3 6 0 1 0 0 0 0 3 7 10
Aug-05 0 0 0 0 0 0 0 0 0 0 0 0 0
Sep-05 0 0 0 4 0 13 0 0 0 0 0 17 17
Oct-05 0 3 3 0 4 0 0 0 0 0 7 3 10
Nov-05 0 0 3 1 3 0 0 0 0 0 6 1 7
Dec-05 0 0 15 57 20 17 0 0 0 0 35 74 109
Jan-06 0 0 0 8 11 0 0 0 0 0 11 8 19
Feb-06 0 0 9 0 8 0 0 0 0 0 17 0 17
Total 1 24 138 292 146 55 9 0 0 3 294 374 668
991
Table 11: Sites of mosquito larvae from Al Taif District
Coordinates (N, E) Mosquito larvae collected
40.17202 21.01426 Cx. univittatus
41.15758 20.4495 An. stephensi An. stephensi Cx. univittatus
40.18139 21.08865 An. stephensi
41.16734 20.43926 Cx. quinquefasciatus Cs. longiareolata
41.16734 20.43926 An. stephensi
41.2627 22.30101 An. d'thali
41.0905 20.45849 An. stephensi Cx. univittatus
41.8159 21.8865 Cx. arbieeni
40.6671 22.096 Cs. longiareolata
41.06824 20.27209 Cx. tigripes Cs. longiareolata An. stephensi
40.96788 21.25021 Cx. univittatus
40.17249 21.25373 Cs. longiareolata
40.16063 21.20094 Cs. longiareolata
40.6672 21.20943 Cs. longiareolata
40.17202 21.01426 An. stephensi
41.17282 21.25042 An. stephensi
41.905 20.45894 Cs. longiareolata An. stephensi
40.08675 20.5092 Cx. tritaeniorhynchus An. subpictus
40.17256 21.2144 An. d'thali An. stephensi Cx. univittatus
40.57243 20.41963 Cx. tritaeniorhynchus An. stephensi
40.1964 21.95932 Cx. pipiens An. stephensi
40.26387 21.40315 An. subpictus
40.26867 21.11713 An. d'thali Cx. univittatus An. turkhudi
40.00656 20.58022 An. d'thali Cx. univittatus
40.27446 21.19623 An. d'thali
41.18498 20.38349 An. d'thali Cx. univittatus
40.26387 21.40315 An. turkhudi Cx. arbieeni
40.26867 21.11713 An. turkhudi
40.00656 20.58022 An. turkhudi
40.27446 21.19623 Cx. pipiens
41.18498 20.38349 An. turkhudi Cx. pipiens
40.1964 21.95932 Cx. pipiens An. turkhudi
40.66722 21.20944 An. d'thali
991
0
100
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400
M
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m
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l
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c
t
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d
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20
30
40
M
e
a
n

t
e
m
p
.

(

C
)
No. of mosquitoes Mean temp.
Fig . 2: Effect of temperature on seasonal abundance of mosquito in Makka al Mukarrama District.
0
100
200
300
400
M
a
r
-
0
5
A
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J
a
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F
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6
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m
o
s
q
u
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o
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c
o
l
l
e
c
t
e
d
0
50
100
M
e
a
n

r
a
i
n
f
a
l
l

(
m
m
)
No. of mosquitoes
Mean rainfall
Fig. 3: Effect of rainfall on seasonal abundance of mosquito in Makka Al Mukarrama District.
Fig. 4: Effect of temperature on seasonal abundance of mosquitoes in Jeddah District.
0
20
40
60
80
100
120
M
a
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a
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F
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N
o
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o
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c
o
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30
40
M
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No. of mosquitoes Mean temp.
991
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40
60
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s
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10
20
30
40
50
M
e
a
n

r
a
i
n
f
a
l
l

(
m
m
)
No. of mosquitoes Mean rainfall
Fig. 5: Effect of rainfall on seasonal abundance of mosquitoes in Jeddah District.
Mosquitoes were collected
throughout the year, but at different
densities depending on the prevail-
ing climatic conditions. Tempera-
ture has no significant effect on
mosquito activity, because the max-
imum and minimum temperature
varied between 25C and 35C,
which was a favorable temperature
range for mosquito activity. Two
peaks were observed, in June and
December when the temperature
was 31C and 35C respectively.
The rainfall in March significantly
increased the number of mosquitoes
collected, and a peak was attained in
June. A second peak was observed
in December, but this peak might be
due to humid winds blowing from
the Red Sea during this time.
Al Taif District: In this site, 1337
mosquito larvae were collected
(Tab.10). Anopheles larvae were the
most abundant 869 (65%) followed
by 303 (22.66%) Culex, 165
(12.34%) Culiseta. Cx. arbieeni was
encountered for the first time in
Saudi Arabia from Al Taif (Tab.
11).
In this study, 388 adult mosqui-
toes were collected from Al Taif
District, 250 (64.43%) were collect-
ed by CDC light traps and 138
(35.57%) by NJ traps. Culex were
the most abundant, 272 (70.1%)
followed by 55 (14.17%) Anophe-
les, 50 (12.89%) Aedes and 11
(2.84%) Culiseta (Tab. 12).
The maximum and minimum
temperatures in this site varied be-
tween 17C and 30C which is a
favorable range of temperature for
mosquito activity. The number of
mosquitoes increased when the
temperature was about 20 C - 25
C, and a peak was attained in May
(Fig 6).
The number of mosquitoes in-
creased during the rainy season
(March- September). Due to the
high rainfall in April, a peak of
mosquito activity was attained in
May.
991
0
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5
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15
20
25
30
35
M
e
a
n

t
e
m
p
.

(

C
)
No. of mosquitoes
Mean temp.
Fig. 6: Effect of temperature on seasonal abundance of mosquitoes in Al Taif District.
0
10
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50
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o
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m
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c
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e
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20
40
60
80
M
e
a
n

r
a
i
n
f
a
l
l

(
m
m
)
No. of mosquitoes Mean rainfall
Fig .7: Effect of rainfall on seasonal abundance of mosquitoes in Al Taif District.
Discussion
The presence of 19 species of
mosquitoes in this region constitutes
a serious health problem, and fur-
ther studies on their ecology and
biology are required before embark-
ing on large scale control projects.
In this study, Culex larvae were
the most abundant (77.17%), and
were collected from various habi-
tats. The widespread of Culex larvae
might be due to the fact that they
can exploit a wide variety of aquatic
habitats for their development and
survival, and can tolerate highly
polluted aquatic environment and
relatively saline water. In fact, be-
side water quality, there are some
other factors which determine the
suitability of various types of aquat-
ic habitats for mosquitoes, like pres-
ence or absence of shade and aquat-
ic vegetations and degree of water
motion and turbidity.
In this study, Cx. arbieeni was
reported for the first time in this
991
region from Al Taif District. The
medical importance and vectorial
capacity of this species requires fur-
ther investigations.
During this survey, Cx. tri-
taeniorhynchus, the main vector of
Rift Valley fever virus in southern
part of Saudi Arabia (Miller et al
2002; Jupp et al 2002) and Ae. ae-
gypti, the main vector of yellow fe-
ver virus, were encountered. The
presence of these two mosquito vec-
tors constitutes a great health prob-
lem, and every effort should be
made to control them so as to pre-
vent the spread of Rift Valley fever
and yellow fever viruses in this re-
gion
Culiseta longiareolata was also re-
ported in this region, but in few
numbers. Adults of this species nev-
er enter houses and rarely bite man
(Salit et al 1994), so this species
appears to be of no medical im-
portance, but its larvae may be can-
nibalistic and prey on aquatic in-
sects and tadpoles (Blaustein and
Margalit, 1994).
In this study, adult mosquitoes
were collected from different parts
of the region, but in different densi-
ties. In fact, the climate in this re-
gion is conducive for development
and survival of mosquitoes, which
makes the control programs very
difficult. Agricultural expansions
and new irrigation canals, pools and
dams and extensive farming may
also may help in wide spread and
occurrence of mosquito larvae
(Sebai et al., 1974).
In general, the numbers of adult and
larvae of mosquito collected was
very low, this might be due to regu-
lar and extensive adulticides and
larvicides applications to control
malaria in the region.
In this study, some species of
Anopheles were collected as larvae
but not as adults, like An. stephensi,
An. subpictus, and An. turkhudi, and
in very few numbers. This might be
due to some differences in the adult
behavior, suggesting that CDC and
NJ light traps are not suitable for
collection of Anopheles mosquitoes.
This is true, because most of malaria
vectors are indoors feeders and they
do not come near light traps which
were placed outside houses. Since
malaria is an endemic disease in
Makka Al Mukarramah Region, the
use of more efficient methods for
sampling Anopheles mosquitoes like
spray sheet method is a must.
Acknowledgement
The financial support from King
Abdul Aziz City for Science and
Technology was highly appreciated.
Thanks are extended to Dr. Ralph
Harbach (Natural History Museum,
London) for confirming the identifi-
cation of the mosquitoes.
References
Abdoon, A.M.M.O.; Alshahrani,
A.M., 2003: Prevalence and distri-
bution of Anopheline mosquitoes in
malaria endemic areas of Asir Re-
gion, Saudi Arabia. East. Mediter.
Hlth. J., 9, 3:240-7.
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Abdullah, M.A.; Merdan, A.I.,
1995: Distribution and ecology of
mosquito fauna in the southwestern
Saudi Arabia. J. Egypt. Soc. Parasi-
tol, 25, 3:815-37.
Alahmed, A.M.; Al Khereiji,
M.A.; Kheir, S.M., 2007: Distribu-
tion and habitat of mosquito larvae
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Al Ghamdi, K.; Alikhan, M.;
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Buttiker, W., 1981: Observation on
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Glick, J.I., 1992: Illustrated key to
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Harbach, R.E., 1988: The mosqui-
toes of the subgenus Culex in South-
western Asia and Egypt (Diptera:
Culicidae). Contribution: Amer.
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onomy of Culicinae Larvae. Lon-
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tees.
Jupp, P.G.; Kemp, A.;
Grobbelaar, A.; Leman, P.; Burt,
F.J.; Alah-med, A.M.; et al., 2002:
The 2000 epidemic of Rift Valley
fever in Saudi Arabia: mosquito
vector studies. Med. Vet. Entomol.,
16:245-52.
Mattingly, P.F.; Knight, K.L.,
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British Museum (Natural History)
Ent., 4, 3:89-141.
Miller, B.R.; Godsey, M.S.; Crab-
tree, M.B.; Savage, H.M.; Al-
Mazrao, Y.; Al-Jeffri, M.H.; et al.,
2002: Isolation and Genetic Charac-
terization of Rift Valley Fever Virus
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Kingdom of Saudi Arabia. Emer.
Infect. Dis., 8, 12:23.
Salit, A.M.; Zakaria, M.; Balba,
M.; Zaghloul, T., 1994: The mos-
quito fauna of Kuwait. J. Kuwait
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Sebai, Z.A.; Morsy, T.A.;
Zawahry, M., 1974: A preliminary
study on filariasis in Western part of
Saudi Arabia. Castellania Tropen-
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12:263-6, Acron Verlag, Berlin.
Wills, W.M.; Jakob, W.L.; Fran-
cy, D.B.; Oertley, R.E.; Anani, E.;
et al., 1985: Sindbis virus isolations
from Saudi Arabian mosquitoes.
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79:63-6
Zaher, A.R., 1973: Review of
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Eastern Mediterranean Region,
WHO report, Geneva.
1015
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 1015 - 1032
FOOD ADDITIVES AND HYMENOLEPIS NANA INFECTION: AN EX-
PERIMENTAL STUDY
By
KHOLOUD A. EL-NOUBY
1
, HALA E. HAMOUDA
2
, MONA A. ABD EL-
AZEEM
3
AND AHMAD A. EL-EBIARY
4
Departments of Parasitology
1
, Medical Biochemistry
2
, Pathology
3
and Forensic
Medicine and Clinical Toxicology
4
, Faculty of Medicine, Tanta University
Abstract
The effect of sodium benzoate (SB) on the pathogenesis of Hymenolepis
nana (H. nana) and its neurological manifestations was studied in the present
work. One hundred and thirty five mice were classified into three groups. GI:
received SB alone, GII: received SB before & after infection with H. nana and
GIII: infected with H. nana. All groups were subjected to parasitological, his-
topathological, immunohistochemical and biochemical assays. The results re-
vealed a significant decrease in IL-4 serum level with a significant increase in
gamma amino butyric acid (GABA) and decrease in zinc brain levels in GI,
while GII showed non significant increase in IL-4 level that resulted in a highly
significant increase in the mean number of cysticercoids and adult worms with
delayed expulsion as compared to GIII. This was reflected on histopathological
and immunohistochemical changes in the brain. Also, there was a highly sig-
nificant increase in GABA and decrease in zinc brain levels in GII to the de-
gree that induced behavioral changes. This emphasizes the possible synergistic
effect of SB on the neurological manifestations of H. nana and could, in part,
explain the increased incidence of behavioral changes in children exposed to
high doses of SB and unfortunately have H. nana infection.
Key words: Sodium benzoate, Hymenolepis nana, IL-4, gamma amino butyric
acid & zinc.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Food additives are described by
the United States Food Labeling
Regulations in 1984 as substances
added or used in small amounts with
food at any stage to preserve flavor,
improve taste, enhance appearance,
improve the nutrient value or pre-
vent damage of food. Salt, sugar and
vinegar were among the first addi-
tives that were used to preserve
food. With the advent of processed
foods in the second half of the 20
th
century, many more additives were
introduced, of both natural and arti-

ficial origin (Boca Raton and


Smoley, 1993).
Food preservatives may be anti-
microbial preservatives inhibiting
the growth of bacteria and fungi,
such as calcium propionate, sodium
benzoate, sodium nitrate, sodium
nitrite, disodium EDTA etc., or an-
tioxidants inhibiting the oxidation of
food constituents such as oxygen
absorbers (Schoenthaler et al.,
1986). The benefits and safety of
artificial food additives was the sub-
ject of debate among academics and
regulators specialized in food sci-
ence and toxicology (Richard and
Pressinger, 1997; Schlosser, 2001).
However, with the advent of pro-
cessed foods, there was a massive
explosion in the chemical adultera-
tion of foods with additives. Con-
siderable controversy has been as-
sociated with the potential threats
and possible benefits of food addi-
tives (Bateman et al., 2004). They
were linked with cancer, digestive
problems and neurological condi-
tions such as attention-deficit hy-
peractivity disorder (ADHD), as
well as heart disease, obesity (Wal-
lis, 2007). Also, food colors, sulfites
and sodium benzoate included in
additives can stimulate allergic re-
sponses and decrease immunity
(McCann et al., 2007).
Benzoic acid and sodium benzoate
were recognized as safe in foods
(United States Food and Drug Ad-
ministration) used as preservatives
for fruit juice, carbonated drinks and
pickles to prevent micro-organisms
growth in acidic foods (Nettis et al.,
2004). The substances naturally oc-
curring in many plants appeared to
be safe for most people, though they
cause asthma, or other allergic reac-
tions in sensitive individuals (Asero,
2006). Sodium benzoate adversely
affected its behavior, especially in
children with ADHD (Bateman et
al., 2004; McCann et al., 2007).
Intestinal parasitic infections are
one of the serious health problems
worldwide especially in developing
countries (Long et al., 2007) as
Saudi Arabia (Shoura and Morsy,
1974), Jordan (Morsy and El
Maridi, 1978) and Egypt (El Naggar
et al., 1978; El-Sobky and El-
Mazar, 2004; El Shazly et al.,
2006a). (Massoud et al. (2007) re-
ported that H. nana was the com-
monest worm infecting the Egyptian
children. The infections are results
of the related inter-social, economic
and cultural factors (Quihui et al.,
2006).
Hymenolepis nana (H. nana) is a
very common infective parasite. An
estimation of 20-50 million persons
were infected worldwide. The rates
of infection are higher in the urban
areas than in rural areas (Lucas et
al., 1979). The rates of H. nana in-
fection were high in institutions as
the day-care centers, orphanages,
the institutions for the mentally im-
paired, outbreaks in some of the
children homes were up to 60%
(Wilson, 1991).
Children are the most vulnerable
to infection due to lack of hygiene
and bad food habits (Mirdha and
Samantary, 2002). It has a faeco-

oral mode of infection due to imme-


diate eggs infectivity favoring auto-
infection and hyper-infection espe-
cially in children as well as devel-
opmenting disabled, psychia-tric
and immunosuppressed patients.
In adults, infection is usually self
limited. In children with moderate
parasite burden, there were loss of
appetite, diarrhea, abdominal pain,
dizziness, headache and localized
sensitivity to touch (Bogitsh et al.,
2005).
In hyperinfection, severe enteritis
with the possible dissemination to
the blood reaching far organs as the
liver was reported (Olson et al.,
2003). None of the gastrointestinal
symptoms were encountered as skin
erythematous eruption, pruritus and
some neurological manifestations
(Markell et al., 1999; Di Lernia et
al., 2004).
A great deal of interest has been
focused on the relationship between
cytokine response of the host and
both protective and pathological
response to parasitic infections
(Abou-Holw, 2005). The response
of the host to intestinal parasitic
infections represents a complex in-
teraction between non specific in-
flammatory mechanisms and specif-
ic immunologically adaptive events.
The type of effector mechanisms
involved depends on the type of
enteric organism (Else and Finkel-
man, 1998).
H. nana is a highly immunogenic
intestinal tapeworm. After a single
oral inoculation of H. nana eggs,
mice acquired a strong protective
immunity to re-infection, which is
proved to be T cell dependent
(Bentwich et al., 1996; Asano et al.,
1998). The T helper cells (T
h
) medi-
ate these protective immune re-
sponses through production of cyto-
kines (Palmas et al., 1997). T
h
1 cy-
tokines (INF-, IL-2) promote de-
fensive reactions mediated by mac-
rophages and other phagocytic cells,
while T
h
2 cytokines (IL-4, IL-5)
promote production & activation of
eosinophils, mastocytosis and in-
crease serum IgE, the hallmark of
active helminthic infection (Udha-
yakumar et al., 1995).
Human cerebral cortical activity
may be influenced by a large num-
ber of neuroactive substances, in-
cluding monoamine neurotransmit-
ter as gamma amino butyric acid
(GABA) and norepinephrine (NE)
and some trace elements as zinc
(Zn). The substances are important
for the normal integrity and function
of the central nervous system (CNS)
which controls a variety of physio-
logical, behavioral and endocrinal
functions (Greengard, 2001). Gam-
ma amino butyric acid acts as the
major inhibitory neurotransmitter in
CNS. GABA mechanisms of brain
function has revolved around its
central role in convulsive disorders
(Kleppner and Tobin, 2001); as
pharmacological manipulations or
any condition that decreases brain
GABA invariably produce convul-
sive disorders (Staley et al., 1995).
Zinc is an essential trace element for
all life forms, and one of neuro-
transmitters contributing too many

important biological processes in-


cluding gene expression, DNA syn-
thesis, enzymatic catalysis, hormo-
nal storage and memorization (Val-
lee and Auld, 1993).
The present work aimed to study
the effect of sodium benzoate on the
pathogenesis of H. nana infection
and its neurological manifestations
in experimental mice. This was as-
sessed by parasitological, histo-
pathological and immunohistochem-
ical examinations of brain tissue,
and biochemical estimation of the
serum level of IL-4 as well as the
brain tissue levels of the GABA and
Zn of all groups was done.
Materials and Methods
Hymenolepis nana was main-
tained in mice in the laboratory of
Parasitology Department, Tanta
Faculty of Medicine. Eggs were
collected from egg donor mice sac-
rificed 4-6 weeks post infection
(P.I.). Infective shell-free eggs were
prepared from gravid segments of
adult worms by stirring egg suspen-
sion with glass beads just before use
(Ito et al., 1991).
One hundred and fifty, parasite
free, male Swiss albino mice (20-25
gm in weight and 6-8 weeks old)
were used. Fifteen mice were served
as a healthy control group. They
were sacrificed at one time for com-
parison. The remaining 135 mice
were subdivided into three groups.
GI: (SB-non infected) included 45
mice that received a diet containing
2% of sodium benzoate (SB) 10
days before and continued through-
out the experiment (Fujian, 1993).
GII: (SB-infected) included 45 mice
that received a diet containing 2%
SB for 10 days before infection with
H. nana and continued throughout
the experiment. GIII: (Infected) in-
cluded 45 mice infected with H.
nana and did not received sodium
benzoate. Mice of Gs II & III were
orally infected with a viable 500
shell-free H. nana eggs/mouse.
Every group was divided into
three subgroups randomly according
to the periods of infection, 15 mice
in each, and sacrificed at five days,
one month and two months P.I. Sera
were collected and kept at -20
o
C
until used for estimation of IL-4.
The small intestines of GII & GIII
mice were removed and subjected to
the following: The upper 10cm of
small intestine of each mouse was
dissected, removed and processed
for counting cysticercoids in the
intestinal wall five days P.I. and
collection with counting of adult H.
nana one and two months P.I. The
remaining parts of the small intesti-
nal tissues were fixed in 10% neu-
tral formalin and embedded in par-
affin for histopathological examina-
tion by haematoxylin and eosin
stain (H&E).
The brains were dissected out and
divided into two parts. One part was
preserved in 10% formalin for his-
topathological examination and
immuno-histochemical examination
for the antigenic deposition by pe-
roxidase-antiperoxidase (PAP)
method. The other part was pro-

cessed for biochemical assay. For


determination of GABA levels:
brain tissue sample was immediate-
ly transferred to a 0.01 mol/L acetic
acid solution containing Na
2
S
2
O
5
(Sodium metabisulfite) and EDTA
at a final concentration of 100g/L
each. After homogenization, sam-
ples were stored at -20
o
C. Analysis
of the supernatants of tissue homog-
enates for GABA was performed
within one month after collection
(Kema et al., 1994). For determina-
tion of Zn levels about 250 mg of
brain tissue were dry mineralized at
450
o
C in an electric oven. After ash-
ing, samples were dissolved in 10ml
of 1M HNO
3
(Brzoska et al., 2002).
Cysticercoids recovery and count-
ing in small intestinal wall: The up-
per 10 cm of the small intestine was
removed and opened longitudinally,
washed in tap water and cut into
2cm pieces. Five randomized pieces
were used for counting cysticercoids
in 10cm of the small intestines
(Friedberg et al., 1979). The other
pieces were preserved in formalin
for histopathological examination.
Adult counting in small intestinal
lumen: The upper 10cm of the small
intestine was removed and opened
lengthwise carefully to avoid injury
of the adults; they were transferred
to a large Petri dish containing
0.85% saline and placed in an incu-
bator at 37
o
C for 30 minutes to
stimulate the worm movement. Ste-
reoscopic examination was per-
formed for counting the adults
(Henderson and Hanna, 1979).
Histopathological examination:
Small intestine and brain tissue
specimens collected from mice of
Gs II & III were fixed in formalin,
embedded in paraffin blocks, sec-
tions (5m thickness) and H&E
stained (Drury and Wallington,
1980).
Immunohistochemical staining of
antigenic deposition in brain tissue:
Formalinfixed, paraffin-embedded
5m brain sections were stained
with PAP technique using chronic
H. nana infected serum as antibody
preparation to specific H. nana anti-
gen in brain tissues. Sections were
incubated at room temperature for
an hour with the antibodies. The
immunoreactions developed with
diaminobenzidine (DAB; Dako) for
5 min, and counterstained with he-
matoxylin.
Preparation of chronic H. nana
infected sera (Hillyer et al., 1979):
Adults were obtained from the small
intestines of infected mice. They
were homogenized in PBS and cen-
trifuged at 500g for 10 minutes. The
supernatant was ultra centrifuged at
100.000g for an hour at 4
o
C. Protein
content of the supernatant was esti-
mated after Bradford (1976). Im-
munization of two adult male (New-
Zeland) rabbits by H. nana antigen
was done (Welch et al., 1983). Rab-
bits were bled and sera were re-
tained as an anti-H. nana antibodies.
Quantitative determination of se-
rum IL-4 by ELISA using Ray
Bio

: Mouse IL-4 ELISA kit is an


in vitro ELISA for the quantitative

measurement of mouse IL-4 in se-


rum using an antibody specific for
mouse IL-4 coated on a 96-well
plate. Standards and samples were
pipetted into the wells and IL-4 pre-
sent in a sample was bound to the
wells by immobilized antibody. The
wells were washed and biotinylated
anti-mouse IL-4 antibody was add-
ed. After washing away unbound
biotinylated antibody, HRP-
conjugated streptavidin was pipetted
to the wells. The wells were again
washed, a TMB substrate solution
was added to the wells and color
developed in proportion to the
amount of IL-4 bound. Stop solution
changed the color from blue to yel-
low and the color intensity was
measured at 450nm (Boulay and
Paul, 1992).
GABA levels were determined by
the reverse-phase high-performance
liquid chromatography (HPLC) with
fluorescence detection after deriva-
tion with 0-phthaldialdehyde (OPA)
after (Kamisaki et al., 1990).
Zn was measured by an atomic
absorption spectrophotometery
(mode l2380; Perkin Elmer), by a
dry ashing tissue samples. Working
standards of Zn containing 0.2, 0.4,
0.6, 0.8 & 1.0 g/ml were prepared
from stock atomic absorption stand-
ard solutions containing 1000g
Zn/ml. The instrument is equipped
with a zinc hollow-cathode lamp, a
hydrogen lamp, a 10-cm air acety-
lene burner, and an adjustable tanta-
lum nebulizer. It was optimized to
minimize background absorption.
The operation conditions were hol-
low-cathode lamp current 7mA,
monochromator slit width 0.5nm,
and acetylene gas flow 2.5 units (2.2
L/min). Zn absorbance was deter-
mined at 213.9 nm. The samples
and standard solutions were injected
into the cone and absorbance values
standard curve was calculated. Ab-
sorbance values for samples were
registered and the actual Zn concen-
trations were calculated from the
standard curve (Palm et al., 1983).
Statistical analysis: Data were ex-
pressed as meanSD. Differences
between groups were analyzed and
compared for significance at
P<0.05* & P<0.001** by students
t test and variance (ANOVA) using
SPSS program, version 13.
Results
There was a highly significant
increase (tab. 1) in number of cysti-
cercoids and adults (P<0.001) with
delayed expulsion in GII (sodium
benzoate (SB) 10 days before infec-
tion and onwards) compared to GIII
(infected without SB).
Small intestinal section of GII
showed high infestation with cysti-
cercoids at villi base (Fig. 1). The
adjacent villi were blunt and fused
together. Some sections showed
submuocosal edema with mucosal
damage and inflammatory infiltrates
(Fig. 2). Others showed adults with
increased lymphocytic infiltration in
the lamina propria (Fig. 3).
Sections of GIII showed the nor-
mal architecture with a relatively
fewer number of cystercercoids ba-

sally. The slight shortening and


broadening of the villi were in some
sections (Fig. 4).
No histopathological changes
were detected in brain sections of
GI (SB control-non infected) and
GIII one month P.I. Only mild
edematous changes were detected
two months P.I. in GI & GIII. The
histopathological changes began to
appear in sections examined from
GII one month P.I. as gliosis with
proliferative gemistiocytic astro-
cytes with globoid shape and abun-
dant eosinophilic cytoplasm (Fig.
5).
Two months P.I., apoptotic pro-
cess occurred in some cells (Fig .6).
Some sections showed multiple
apoptotic cells with condensed
chromatin, pyknotic nuclei and
dense eosinophilic cytoplasm shrun-
ken away from the surrounding tis-
sues (Fig. 7). No aberrant cysticer-
coids were in all brain sections.
Brain tissues of GII showed H.
nana antigen deposition that started
to appear in the blood vessels wall
one month P.I. Two months P.I.,
antigenic deposition in brain stroma
was in form of cytoplasmic positive
astrocytes (Fig. 8). No deposition
was in GIII till the end of the study.
There was a gradual increase (tab.
2) in serum levels of IL-4 in GII &
G III to a peak at one month P.I.
compared to others. This increase
was highly significant in GIII
(P<0.001) without significance in
GII compared to control (P>0.001).
There was a gradual decrease in
IL-4 level in GI as compared to oth-
er groups. The difference was sig-
nificant compared to GII & GIII
(P<0.05), but without significance
as compared to control (P>0.05).
There was a progressive (tab 3, 4)
significant increase in GABA in GI
& GII reached higher level 2
months P.I. as compared to healthy
control and GIII (P<0.001). No sig-
nificant difference was between GI
& GII. GIII showed non significant
increase compared to control.
Zinc showed a gradual significant
decrease in GI & GII at two months
P.I. compared to others (P<0.05).
No significant difference was be-
tween GI & GII. GIII showed non
significant decrease compared to
control (P>0.05).
Table 1: Cysticercoid and adults count of groups at different duration P.I.
Infection
duration
Cysticercoid count Adult worm count
5 days P.I. 1month P.I. 2months P.I.
Group MeanSD Range MeanSD Range MeanSD Range
GII 207.56.7 195-220 1052.58 95-110 43.52.91 32-55
GIII 98.58.86 85-112 37.56.9 30-45 41.131 0.00-8
P <0.001** <0.001** <0.001**

P<0.001** = highly significant (GII versus G III).


Table 2: Serum levels of IL-4 of groups at different duration P.I.
Infection
duration
IL-4 levels
5 days P.I. 1month P.I. 2months P.I.
Group Range MeanSD Range MeanSD Range MeanSD
GI 0.06-0.92 0.460.31 0.03-0.67 0.320.16 0.01-0.52 0.260.33*
GII 0.21-0.38 0.410.13 0.77-1.04 0.850.13 0.59-0.73 0.660.02
GIII 0.57-0.45 0.510.01 1.12-1.83 1.350.03** 0.68-0.98 0.830.41**
IL-4 level in control ranged from 0.03-1.01 with a meanSD=0.360.43, P<0.001** =highly
significant {GIII versus Gs I, II & control}, P<0.05* =significant {G I versus Gs II & III}.
Table 3: GABA in brain tissue at different durations (ug/gm wet tissue mass).
Infection
duration
5 days P.I. 1 month P.I. 2 months P.I.
Group Range MeanSD Range MeanSD Range MeanSD
GI 412-499 45542 868-1015 93755** 896-1179 103844**
GII 400-542 47161 960-1212 108698** 905-1420 1162.521**
GIII 408-506 45764 445-582 51386 458-600 52943
GABA in control from 400-520 with a meanSD = 4600.33, P<0.001** = highly significant {Gs I & II
versus GIII and control}.
Table 4: Zinc level in brain tissue of groups at different durations.
Infection
duration
5 days P.I. 1month P.I. 2months P.I.
Group Range MeanSD Range MeanSD Range MeanSD
GI 9.1-10.8 9.91.23 8.9-10.1 9.051.06* 7.7-9.9 8.60.12*
GII 9.3-11.4 10.351.13 8.7-10.5 9.61.03* 7.8-9.1 8.90.87*
GIII 10.1-12.8 11.41.22 9.8-11.4 10.61.1 8.8-11.9 10.31.04
Zinc level in control from 10.3-12.4 (ug/gm wet tissue mass) with a meanSD = 11.30.26
P<0.05* =significant {Gs I & II versus GIII and control}.
Discussion
H. nana was the commonest zo-
onotic parasite in the world (Roberts
and Janovry, 2000). Children were
the most vulnerable age group to
infection in Egypt owing to the lack
of hygiene together with bad food
habits (Mirdha and Samantary,
2002; El Shazly et al., 2006b). They
are at higher risk from the neurolog-
ical effects of the disease such as
headache, dizziness and convulsions
(Chandraskhar and Nagesha, 2003).
Nowadays, the fast rhythm of our
life increases the chance of con-
sumption of food containing pre-
servatives which may evoke health
problems. One of these problems
that has appeared recently and affect

children is the behavioral disturb-


ances either in the form of the hy-
peractivity or the irritability (Mc-
Cann et al., 2007).
Sodium benzoate (SB) is one of
the commonest food preservatives
used for fruit juice, carbonated
drinks and pickles to prevent the
growth of micro-organisms in acidic
foods. The effect of exposure to
high dose of SB caused repeated
episodes of acute urticaria and pru-
ritus (Nettis et al., 2004; Tokunaga
et al., 2004), in addition to neurobe-
havioral abnormalities (Bateman et
al., 2004; Wallis, 2007). However,
none regarding the effect of expo-
sure to high doses of SB on the
course of parasitic diseases
No doubt, helminthes evoke T
h
2
cell response in the mammalian host
(Yazdanbaksh et al., 2001). IL-4 is
one of T
h
2 cytokines that has multi-
ple stimulatory and regulatory ef-
fects, and it was strongly suggested
as the most important and essential
cytokine in protective immunity to
H. nana (Abou-Holw, 2005). It can
promote growth and differentiation
of B cells, T cells and mast cells.
IL-4 affects a wide variety of cell
types as macrophages, eosinophils,
basophils and endothelial cells
(Kelly et al., 2004), and it is im-
portant in the rapid expulsion of the
parasites from the intestine (Meeu-
sen and Balic, 2000). On this con-
text, IL-4 serum levels were meas-
ured to study its effect of exposure
of mice to SB alone or with H. nana
infection on the immune response
elicited in mice.
In the present study, significant
decrease in IL-4 serum levels was in
SB-non infected GI. However, sig-
nificant increase in serum levels of
IL-4 was in H. nana infected GIII as
compared to other groups. This
could be due to exposure of the
mice to a high dose of SB resulting
in marked suppression of the im-
mune system, evidenced by signifi-
cant decrease in IL-4 levels in SB-
non infected mice. However, infec-
tion with H. nana is well known to
stimulate T
h
2 response with signifi-
cant increase of IL-4 serum levels
that reached the peak one month P.I.
Exposure of the mice to high dose
of SB resulted in reduction of serum
IL-4 levels prior to infection but
after exposure of the mice to infec-
tion with H. nana, the immune sys-
tem suffered from two antagonistic
stimuli; one is the suppressive effect
of SB and the other is the stimulat-
ing effect of H. nana infection. This
resulted in non significant increase
of the serum levels of IL-4 in SB-
infected group and could explain the
significant increase in their levels in
infected groups. Similarly,
Conchedda et al. (1997) on studying
cytokine production during H. nana
infection reported that T
h
2-like re-
sponse (IL-4, IL-5 secretion) was
stimulated 4-5 days P.I., when the
parasites were thought to begin their
luminal phase. Besides, Abou-Holw
(2005) and Ajami and Rafiei (2007)
found significant increase in IFN-,
IL-4 & IL-5 serum levels in H. nana
infected patients as compared to
control.

The present histopathological ex-


amination of the small intestine of
SB-infected mice showed high rate
of cysticercoids with changes in the
form of blunt and fused villi togeth-
er with submuocosal edema and
mucosal damage in some sections.
The infected ones without SB
showed a lower parasitic load and
complete expulsion of the adults
from the small intestine two months
P.I., with preservation of the normal
architecture as compared to SB-
infected group. Asano et al. (1990)
found that H. nana proved to be
immunogenic and induced long last-
ing resistance to the re-infection
through a T-cell dependent mecha-
nism. So, exposure to a high dose of
SB induced a state closely similar to
immunosuppression proved by sig-
nificant decrease in IL-4. This was
responsible for re-infection, autoin-
fection and hyperinfection, with
large number of cysticercoids in
villi that led to marked histopatho-
logical changes in the small intesti-
nal wall. These findings agreed with
Makled et al. (1994) who found that
cortisone-induced immunosuppres-
sion in H. nana infected mice
caused the development and persis-
tence of high number of cysticer-
coids and delayed expulsion of the
adults up to four months P.I.
The histopathological examination
of brain revealed only mild edema-
tous changes detected 2 months P.I.
in SB-non infected group and in-
fected mice. But, in the SB-infected
ones, histopathological changes be-
gan to appear in sections examined
one month P.I. as gliosis with pro-
liferative gemistiocytic astrocytes
with globoid shape and abundant
eosinophilic cytoplasm. But, two
months P.I., an apoptotic process
occurred in some cells. Some sec-
tions showed multiple apoptotic
cells with condensed chromatin,
pyknotic nuclei and dense eosino-
philic cytoplasm shrunken away
from the surrounding tissues. The
results agreed with El-Kowrany and
El-Shafey (2000) who found that
brains of immunosuppressed H.
nana infected mice showed moder-
ate perineural oedema and mononu-
clear cellular infiltration with fibril-
lary glial background at 2
nd
month
P.I. This could be due to the expo-
sure of the mice to a high dose of
SB that may result in hyperinfec-
tion. Dissemination of H. nana tox-
ins in the brain may be responsible
for the histopathological changes.
No aberrant cysticercoids were de-
tected in all brain sections. This
agreed with El-Kowrany and El-
Shafey (2000) who did not detect
any cysticercoids in brains of im-
muno-suppressed H. nana infected.
But, Morozov (1967) reported the
presence of H. nana eggs in the cer-
ebral hemisphere of a brown rat.
Concerning the immunohistochemi-
cal examination of the brain tissues
of SB-infected mice examined by
peroxidase anti-peroxidase (PAP)
technique, there were H. nana anti-
gen deposits in the walls of blood
vessels one month P.I.. Two months
P.I., antigenic depositions of brain
stroma were detected in the form of

cytoplasmic positivity of the astro-


cytes. No antigenic deposition could
be detected till the end of the exper-
iment in infected group. This could
be attributed to exposure to high
dose of SB that resulted in immuno-
suppression, leading to heavy infec-
tion and high number of adult
worms in the small intestines that
produce toxins which may be car-
ried by the circulation to reach the
brain. This agreed with El-Kowrany
and El-Shafey (2000) who reported
deposition of H. nana antigens in
the walls of blood vessels and the
stroma of the brain of immunosup-
pressed mice by indirect immuno-
fluorescence test. They added that
the toxins derived from the high
number of adult worms in the small
intestines of immunosuppressed
mice may be absorbed and carried
by the circulation to reach the brain
and become deposited there. The
source of the H. nana antigens was
discussed by Gomez-Priego et al.
(1991) who detected circulating an-
tigens derived from the adult worms
whether, surface, somatic or meta-
bolic antigens or from the tissue
cysticercoids in disseminated infec-
tions in the immunosuppressed.
In the central nervous system,
monoamine neurotransmitters in-
cluding gamma amino butyric acid
as well as some trace elements as
zinc can control a variety of physio-
logical, behavioral and endocrinal
functions (Kamisaki et al., 1990).
Thus the changes in neurotransmit-
ter concentration in the brain are
considered as a cornerstone of intra-
cellular communication that drives
the brain function. There are grow-
ing numbers of clinicians and basic
scientists who are convinced that
food additives alter the brain levels
of neurotransmitters and zinc, and
play a critical role in the develop-
ment of several neurological disor-
ders including seizures, abnormal
neural development and certain en-
docrinal and neurodegenerative dis-
eases (Engel et al., 2004).
Therefore, a point worthy of dis-
cussion in the present study is the
highly significant increase in GABA
level in SB-non infected group and
SB-infected groups at 2 months P.I.
as compared to other groups. The
level was higher in SB-infected
group as compared to SB-non in-
fected group. This could be ex-
plained as exposure to SB alone
increases the GABA level, but when
this exposure is associated with H.
nana infection the GABA level is
markedly increased, which empha-
sized the possible synergistic effect
of both SB and H. nana on the brain
GABA level.
The increase was surprising as it
has been documented that patients
with H. nana infection suffer from
increased brain excitability and even
convulsion, while GABA is a well
known fast inhibitory neurotrans-
mitter in the brain. It was noted that
although GABA is the principal
mediator of synaptic inhibition,
GABA receptors, yet, when inten-
sively activated, excite rather than
inhibit neurons (Saliba et al., 2009),
and this may explain the occurrence

of convulsions in spite of high


GABA levels. This may also ex-
plain the behavioural disturbances
that have been increased nowadays
in children due to increased con-
sumption of preserved food, which
does not only increase GABA level,
but also the incidence of hyperinfec-
tion if the child is unfortunately in-
fected with H. nana.
The link between behavioural dis-
turbances and SB consumption de-
tected in this study goes with many
studies. Bateman et al. (2004) stud-
ied the effect of artificial colouring
and benzoate preservative challenge
on hyperactivity in a general popu-
lation sample of the preschool chil-
dren, and they found hyperactivity
and behavioural disturbances of 3
years old children that were detect-
ed by the parents but not by simple
clinical assessment. Wallis (2007)
and McCann et al. (2007) confirmed
a long-suspected link between hy-
peractivity and food additives. But,
Tariq et al. (2004) found that SB
induced the imino-dipropionitrile
behavioural syndrome in rats as
dyskinetic head movements, circling
and tail hanging righting reflex.
The role of Zn in the CNS has
remained enigmatic for several dec-
ades. This divalent cation is accu-
mulated by specific neurons into
synaptic vesicles and can be re-
leased by stimulation in calcium-
dependent manner (Anderson et al.,
1996).
In the present work, zinc showed a
gradual decrease in its level, which
became significant in SB-non in-
fected mice and SB-infected ones
examined two months P.I. in com-
parison to the level in the other
groups. The difference between
both groups was non significant.
Zinc may have an important neu-
romodulatory action that is mediat-
ed, in part, by effects on GABA
transporters. This may explain the
increased GABA levels in groups
that showed reduced Zn levels, as
Zn is important for GABA trans-
porters action to maintain low level
of GABA (Gibbs et al., 2000). In
the current work, Zn deficiency may
be caused by chronic diarrhoea as
well as the marked injurious effect
of the high count of adult H .nana
on the small intestinal mucosa in
SB-infected mice. Zinc is the se-
cond-most abundant transition metal
within cells and is an essential mi-
cronutrient. As adequate Zn is es-
sential for the cellular function
(Wiseman et al., 2009), so the re-
duced level in SB-infected group in
the present study may explain the
appearance of apoptotic changes
that were observed in the histo-
pathological examination of the
brain tissues. It is well known that
the maintenance of discrete subcel-
lular pools of Zn is critical for the
functional and structural integrity of
cells and contributes to the regula-
tion of apoptosis, and Zn deficiency
in vivo may increase the sensitivity
of animals to toxins (Anderson et
al., 1996; Wiseman et al., 2009).
The fast rhythm of life paves the
way to the take away food. The ex-
cess daily intake of SB in various

types of food and beverages on the


long run may accumulate in the
body and carry as risk as exposure
to a high dose.
Conclusion
Mice exposed to a high dose of
SB led to decreased immunity as
proved by significant reduction of
serum levels of IL-4, the most im-
portant cytokine in protective im-
munity against many parasites espe-
cially H. nana. This resulted in ful-
mination of H. nana was reflected
on histopathological and immune-
histochemical changes. Exposure to
SB caused significant increase in
GABA and decrease in zinc levels.
The changes were so manifested
when mice were exposed to high
doses of SB together with H. nana
to a degree of causing behavioral
disturbances. Emphasized possible
synergistic effect of SB on the neu-
rological manifestations explained
the increased incidence of behavior-
al disturbance in infected children
exposed to high doses of SB.
So, it is recommended to return
back to healthy food pattern espe-
cially for children, and to prevent or
at least decrease consumption of
food containing SB particularly in
countries where zoonotic parasitosis
are common.
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Legend of figures

Fig. 1: T.S. in a mouse small intestinal wall of GII one month P.I., showing many cysticercoids
at base of villi (H&E, X160).
Fig. 2: T.S. in a mouse small intestinal wall of GII one month P.I., showing many cysticercoids
at base of villi with submuocosal edema, mucosal damage and inflammatory infiltrates (H&E,
X250).
Fig. 3: T.S. in a mouse small intestinal wall of GII two months P.I., showing adults with in-
creased lymphocytic infiltration in lamina propria (H&E, X160).
Fig. 4: T.S. in a mouse small intestinal wall of GIII one month P.I., showing few cysticercoids at
base of villi. Some showed slight shortening and broadening (H&E, X160).
Fig. 5: T.S. in a mouse brain of GII one month P.I., showing gliosis with proliferative gemistio-
cytic astrocytes with globoid shape and abundant eosinophilic cytoplasm (H&E, X400).
Fig. 6: T.S. in a mouse brain of GII two months P.I., showing apoptotic process starts to occur in
some cells (H&E, X400).
Fig. 7: T.S. in a mouse brain of GII two months P.I., showing multiple apoptotic cells with con-
densed chromatin, pyknotic nuclei and dense eosinophilic cytoplasm shrunken away from sur-
rounding tissues (H&E, X400).
Fig. 8: T.S. in a mouse brain of GII two months P.I., showing antigenic deposition of brain stro-
ma in the form of cytoplasmic positivity of astrocytes (PAP, X400).
1015
1033
Journal of the Egyptian Society of Parasitology, Vol. 39, No. 3, December 2009
J. Egypt. Soc. Parasitol., 39 (3), 2009: 1033 - 1047
PROPHYLACTIC EFFECT OF BOVINE LACTOFERRIN AGAINST
ACUTE TOXOPLASMOSIS IN IMMUNOCOMPETENT AND
IMMUNOSUPPRESSED MICE
By
SHEREEN F. MOSSALLAM
Department of Parasitology, Faculty of Medicine, Alexandria University,
Alexandria, Egypt
Abstract
This study evaluated the immune-potentiating effect of administrating bovine
Lactoferrin (LF) to immunocompetent (IC) and immunosuppressed (IS) mice
prior to infection with tachyzoites of T. gondii RH strain. Mice were IS with
cyclophosphamide. LF was given in seven of them as oral doses on alternate
days. Immunological and parasitological assessments showed that LF induced
statistical significance comparable resistance against acute toxoplasmosis in IC
and IS mice. This was verified by elevated splenic CD4
+
T lymphocytes,
reduced tachyzoites' viability and infectivity, with diminished parasite burdens.
So, mice mortality declined and their survival was prolonged. This indicated
that LF have prophylactic efficacy against human toxoplasmosis in risky
persons with alleviating immune balance.
Keywords: Toxoplasmosis, prophylaxis, Lactoferrin, cyclophosphamide,
immunosuppression, CD4
+
T lymphocytes, immune-modulation.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction
Toxoplasma gondii (T. gondii) is a
universally distributed obligate int-
racellular protozoan of the coccidian
family that multiplies in any
nucleated cell of vertebrate hosts
(Feldman, 1968). This protozoan
triggers both humoral and cellular
immune responses, with the later
being more important for the
development of protective and
persistent immunity, by producing
high levels of host protective
cytokines especially gamma interfe-
ron (IFN-). This cytokine is
responsible for resistance, during
acute and chronic phases of the
disease, through the activation of
macrophages which promote intra-
cellular death of the parasite.
Natural killer (NK) cells, CD4
+
&
CD8
+
T cells are the major sources
of the IFN- that controls initial
parasite replication (Gazzinelli et.
al., 1991; Denkers and Gazzinelli,
1998). The CD4
+
T lymphocytes
mediated early resistance to
infection even in the absence of NK
1033
& CD8
+
T cells (Araujo, 1991; Sch-
arton-Kersten et. al., 1998).
Infection is benign in people
having normal immune function,
and spontaneous recovery is the
usual rule. However, about 20%
acute infection was accompanied by
febrile lymphadenopathy, asthenia,
lymphomonocytosis, and occasional
eye involvement, with the course of
self-limited infection (Feldman,
1968). Conversely, toxoplasmosis is
of great clinical importance in two
major situations; as a cause of
congenital infection, and as an
opportunistic infection in immuno-
suppressed (IS) individuals, especi-
ally those with deficient cellular
immunity (Ferreira and Borges,
2002). These are at risk of occurre-
nce of fulminating course, in the
form of disseminated disease or
toxoplasmic encephalitis with repor-
ted mortality rate exceeding 30%
(Lang et. al., 1989; Dannemann et.
al., 1991). Since the sixties, the
number of IS patients is increasing
and has culminated in the advent of
acquired immunodeficiency syndr-
ome (AIDS) at the beginning of the
eighties. Nowadays, the increasing
use of organ transplants and
immunosuppressive drugs have led
to the occurrence of a large number
of individuals with chronic immun-
osuppression, who are predisposed
to developing opportunistic infect-
ions; toxoplasmosis being ranked
among the most frequent (Young,
1981; Ferreira and Borges, 2002).
Thus, hopes are directed towards
immune-potentiating elements in
trial to save the fatal outcomes of
this opportunistic protozoan among
these patients.
Recent studies have provided
evidence that Lactoferrin (LF), an
80-kDa milk-derived iron-binding
glycoprotein, is a promising imm-
une-regulator which is currently
isolated and purified on an industrial
scale from cheese whey and skim
milk (Tomita et. al., 2002). LF has
multiple known biological activities,
including iron regulation, anti-
inflammatory activity, antimicrobial
defense, and cancer protection,
without any reported adverse effect
(Ward et. al., 2005). It is now
recognized that LF plays an impor-
tant role in host defense against
infections and excessive inflamm-
ation, in a manner dependent on the
host's immune status (Fischer et. al.
2006; Actor et. al., 2009). The
antimicrobial role of LF is either
related to its ability to sequester iron
in biological fluids, or through iron-
independent direct microbicidal
activities (Yamauchi et al., 2006).
The protective role of LF against
various types of pathogens such as
bacteria, viruses, and fungi is well
established (Artym et. al., 2004;
Takakura et al. 2004; Ueno et al.,
2006). The potency of this protein
against some protozoal infections
was reported, including Eimeria
stadeai, Pneumocystis carinii, T.
gondii, and intestinal protozoa
(Tanaka et al., 1995; Cirioni et al.,
2000; Omata et al., 2001; Ali and
Nouh, 2005). Its efficacy against
intestinal protozoa in IS mice (Ali
1033
and Nouh, 2005), but, little is
known about its influence on
toxoplasmosis in IS models.
This study aimed at evaluating the
immune-potentiating activity of
orally administered bovine LF
against acute T. gondii infection in
mice with intact immunity, as well
as in IS mice.
Material and Methods
Tachyzoites of T. gondii RH
strain maintained in Department of
Parasitology, Alexandria Faculty of
Medicine were used. Intraperitoneal
(I.P) passage of tachyzoites in Swiss
albino mice was carried out every
three days (Johnson et. al., 1979),
as10
4
tachyzoites/mouse (Guimar-
es et. al., 1997).
Lactoferrin: Bovine milk LF
(Sigma; L9507) was administered
per mouse, on alternate days by a
feeding syringe, in a dose of
1mg/dose, altogether seven doses
(Artym et. al., 2003).
Cyclophosphamide: Mice were IS
by single I.P injection of 250 mg/kg
body weight Cyclophosphamide
(CP=Endoxan

) after Artym et al.


(2004).
A total of 100 clean laboratory
bred mice, 4-6 weeks old fed on
standard pellet food, water and
lipitum were divided into two
groups: GI contained 60 mice
subdivided into six subgroups: SG
I-a (Normal control): 10 non-
infected non-treated mice. SGI-b
(Infected control): 10 infected mice.
SGI-c (IS control): 10 IS mice. SGI-
d (IS infected control): 10 IS mice,
infected 24 hours after CP injection.
SGI-e (LF control): 10 mice treated
with LF regimen. SGI-f (IS Lf-
treated control): 10 IS mice treated
with LF regimen 24 hours after CP
injection.
GII contained 40 mice subdivided
into two subgroups: SGII-a (LF
infected): 20 mice treated with LF
regimen then infected 24 hours after
the last dose of LF. SGII-b (IS LF-
treated infected): 20 IS mice, treated
with LF regimen 24 hours after CP
injection, then infected 24 hours
after the last dose of LF.
Scarification of SGI-b, SGI-d,
SGII-a & SGII-b was on 9
th
day post
infection (P.I). Scarification of SGI-
c & SGI-f was on 23
rd
day post
immunosuppression, and of SGI-e
was on 22
nd
days post LF first dose.
Each group of mice was sacrificed
with corresponding normal control.
Direct immune fluorescence (DIF)
of splenic CD4
+
T lymphocytes:
Spleens isolated from various
subgroups were individually pressed
through wire mesh sieves to obtain
single cell suspensions, by recurrent
washes in RPMI 1640 culture
medium (Sigma; R8755). Lympho-
cyte monolayers were separated
from each cell suspension by triple
centrifugations in Ficoll-Hypaque
(Sigma; 1077-1) at 400Xg, for ten
minutes. Four l Fluorescin-iso-
thiocyante (FITC) anti mouse-CD4
monoclonal antibody (Sigma;
F7400) were mixed with each
resulting cell pellet, and washed
thrice by centrifugation in two ml
monoclonal diluent at 250Xg for ten
1033
minutes. Each cell pellet was
resuspended in 0.5 ml diluent, to be
examined by fluorescent microscope
(Leitz-Dialux 22EB). Fluorescently
stained CD4
+
T lymphocytes (viable
& non-viable) were counted, then
total lymphocytes were counted
under phase contrast for calculating
the CD4
+
T cell percentage: fluores-
cent lymphocytes/total lymphocytes
number X 100 (Pierres et al., 1984).
Mortality rates and survival time
of mice were estimated according to
the times of scarification of each SG
(Guimares et. al., 1997). Subgrou-
ps spontaneous died during the
study, were followed up daily for
survivors till date of scarification.
Each infected mouse was micro-
scopically checked for Toxoplasma
parasite in peritoneal fluid, and the
mean number of tachyzoites/ml was
assessed by the Haemocytometer
chamber (Bauer, 1963). The mean
number of tachyzoites/10 high
power fields of Giemsa-stained liver
impressedsmears of each infected
SG was performed (Guimares et.
al., 1997). Determination of viabil-
ity of tachyzoites harvested from
peritoneal cavities of infected mice
was done by using 0.2% trypan blue
stain (Sigma; T5891). The viability
percentage was determined by
counting mean number of viable
tachyzoites/100 organisms (Omata
et al., 1999).
Determination of tachyzoites
infectivity of infected subgroups
of mice was done by I.P injection
into naive Swiss albino mice (six
for each subgroup). Four days
P.I, peritoneal fluid was checked
for tachyzoites. Infectivity rate
and mean number of tachyzoites/
ml peritoneal fluid of these sub-
groups were calculated (Omata et
al., 1999). The %R= (C-E) / C X
100) was calculated by compar-
ing each individual experimental
(E) subgroup versus correspond-
ing control (C). The %R of SGII-
a was calculated in relation to
SGI-b, and %R of SGII-b was
calculated in relation to GI-d.
Statistical analysis: Differences
between groups were analyzed by
Student's unpaired t-test and Fisher
Exact test. P values of p 0.05 were
considered significant (Castle,
1977).
Results
DIF staining of splenic CD4
+
T
lymphocytes, using FITC anti-
mouse CD4 monoclonal antibody,
recognized the CD4-receptor expr-
essed on the surface of mouse T
cell. Staining viable cells confined
the antibody to the external surface
membrane, without staining the
internal cell structure. Viable CD4
+
T cells showed brilliant ring of
fluorescence on surfaces, while non-
viables showed homogenous intra-
cytoplasmic fluorescence (Fig.1).
For calculating the CD4
+
T cell
percentage, viable and non-viable
CD4
+
T lymphocytes were counted,
versus total lymphocytes count
under phase contrast microscopy
(Fig.2).
1033
The mean percentage of splenic
CD4
+
T lymphocytes of SGIa was
23.66%1.28, which statistic-ally
increased to 26.27%0.11 in SFI-b
(t=6.424, p<0.001), but was drama-
tically reduced in a statistical
manner in SGI-c 11.57%0.52, (t=
27.672, p<0.001). The CD4 value of
IS SGI-d statistically declined to
11.29%2.77 compared to infected
controls (t=17.088, p<0.001) but
lack of significance was quite
evident between SGI-d & SGI-c
(t=0.314, p=0.757). Conversely
administration of the LF regimen to
SGI-e resulted in a significant
elevation of CD4 pool (50.51%
2.61) versus normal controls,
(t=29.208, p<0.001). In SGI-f, treat-
ment of IS mice with LF increased
CD4 value to 38.30%3.18, with
statistically significant in relation to
SGI-c & SGI-e (p<0.001). The
highest CD4 (56.42%1.49) was
achieved from SGII-a when LF
preceded infection and was statistic-
ally higher than in SGI-b & SGI-e
(p<0.001). CD4% (45.17%2.43)
obtained by infecting IS LF-treated
SGII-b, a value was statistically
elevated than SGI-d & SGII-a
(p<0.001). Fig.3-a demonstrated the
splenic CD4
+
T lymphocyte enrich-
ment in LF-treated mice, in compa-
rison to untreated ones (Fig.3-b).
MR and the survival time of mice:
Until the sacrifice date, none died
from SG I-a, SGI-e & SGI-f. But,
80% of SGI-b died within 6-8 days
P.I, appearing ill with ruffled fur
and hunched postures. While 60 %
of SGI-c mice died within 5-6 days.
Mice of SGI-d were ill and none
survived more than 3-4 days P.I.
without significant elevation in MR
to 100%, versus SGI-b (FEp=-
0.474). In SGII-a, only one mouse
died before the scarification time
(5%MR) and those survived were
followed up daily and were sleek,
active and died within 20-22 days
P.I. Whereas, two mice died from
SGII-b (10%MR), and the 18
survivors lived for up to 17-18 days
P.I. %R in the MR of the experi-
mental subgroups were 93.75 &
90%, respectively. No signifycance
was noted in mortality rates between
SGII-a & SGII-b (FEp =1.000), but
there were statistical differences
between mortality rates in
correspondence to SGI-b &SG I-d
(FEp < 0.001). The mean number of
tachyzoites per one ml peritoneal
fluid of SGI-b was 6.85 millions
0.03, and it reached up to 9.58
millions 2.95 in SGI-d, with
significant differences between both
controls (t=2.926, p=0.009). The
value dropped to 0.25 million1.19
in SGII-a, and 0.39 million0.83 in
SGII-b, reflecting that LF caused
notable percentage reduction in the
peritoneal parasite burden by
96.35% & 95.92% in experimental
subgroups, respectively. There was
no significant difference in the mean
number of peritoneal tachyzoites
between the two experimental
subgroups (t= 0.432, p=0.669), yet
significance existed between SGII-a
versus I-b (t=7.382, p<0.001), and
between SGII-b versus SGI-d
(t=13.133, p<0.001).
1033
Table: Immunological assay and parasitological parameters in control and
experimental groups:
G
r
o
u
p
s
No.
Immunology Parasitology
Splenic CD4
+
T
cells%
Mice Death
Survival
time
Tachyzoites (X SD)
Tachyzoite
viability%
Infectivity of
harvested
tachyzoites
No MR
P.F.
(10
6
/ml)
Liver (/10 HPF) I.R
10
6
tachyzoites
/ml P.F.
SGI-a 10 23.66%1.28 0 0% ST -- -- -- -- --
SGI-b 10 26.27%0.11 8 80% 6-8 d. 6.850.03 17.202.00 92.69%1.97 100% 6.832.76
SGI-c 10 11.57%0.52 6 60% 5-6 d. -- -- -- -- --
SGI-d 10 11.29%2.77 10 100% 3-4 d 9.582.95 22.351.00 88.37%3.9 100% 8.552.12
SGI-e 10 50.51%2.61 0 0% ST -- -- -- --
SGI-f 10 38.30%3.18 0 0% ST -- -- -- -- --
SGII-
a
20 56.42%1.49 1
5%
(%R
=93.75%)
20-22 d.
0.251.19
(%R =
96.35%)
0.740.01
(%R = 95.69%)
10.34%1.42
(%R =
88.84%)
16.67%
(%R =
83.33%)
0.201.00
(%R =
97.07%)
SGII-
b
20 45.17%2.43 2
10%
(%R =
90%)
17-18 d.
0.390.83
(%R
=95.93%)
2.161.50
(%R = 90.33%)
9.29%0.37
(%R =
89.49%)
16.67%
(%R =
83.33%)
0.410.73
(%R
=95.20%)
Test of
significance
t1: 6.424
*
, p<0.001
t2: 27.672
*
, p<0.001
t3: 17.088
*
, p<0.001
t4: (0.314), p=0.757
t5: 29.208
*
, p<0.001
t6: 46.207
*
, p<0.001
t7: 9.386
*
, p<0.001
t8: 63.343
*
, p<0.001
t9:7.937
*
, p<0.001
t10:34.383
*
,
p<0.001
t11:13.369
*
,p<0.001
FEp3:
(0.474)
FEp8:
<0.001
*
FEp10:
<0.001
*
FEp11:
(1.000)
t3: 2.926
*
,
p=0.009
t8: 7.382
*
,
p<0.001
t10:13.133
*
,
p<0.001
t11:(0.432) ,
p= 0.669
t3: 7.283
*
, p<0.001
t8: 98.406
*
,
p<0.001
t10:38.346
*
,p<0.001
t11:
(4.234),p=0.570
t3: (3.127),
p=0.06
t8: 62.743
*
,
p<0.001
t10:176.366
*
,
p<0.001
t11:(3.200),
p=0.03
FEp3: ---
-
FEp8:
0.015
*
FEp10:
0.015
*
FEp11:
(1.000)
t3: (1.654) ,
p=0.116
t8: 0.681*,
p<0.001
t10: 9.681*,
p <0.001
t11: (0.759)
,p=0.453
GP: Group, EXP: Experimental, IN: Infected, IS: Immunosuppressed, LF: Lactoferrin- treated, No.:
Number, X: Mean, SD Standard deviation, MR: Mortality rate, %R: % reduction, ST: Sacrifice time, I.R:
infectivity rate, , HPF: High power field, P.F. Peritoneal fluid, 10
6
/ml: Million/ml, t: Student t-test, * :
Statistically significant at p 0.05, ( ): Non significant, t
1: Gp I-b vs I-a, t2: Gp I-c vs I-a, t3: Gp Id vs I-b,
t4: Gp Id vs I-c, t5: Gp I-e vs I-a, t6: Gp I-f vs I-c, t7: Gp I- f vs I-e, t8: Gp II-a vs I-b, t9: Gp IIa vs I-e, t10: Gp
II-b vs I-d, t11: Gp II-b vs II-a , FEp : p value for Fisher Exact test, FEp3: Gp Id vs I-b, FEp8: Gp II-a vs I-
b, FEp10: Gp II-b vs I-d, FEp11: Gp II-b vs II-a.
1033
Fig 4-a and 5-a demonstrated
numerous T. gondii tachyzoites in
peritoneal fluid of infected untreated
mice, while Fig 4-b & 5-b showed
the reduction in peritoneal tachyz-
oites in LF-treated infected mice.
The mean number of tachyzoites
per 10 high power fields in smears
of livers of mice of SGI-b was
17.202.00, and in SGI-d it was
22.351.00 with significant differ-
ence between them (t=7.283, p<
0.001). While in SGII-a, it was
0.740.01 (reduced by 95.69%), and
in SGII-b, it was 2.161.50 (red-
uced by 90.33%). No significance
existed between SGII-a & SGII-b
(t=4.234,p=0.570), but evident sig-
nificance was on comparing SGII-a
versus SGI-b (t=98.406, p<0.001),
and SGII-b versus I-d (t= 38.346,
p<0.001). Fig 6-a&b. showed
Giemsa-stained T. gondii tachyz-
oites in liver impression smears of
infected untreated and LF-treated
infected mice, in succession.
Differentiation between living and
dead tachyzoites harvested from the
peritoneal fluid of infected mice was
done using 0.2% trypan blue stain.
Fig 7-a showed that living T. gondii
tachyzoites appeared clear blue in
color, while dead ones were homo-
genous blue in color, with undetect-
able SGI-d it was 88.37% 3.9,
reflecting the lack of significant
differences between them (t=3.127,
p=0.06). The least viable tachyz-
oites% was in SGII-b (9.29%
0.37), followed by SGII-a (10.34%
1.42), resulting in a marginal %R
in their viability (88.84 & 89.49%,
respectively). A statistically signify-
cant differences was between viabil-
ity of SGII-a & SGII-b, in compar-
ison to SGI-b & SGI-d, respectively
(p<0.001), without significant (t=
3.200, p=0.03).
Among the naive mice that were
infected with tachyzoites gathered
from the peritoneal cavities of the
experimental subgroups, just one
(out of six) from each subgroup
caught the infection, resulting in
significant diminution of infectivity
rates of both subgroups (16.67%),
being compared to the 100%
infectivity rates in SGI-b & SGI-d
(FEp=0.015). Tremendous numbers
of tachyzoites (6.832.76& 8.65
2.12 millions/ml) were consecut-
ively harvested from the peritoneal
cavities of SGI-b & SGI-d, without
significant differences (t=1.654,
p=0.116). The mean number of
tachyzoites from peritoneal cavities
of SGII-a infected mouse was 0.20
1.00 millions/ml, as compared to
SGI-b (%R =97.07%). Likewise,
0.410.73 million tachyzoites were
collected from thSGII-b, with a
percentage reduction of 95.26%
against SGI-d. Significant differe-
nces was between the infectivity in
SGII-a& SGII-b as compared to
SGI-b & SGI-d, respectively
(p<0.001). Difference between exp-
erimental subgroups statistically
insignificant (t=0.759, p=0.453).
Discussion
Among those who are infected
with T. gondii, few have symptoms
because a healthy person's immune
1033
system usually keeps the parasite
from causing illness. So, individuals
at risk should be cautious; for them,
Toxoplasma infection could cause
serious health problems (Ambroise-
Thomas and Pelloux, 1993). Adv-
ances of modern medicine increase
the demand for new pharmacolo-
gically active compounds of stimu-
latory properties within the immune
system (Zimecki et. al., 2007). LF
has gained increasing interest as a
result of its protective effects
against a variety of diseases via its
immune-regulatory properties (Fis-
cher et. al. 2006). Hence, in this
study it has been worthy trying the
possible immune-potentiating effect
of this protein in both immuno-
competent and IS mice, infected
with acute toxoplasmosis.
The phenotypic analysis of splenic
CD4
+
T lymphocytes of SGI-b was
26.27%, as compared to normal
controls. Although difference bet-
ween the subgroups was marginal,
yet there was statistically signi-
ficant, denoting that the parasite
affecting these cells was slightly
stimulatory. This resulted from a
direct activity of the tachyzoite in
inducing preliminary proliferation
of CD4
+
T cells (Nguyen et. al.,
1998; Scharton-Kersten et. al.,
1998). Other possibility was that
interleukin (IL)-12 burst occurred
soon after host invasion by the
parasite and in turn the non-
specifically triggers CD4
+
T cells
(Wu et al., 1993; Gazzinelli et al.,
1994; Nguyen et al., 2003). Unfo-
rtunately, this immune response was
not enough to eliminate infection, as
80% mortality rate was within 6-8
days, together with the outstanding
mean number of tachyzoites in
peritoneal fluid and livers. Besides,
all tachyzoites from this subgroup
were infective to naive mice, and
92.69% showed intact viability.
Tachyzoites of Toxoplasma RH
strain caused extensive tissue infes-
tation with significant early mice
mortality from 80% to 100% (Dero-
uin et. al., 1992; Mahmoud, 1999;
Nguyen et. al., 2003).
In IS controls (SGI-c) administ-
ration of CP resulted in substantial
significant reduction of splenic
CD4
+
T cell population (11.57%)
regarding normal controls. 60% of
IS controls died within 5-6 days
post immunosuppression. Cyclo-
phosphamide is an alkylating agent
used for treating malignant and non-
malignant immune-mediated infla-
mmatory disorders in humans. It is
also known as a potent immuno-
suppressive drug in humans and in
experimental animals. Appropriate
concentration this drug caused T
cell depletion, with consequent low
production of immunoregulatory
lymphokines such as IFN- (Artym,
2003; Artym et al., 2003, 2004;
Zimecki et al., 2007). This is similar
to cellular compartment defects of
the immune system in patients
suffering from AIDS and lympho-
proliferative neoplasias, explaining
the high frequency of infection
caused by intracellularly multiply-
ing agents such as T. gondii in these
1033
patients (Fauci, 1984; Ferreira and
Borges, 2002).
In the current study, all SGI-d
mice, which were IS, then inocu-
lated with tachyzoites died within 3-
4 days P.I. This was associated with
extensive significant diminution of
their splenic CD4
+
(11.29%), in
comparison to SGI-b, while this
value was statistically comparable
to IS controls. Infectivity of tachyz-
oites of the IS infected controls did
not alter (100%), and substantial
peritoneal tachyzoites were isolated
from these mice. An evident lack of
significance in tachyzoites' viability
existed between this subgroup and
the infected one, which could be
likely because immunosuppression
has no effect on either parameter. A
significant difference was on
comparing the parasite burdens in
the peritoneal fluid and livers in
SGI-b & SGI-d. This reflected the
probably of immunosuppression
exacerbated infection in SGI-d. This
agreed with others who emphasized
that immunosuppression worsens
the outcome of toxoplasmosis
resulting in early anticipation of
infection due to T-cell subsets
reduction, in accordance with a
marked suppression of the endo-
genous IFN- production (Sumyuen
et al., 1996; Guimares et al., 1997;
Khalifa et al., 2000).
In SGI-e, administration of oral
LF regimen caused significant
elevation in splenic CD4
+
T cells
(50.51%) compared to normal
controls, and caused their survival
up till to scarification date. Kuhara
et al. (2000) found that oral LF
induced intestinal epithelial cells to
produce IL-18, in turn, enhanced
Th1 functions, resulting in increa-
sing CD4
+
cells in mice peripheral
blood and small intestine. After
being absorbed from the intestinal
barrier, LF possessed direct immu-
notropic properties for immature
negative thymocytes, through the
recognition of specific LF binding
sites leading to induction of CD4
antigens, and promoting their
phenotypic and functional matur-
ation (Zimecki et al., 1991, 2007).
Oral LF provoked both mucosal and
systemic immune responses and the
effect was stimulatory within the
immune system (Debbabi et al.,
1998; Sfeir et. al., 2004).
In the present study, in IS mice
treated with LF (SGI-f) CD4
+
T cell
pool was 38.30% with a significant
elevation in relation to IS controls.
Comparison between the immune-
stimulatory effects of LF in SGI-e &
SGI-f showed significant difference
between both. In SGI-f, the strong
preliminary reduction of the CD4
percentage induced by CP was up-
regulated by LF that prolonged there
survival till the experimental end.
Restoration of the splenic CD4
+
cells in IS mice by oral LF treatment
was reported (Artym, 2003; Artym
et. al., 2003).
This protein accelerates the
renewal of the number and function
of immunocompetent T cells in IS
mice, mostly, through modulating
iron supply to the spleen (Zimecki
et al., 2007), or by inducing
1033
cytokine production from other cell
types as macrophages and mainly
directed to repopulation of circula-
ting blood and splenic lymphocytes
(Artym et. al., 2003).
In the present study, the highest
CD4
+
T cell yield was in SG II-a
LF-treated prior to infection. The
significant elevation of the CD4
pool (56.42%) was quite evident
versus infected controls. The LF
initially stimulated the activation
and differentiation of T cells from
immature precursors by induction of
the CD4 antigen (Tanaka et al.,
1995). The incoming tachyzoites
activated the host defense system
(Isamida et. al., 1998). So, LF and
the parasite might have exerted dual
stimulatory effect on the immune
system with significant CD4 rise in
this subgroup versus controls. The
same scenario of events might have
occurred in SGII-b, in which treat-
ment of IS mice with LF before
infection, significantly reconstituted
the CD4 population as compared to
IS-infected controls (45.17% &
11.29% respectively). The signific-
ant difference in the CD4 level
between SGII-a & SGII-b was
probably due to LF consumption in
up-regulating the strong preliminary
CD4 reduction induced by CP in
SGII-b.
In the experimental subgroups,
after stimulating the CD4 response,
LF was bound to tachyzoites as a
cationic peptide capable of attaching
to anionic components on the bio-
logical membranes surface (Tanaka
et al., 1995). Since the cell surface
of the tachyzoite has a strong
negative charge, it binds cationic
substances, and this interaction led
to disruption of the function of the
parasites' membrane (Isamida et. al.,
1998). The %R viability in the
experimental subgroups was 88.84
& 89.49% respectively. Tanaka et
al. (1995) reported that viability
depended on membrane's integrity.
The %R in the infectivity rates of
tachyzoites in both experimental
subgroups was 83.33%. Peritoneal
parasite load produced by the
tachyzoites was reduced by 97.07%
& 95.20% in SGII-a, and SGII-b
successively. These data could
reinforce the previously proven
ability of LF to diminish host cell
penetration capacity of the parasite
(Omata et al., 2001).
Consequently, the striking inhibit-
tory effect of LF on the viability and
infectivity of tachyzoites, together
with the CD4 pool was responsible
for the clearance of tachyzoites from
host tissues (Denkers and Gazzin-
elli, 1998), might have simultaneo-
usly augmented in controlling the
outcome of infection in both experi-
mental subgroups. This accounts for
the significant decline in the %R in
the MR of SGII-a to 93.75%, and
the prolongation in their survival
time up till 20-22 days, in conjun-
ction with the %R in the peritoneal
(96.35%), and liver parasite loads
(95.69%), as compared to infected
controls. Thus, in SGII-b treatment
of IS mice with LF prior to infec-
tion, led to profound reduction in
mortality rate (90%) and increased
1033
survival time to 17-18 days,
together with the significant %R in
peritoneal, and liver parasite loads
by 95.92 & 90.33% as compared to
the IS-infected non-treated controls.
So, LF feeding to IS mice resulted
in their immune restoration and led
to an inhibitory effect on infection
in a manner statistically comparable
to what it did to infected mice with
normal immune function. Prophyla-
ctic activity of LF against a number
of infections has been shown using
IS murine models. It was found that
the alleviation of such infections by
LF feeding may correlate with the
enhancement of the number of
lymphocytes and their cytokine
responses (Artym, 2003; Artym et
al., 2004; Takakura et al., 2004; Ali
and Nouh, 2005).
Conclusion
The oral bovine LF can induce
resistance to acute infection with T.
gondii RH strain regardless the
hosts immune status. This could be
correlated to its ability to enrich
splenic CD4
+
T cell population, a
step which effectively contributes in
conferring resistance to infection in
IS mice, as well as those with intact
immunity. Such findings pave the
way to study the outcome of this
promising antimicrobial, immune-
modulator on cyst forming strain of
T. gondii to determine its efficacy in
controlling human toxoplasmosis.
This may provide a basis for
applying this multifunctional milk-
derived protein as an efficacious
prophylactic therapy in populations
at high risk for acquiring toxoplas-
mosis, or those prone to developing
severe form of infection, through
alleviating their immune balance.
These include pregnant mothers
who are first exposed to infection
during pregnancy or several months
before, persons handling cats espe-
cially children, and most important-
tly those with weakened immune
systems who have impaired activity
of their T cell function.
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1033
Legend to Figures
Fig. 1: DIF staining of splenic tissue showing viable CD4
+
T lymphocyte (V) with brilliant ring of fluorescence on surface, and non-viable CD4
+
T lymphocyte (NV) showing homogenous intra-cytoplasmic fluorescence (X1000).
Fig. 2: Total splenic lymphocytes viewed by phase contrast (X 400).
Fig. 3a: DIF of LF-treated mice reflecting the CD4
+
T lymphocytes enrichment (X 400).
Fig. 3b: DIF of CD4
+
T lymphocytes of untreated mice (X 400).
Fig. 4a: Numerous unstained T. gondii tachyzoites in the peritoneal fluid of untreated infected mice (X 400).
Fig. 4b: Few unstained T. gondii tachyzoites in peritoneal fluid of LF-treated infected mice (X 400).
Fig. 5a: Abundant T. gondii tachyzoites in peritoneal fluid of untreated infected mice stained with Giemsa (X 1000).
Fig. 5b: Scanty T. gondii tachyzoites in peritoneal fluid of LF-treated infected mice stained with Giemsa (X 1000).
Fig. 6a: T. gondii tachyzoites in liver impression smear of untreated infected mice stained with Giemsa (X 1000).
Fig. 6b: T. gondii tachyzoites in liver impression smear of LF-treated infected mice stained with Giemsa (X 1000).
Fig. 7a: Living tachyzoites of T. gondii stained with 0.2% trypan blue, appearing clear blue in color (x1000).
Fig. 7b: Dead T. gondii tachyzoites stained with 0.2% trypan blue, taking homogenous blue color, with
undetectable internal structure (x1000).
1
2
3-a
4-a
4-b 3-b
1033
5-a 5-b
6-b
6-a
7-b 7-a
1052
WHO IS WHO (April, 2009)
****************
Name: P. Dr. Mohamed Safwat Seif El Nasr
Family name: Seif El-Nasr
Birth date: October 1943
Nationality: Egypt
Marital state: Married, with a daughter and a son (both university staff)
Post: Professor of Internal Medicine, and Endocrinology Consultant,
Department of Tropical Medicine, Faculty of Medicine, Al-Azhar
University, Cairo, Egypt
Fax (202)33467381, Mobile (202) 0122285274
Scientific Society Membership:
1- The American Diabetes Association (ADA)
2- Selected for the ADA Council
3- The International Diabetes Federation (IDF)
4- EMA
5- The New York Academy of Sciences
6- The American Association for Advancement of Science
7- Advisor, Research Board, the American Biographical Institute
8- Consultant Board, the Egyptian Society of Parasitology
Relevant Facts:
1- Referee WHO/EMRO
2- WHO is WHO (Diabetes, Education, Research & Treatment)
ADA from 1998 - 2009
3- Who is Who, the American Biographical Institute
4- Sixty papers published in national and international Journals
5- Sharing in supervision and/or external examiner of 120 M.Sc.
& MD theses
6- Shared in many local, national and international congresses
1050
) * ISC (
..
. . ..
. . . .
.
*

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*



*



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NOTICE TO CONTRIBUTORS
This is a regular journal published by the Egyptian Society of Parasitology and is
concerned with the publication of research papers in the field of Parasitology and
related subjects. Three volumes are published annually April, August and December.
Two types of communications appear regularly; papers and correspondence. Letter to
the editor is also published. Publication is not restricted to Fellows of the Society but
non-Fellows can also publish on their own expense. Manuscripts should be signed by
the author or authors concerned and sent to the Chief Editor. All the manuscripts are
peer reviewed. However, the authors alone are responsible about all data and
scientific views they expressed.
Instructions for preparation of manuscripts: Paper should be sent in type-
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in relation to others and not in any way, a review of literature or aim of the work.
References should be listed at the end of the paper and arranged in an alphabetical
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as in the World List of Scientific Periodicals, 4
th
edition, 1964. The M.Sc. or Ph.D.
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Abstracts of this journal are published in Helminthological Abstracts, Series A,
Protozoological Abstracts (Commonwealth Institute of Parasitology), Veterinary
Bulle-tin and Index Veterinarius (Commonwealth Bureau of Animal Health), Tropical
Dis-eases Bulletin, Review of Applied Entomology, Quarterly Bibliography of Major
Tropical Diseases, US Medline database (Index Medicus ISSN: 0253-5890) and
WHO Eastern Mediterranean Index Medicus (ww.emro.who.int/His/VHSL/
Imemr.htm).
--------------------------------------------------------------------------------------------------
Al-Taawen Press: Cairo, Egypt. Mobile : 012/7338132
Consultant Board
Acarology,
Prof. Dr. Kawther M. El-Kammah, Faculty of Agriculture, Cairo University.
Community, Environmental and Occupational Medicine:
Prof. Dr. Aly Abdel-Hady Massoud, Faculty of Medicine, Ain Shams University.
Dermatology and Venaerology:
Prof. Dr. Mohammad Amin Amer, Faculty of Medicine, Zagazig University.
Entomology:
Prof. Dr. Bahira Mahmoud El-Sawaf, Faculty of Science, Ain Shams University.
Forensic Medicine and Toxicology:
Prof. Dr. Mohamed Safwat Soliman, Faculty of Medicine, Ain Shams University.
Gastroenterology, Hepatology and Infectious Diseases:
Prof. Dr. Ahmad Mohamed A. Massoud, Faculty of Medicine, Al-Azhar University.
Brigadier Dr. Mamdouh M. El-Bahnasawy, Military Medical Academy.
Histology:
Prof. Dr. Prof. Dr. Ahmed Said El Morsy, Faculty of Medicine, Ain Shams University.
Internal Medicine:
Prof. Dr. Mohamed Safwat Seif-El Nasr, Faculty of Medicine, Al-Azhar University.
Ministry of Health:
Prof. Dr. Mohamad Mustafa, General Director Endemic Diseases and Schisto Control.
Prof. Dr. Zeinab Mohamed Youssef, Undersecretary for Endemic Diseases.
Pathology:
Prof. Dr. Amal Mohamed Mangoud, Faculty of Medicine, Zagazig University.
Prof. Dr. Badawiya Bayoumi Ibrahim, Faculty of Medicine, Cairo University.
Pediatrics:
Prof. Dr. Hamed Mahmoud Shatla, Faculty of Medicine, Ain Shams University.
Pharmaceutical Science:
Prod. Dr. Neiven M. Abdel-Hady, Faculty of Pharmacy, Al-Azhar University for Girls.
Plant Protection and Rodent Control:
Prof. Dr. Awad Farahat A. El Bahrawy, Faculty of Agriculture, Suez Canal University.
Public Health and Behavioral Medicine:
Prof. Dr. Saad Mohamed Motawea, Faculty of Medicine, Mansoura University.
Zoology and Malacology:
Prof. Dr. Fathy A. Abdel-Ghaffar, Faculty of Science, Cairo University.
*****
Past Presidents since the Foundation of the Society
1969-1987: Prof. Dr. Mahmoud Hafez, Faculty of Science, Cairo University.
1988-1996: Prof. Dr. Hamid M. Khalil, Faculty of Medicine, Ain Shams University.
1997-2002: Prof. Dr. Nabil Taha Nasr, Faculty of Medicine, Cairo University.
2003-2009: Prof. Dr. Mohsen M. Hassan, Faculty of Medicine, Zagazig University.
JOURNAL OF THE EGYPTIAN SOCIETY OF PARASITOLOGY
(WORLD FEDERATION OF PARASITOLOGISTS, MEMBER AT
LARGE, AFRICA)
http://www.parasitology.eg.net
* * * * *
THE EXECUTIVE AND ADVISORY BOARD 2009
President: Prof. Dr. Abdel Hameed Abdel Tawab Sabry
Vice President: Prof. Dr. Aly Awad Aly Hassan
Treasurer: Prof. Dr. Atef Mohamed Aly El Shazly
Secretary General: Prof. Dr. Tosson Aly Morsy
Members: Prof. Dr. Ahmad Aly Aly Sabah
Dr. Fouad Mohamed Sayed Haridy
Prof. Dr. Magdy Abdel Latif Saleh Arafa
Prof. Dr. Mohamed El-Husseiny Fayad
Prof. Dr. Sanaa Nageeb Antonios Boctor
* * * * *
Honorary Members: Prof. Dr. Ebtesam M. Al-Mathal (East Saudi Arabia),
Prof. Dr. Saeed A. Al-Harthi (West Saudi Arabia),
Prof. Dr. Gehan Gamal-Eldin El Fandy (Egypt),
Prof. Dr. Mary E. Wilson (USA),
Prof. Dr. Patrice Bouree (France),
Prof. Dr. Reda L. Roufael El-Gamal (Egypt),
Prof. Dr. Robert W. Ashford (United Kingdom),
Prof. Dr. Safiya S.M. Khalil (Egypt),
Prof. Dr. Santiago Mas-Coma (Spain),
Prof. Dr. Sayeda Aly Gafaar (Egypt).
* * * * *
The Chief Editor: Prof. Dr. Tosson Aly Morsy
Editors: Prof. Dr. Abdallah Michael Boghdadi (Cairo University),
Prof. Dr. Amany Helmy M. Lashin (Benha University),
Prof. Dr. Heba M. Abdel Aaty (Ain Shams University),
Prof. Dr. Faiza Hussein Mohammad (Al-Azhar University for Girls),
Prof. Dr. Manal Farouk El Garhy (Cairo University),
Prof. Dr. Manal Salah-Eddin Mahmoud (Ain Shams University),
Prof. Dr. Mohammad El-Salahy M.M. Moneib (Assuit University),
Prof. Dr. Salwa Talaat El Mansoury (Alexandria University),
Prof. Dr. Samy El-Sheikh Tayel (Al-Azhar University),
Assist-Prof. Dr. Nahla M. Shoukry (Suez Canal University).
* * * * *
For Communications: Professor Dr. Tosson A. Morsy, Department of Parasitology,
Faculty of Medicine, Ain Shams University, Cairo 11566, Egypt.
Fax: (+202)24036497 Mobile: (+2) 0101331999
E-mail:morsyegypt2000@yahoo.com or morsy2000@hotmail.com
* * * * *
The Egyptian Book Center
Deposit No. 695/1978

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