) (
) (
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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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
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)
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-
(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
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
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
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
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,
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 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
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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932
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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
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
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
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
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%)
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-
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
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
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M
e
a
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t
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(
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No. of mosquitoes Mean temp.
Fig . 2: Effect of temperature on seasonal abundance of mosquito in Makka al Mukarrama District.
0
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100
M
e
a
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r
a
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f
a
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(
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
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No. of mosquitoes Mean temp.
991
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0
10
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30
40
50
M
e
a
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r
a
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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
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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
20
30
40
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(
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,
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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-
6
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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