J. R. BAKER
Cambridge, England
and
R. MULLER
International Institute of Parasitology
St Albans. England
VOLUME 31
ACADEMIC PRESS
Harcourt Brace Jovanovich, Publishers
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V
PREFACE
relate the new understanding to the prospects for improved therapy and
vaccination.
Gunter Schaub, now at the Ruhr University of Bochum but until recently
at the Albert-Ludwigs University in Freiburg, reviews a rather neglected
aspect of parasitic protozoology-the effects of trypanosomatids on their
insect hosts. Dr Schaub discusses not only the well-known “two-host’’
trypanosomatids of medical and veterinary importance ( Trypanosoma and
Leishmania) but also those much less studied members of the family which
parasitize insects only, a topic to which he has himself contributed greatly.
Most of these organisms are examples of the truly “successful” parasites,
those which cause little or no harm to their hosts, but one, Blastocrithida
triatomae, causes considerable damage to its hosts (reduviid bugs, the
vectors of Trypanosoma cruzi) and Dr Schaub discusses the possibility of its
being used as an agent of biological control of the bugs in areas where
Chagas disease is endemic (biological control of insects is also dealt with in
the last chapter of this volume).
Bruno Gottstein then reviews the immunology and immunodiagnosis of
infection with Echinococcus multilocularis. Dr Gottstein is at the University
of Zurich, which under Professor J. Eckert is undoubtedly the leading world
centre for research into this organism. Although E . multilocularis does not
have such widespread economic importance as E. granulosus, principally
because the latter utilizes ubiquitous domestic herbivores as intermediate
hosts, when alveolar echinococcosis does occur in man it is one of the most
serious parasitic diseases known. Variation within the genus Echinococcus
has already been reviewed by Drs Thompson and Lymberry in Volume 27,
but the present review outlines some very elegant work on the differential
diagnosis of the two species carried out in Zurich. Recent progress has
focused on early diagnosis of pre-clinical cases in endemic areas and this,
together with new treatment procedures, has dramatically changed the
prognosis of the disease. New purified antigens are now available, including
what may prove to be the first commercially available recombinant helminth
antigen, and the exciting prospects for a vaccine are outlined.
The final chapter, by Irene Popiel of Paravax Inc. and William Hominick
of Imperial College, London, is a continuation of that by James Petersen in
Volume 24 which reviewed the mermithids as biological control agents. The
present contribution deals mainly with the other nematode groups-the
allantonematids, steinernematids and heterorhabditids-but also briefly
reviews recent information on the mermithids in the ensuing seven years.
Following sections on morphology and taxonomy and on the biology of the
groups, stressing the population dynamics and environmental limitations,
there is a stimulating discussion of their commercial status and prospects for
the future. The techniques of mass propagation, production and storage of
PREFACE ix
I. Introduction .............................................. I
11. Epidemiologi r Sex Differences in Parasite Prevalence, Densit
and Clinical Disease Mani .......................... 2
A. Helminthic infections ...................................... 2
B. Protozoal infections ........................ 5
111. Evidence for Sex Differences Attributed to Exposure . . . . . . . . . . . . . . 9
A. Behavioural observations ............................... 9
B. Immunological observations . . . . . . . . . . . .......... 14
C. Effect of sex on the host immune response to chemotherapy . . . . . 20
IV. Evidence for Sex Differences Attributed to Hormonal and Genetic Factors . . . . 23
ntributing to sexual dimorphism in human
....................................... 24
parasitic disease contributing to sexual
26
............ 31
32
33
........... 31
C. Infection of the foetus and n ........................... 39
D. Foetal and infant immunity ........................... 49
VII. Conclusions ................................... 56
A. Maternakhild health .......................................... 51
B. Vaccine development . . . . . . . .................... 51
C. Drug treatment ..................................... 58
59
............. 60
.......................................... 60
I. INTRODUCTION
behaviour and work patterns of males and females, which are frequently
distinct; (ii) immunity to infection and response to treatment may differ
between the sexes; (iii) pregnancy alters susceptibility to infection and risk of
disease which can lead to deterioration in maternal health; (iv) infections
during pregnancy frequently influence the outcome of pregnancy; and (v)
maternal immune status relates to the development of infant immunity.
These factors are integral to our awareness of how control of parasitic
diseases in communities can be influenced by an understanding of the
pattern of infection in women.
The infections reviewed in this chapter are primarily those identified by
the Special Programme for Research and Training in Tropical Diseases
(World Health Organization) as being of particular public health concern
(onchocerciasis, filariasis, schistosomiasis, malaria, African trypanosomiasis
and leishmaniases), although reference to other infections is made where
relevant.
EVIDENCE
11. EPIDEMIOLOGICAL FOR SEX DIFFERENCES
IN PARASITE
PREVALENCE, DENSITY
AND CLINICAL
DISEASE
MANIFESTATIONS
A. HELMINTHIC INFECTIONS
I, Lymphatic jilariasis
One problem facing the interpretation of prevalence data is the relative in-
sensitivity of blood slide examination in detecting low density parasitaemias.
In Tonga, for example, 68% and 74%, respectively, of men and women aged
21-50 years were positive by a sensitive filtration method, but only 33% of
males and 26% of females by blood slide examination (Desowitz and
Hitchcock, 1974). If women are less exposed, or have lighter infections,
lower prevalence in the reproductive age may reflect the insensitivity of
blood slide techniques. A similar problem is faced when interpreting the
observed plateau effect in prevalence curves, i.e. a flattening of the curve
with increasing age, which is thought to indicate belated development of
host resistance (Piessens and Partano, 1984). This latter problem has been
addressed by two recent papers which analysed microfilarial frequency
distributions by fitting various statistical models (Das et al., 1990; Grenfell et
al., 1990). One finding was that a large proportion of observed microfilariae-
negative individuals may be truly negative, due to the absence of adult
worms or the presence of unmated adults only, rather than the result of an
inaccurate blood sampling process. The models also provided indirect
evidence for the operation of density-dependent limitations on parasite
burden, as reflected in microfilarial counts which might be attributable to
acquired immunity. Mechanisms acting to reduce microfilarial infection in
women of reproductive age may differ from those operating to diminish
microfilaraemia with increasing age.
2. Onchocerciasis
1- -1
0- 5- 10- 15- 20- 25- 30- 35- 40- 45- 50- 55- 60- 65-
Age (years)
FIG. I . Geometric mean microfilarial density by age and sex in 65 villages in West
Africa and in seven Guatemalan fincas (estimated from Kirkwood et al., 1983a;
Brandling-Bennett et al., 1981). Reproduced with permission from Brabin, L.
(1990b), Acta Leidensia 59, 413426.
Ocular lesions and blindness are less frequent in females. The severity of
ocular onchocerciasis is known to be related to the intensity of infection
(Thylefors and Brinkman, 1977) and the community microfilarial load
(CMFL) has been developed as an index of the community level of infection
(Remme et a/.,1989). In 33 West African savanna villages, mean microfilar-
ial loads in the anterior chamber of the eye and in the cornea showed a linear
relationship with the CMFL and the relationship appeared to be the same
for both sexes. None the less, the prevalence of posterior segment lesions was
higher in males-a difference which remained statistically significant after
correction for intensity of infection. With a different approach, Kirkwood et
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 5
al. (1983b) found that ocular lesions were more common in males after
applying a logistic regression analysis to data from 53 OCP villages in order
to control for sex and duration of exposure at a given microfilarial load. The
results indicated that males were about I .5 times more likely to be blind than
females of the same age and same level of infection (P c 0.001). One
disadvantage of this study was that it did not control for non-onchocercal
eye disease, but it does suggest that community level indicators may obscure
differential sex risks.
B. PROTOZOAL INFECTIONS
2. Visceral leishmaniasis
From very early, sex differences in visceral leishmaniasis were noted and a
number of studies of kala-azar in India addressed the question of why more
cases were diagnosed in boys than in girls (Balasubramanian, 1920-1 92 1;
Cunningham and Pundit, 1924-1925). Poor case detection was suspected in
a society where women’s mobility was restricted by seclusion in purdah
(Turkhud et al., 1925-1926). Napier and Das Gupta (1931-1932) showed
that both the age and sex distributions of kala-azar were affected by distance
from a dispensary. In the Bihar epidemic in 1977-1978 the ma1e:female ratio
of confirmed reported cases of kala-azar was 5.5:l (Thakur, 1984). Given
that family clustering of infection is characteristic of Indian kala-azar
(Michael, 1925-1926; Nandy et al., 1988), it would be surprising if females
were not frequently exposed and more cases in girls were not found by active
case detection.
TABLE1 Age an2 sex distribution of visceral leishmaniasis cases in Kenya, south
Ethiopia and Sudan (Jahn et al., 1986; Ayele and Ali. 1984; Van Peenen and Reid,
1963, respectively)
Totals 95 69 24 10 1I9 85
3. Malaria
Malaria parasite rates are often not reported by sex and there is a general
belief that parasite prevalence in males and non-pregnant females is similar.
This was not the finding of the Garki project, where lower parasite rates and
densities were found for all age groups of females above 4 years of age for
both Plasmodium falciparum and P . malariae (Molineaux and Gramiccia,
1979). In children aged 4 years and under, parasite rates were similar for
both sexes. Spleen and parasite indices for males aged 5-14 years were
significantly higher than those in females in another large study (4500
subjects) in Pattukkotai, south-east India (Russell et al., 1938). Several
smaller studies have shown higher parasite densities in infant girls (McGre-
gor, I. A., 1964; Hendrickse et al., 1971). Considering that mortality from
malaria is greatest in children under 5 years old in holo- and hyperendemic
areas, any advantage to females in parasite clearance appears to be slight.
Females experience increased susceptibility to malaria in their first preg-
nancy (Brabin, B. J., 1983; Brabin, B. J. et al., 1988) and malaria-associated
anaemia has more serious consequences in adolescent girls and women
(Brabin, B. J., 1990). In an early study, higher rates of albuminuria were
noted in females in Surinam although rates of P . malariae and P. falciparum
nephritis were higher in males (Van der Kuyp, 1950). Since this was a
hospital-based study, the results may be due to case selection bias.
Sex differences are apparent in clinical syndromes associated with malaria,
such as hyper-reactive malarious splenomegaly and Burkitt’s lymphoma. In
Papua New Guinea, where hyper-reactive malarious splenomegaly occurs
with varying degrees of severity (Brabin et al., 1989), higher spleen rates
have been found in women. In a total village survey, Crane and Pryor (1971)
observed a higher spleen rate (males 72%; females 88%) and larger average
enlarged spleen size in women, and this confirmed similar results from
earlier, non-random samples from the Sepik region (Mackerras and Aber-
deen, 1945; Peters, 1960). Comparable data were reported by Schofield
(1962) in two complete village surveys and, more recently, from Madang
(Brabin, L., 1988).
Burkitt’s lymphoma, a cancer associated with both malaria and Epstein-
Barr virus (EBV) infection, has been observed more frequently in boys than
8 L. BRABIN AND B. J. BRABIN
girls in Uganda and Papua New Guinea. In the Mengo district of Uganda
the incidence rate among males was 1.5 that of females. Incidence was higher
in females under 5 years of age but was considerably higher in males over 15
years (Morrow et al., 1976). In the Lango and Acholi districts the incidence
rate was twice as high in males and the ma1e:female ratio was higher in the
younger age groups (Morrow et a[., 1977). In Papua New Guinea, Burkitt
and O’Conor (1 96 1) reported a male predominance of 2 to 1 but noted that
this was also the approximate ratio of male to female patients in general
hospital admissions. In a later study the ratio was noted to be 1.8: 1 (Reay-
Young and Chir, 1974). In contrast, in North Mara, Tanzania, from 1964 to
1983 the male:female ratio was similar overall but showed a marked
difference in the 6-7 years age group, where there was a large excess of
female cases and the ma1e:female ratio was 0.29:l (Geser and Brubaker,
1985).
The factors which affect the age and sex distribution of Burkitt’s lym-
phoma and how both malaria and EBV affect the distribution of cases are
not clear. In North Mara females had higher geometric mean titres (GMT)
for EBV but it is of interest that males bearing the X-linked lymphopro-
liferation gene are predisposed to EBV infection, and in them a variety of
lymphoproliferative diseases develop, such as malignant lymphomas and
malignant mononucleosis (Barnabei et al., 1982). One explanation for sex
differences may be related to differential susceptibility to EBV infection
rather than to malaria. The higher frequency of Burkitt’s lymphoma in boys
than in girls, which is the most frequent observation from endemic areas, has
posed a dilemma for the hypothesis that malaria causes Burkitt’s lymphoma
since sex differences in malaria prevalence are not recognized (Geser et al.,
1989). In North Mara, the sex ratio of Burkitt’s lymphoma and malaria were
said to be similar (although malaria antibody data by sex and age were not
presented), and there appeared to be no conflict with the possibility of a
causal role for malaria (Geser et al., 1989). This problem will not be resolved
until the questions of sex differences in malaria prevalence, and whether this
is affected by the level of malarial endemicity, are addressed.
4. African trypanosomiasis
Males Females
Age group (years) Number" Percentage Number Percentage
CL-4 158 1.3 180 2.8
5-9 209 0.5 186 2.6
Is14 20 1 2.0 168 4.8
15-19 124 4.0 107 4.7
2&29 I22 5.0 136 8.8
3w9 163 8.0 225 8.0
111. EVIDENCE
FOR SEX DIFFERENCES
ATTRIBUTED
TO EXPOSURE
A. BEHAVIOURAL OBSERVATIONS
Exposure is affected by the division of labour, age, family size and labour
requirements, economic and social status, but womens' activities are usually
delineated from male activities. The importance of documenting patterns of
This terminology has been used to avoid commitment to the precise taxonomic status of the
names rhodesiense and gambiense [eds].
10 L. BRABIN AND B. J. BRABIN
1. Helminthic infections
many areas, water contact by males decreases with age whereas it remains
more constant for females. Increased susceptibility of young girls was
implied in a study of Schistosoma mansoni in Machakos, Kenya, where girls
less than 9 years of age were judged to have less water contact than boys but
higher mean egg counts of S. mansoni (arap Siongok et al., 1976). In
Zanzibar, in a high prevalence area for S. haematobium, schistosomiasis-
related morbidity was measured in schoolchildren by ultrasound (Hatz et al.,
1990). A striking feature of the results was the relatively low risk of uropathy
among females when compared with egg counts and haematuria. The highest
proportion of uropathy and haematuria was in girls aged M years, the
reverse of the situation for boys of the same age. The lowest proportion of
female uropathy and haematuria cases occurred in women of reproductive
age (26-40 years). Menstruation did not appear to influence the predictive
potential of microhaematuria. By contrast, in an area of low endemicity
studied by Hatz et al. (1990) (Mauritius), the most severe uropathy was
found in adult women. No sex difference was found in the incidence of
squamous cell carcinoma of the bladder in Zimbabwe, but these obser-
vations could not be correlated with exposure since data were retrospectively
based on medical records (Thomas, J. E. et al., 1990).
The availability of macrofilaricidal drugs permits quantitative studies of
reinfection rates and water contact which can be analysed by sex. In The
Gambia, girls had significantly higher reinfection rates of S. haematobium
following treatment with praziquantel than boys of the same age range, and
these differences remained after controlling for levels of exposure and
eosinophil counts (Hagan et al., 1985). In women ( > 15 years), intensities of
infection following treatment were 100 times lower than in men, although
levels of exposure were only five-fold less (Wilkins et al., 1987). Differences
were not observed in 72 schoolchildren in Tanzania (Hatz et al., 1990), but
water contact studies were not done. These results seem to indicate differ-
ences in the immune response of females of different ages as well as
differences due to sex.
2. Protozoal infections
(Desjeux et al., 1974). and soldiers have been particularly affected in French
Guyana (Dedet et al., 1989).
Females are at risk in some areas. Since the 1980s, the migration of entire
families to new residential areas in endemic areas of Panama has increased
infection rates (Arias, 1988). In Brazil, cutaneous leishmaniasis is now
observed close to metropolitan regions in areas where forest was cleared
many years previously. In one study area 35 km from Rio de Janeiro city
centre, cases due to Leishmania braziliensis braziliensis were found frequently
in women and children (Oliveira-Net0 et al., 1988). Similarly, in the north
Amazon jungle, villages are likely to be well inside the forest, bringing the
total population into a risk zone (Guerra, 1988). In Costa Rica, cases of
cutaneous leishmaniasis reported by province show an equal distribution
between the sexes (Hidalgo, 1988).
From regions outside the Americas, detailed data on sex differences are
limited. In a retrospective study of cutaneous leishmaniasis in Meta Abo,
Ethiopia, similar rates were found for all ages and both sexes (Wilkins,
1972). However, of 33 patients with diffuse cutaneous leishmaniasis studied
by Bryceson (1969), 21 were male and 12 female. In Khartoum, up to 1975,
51 cases of mucocutaneous leishmaniasis had been reported, all in adult
males (El Safi, 1988). Of 9657 cases of cutaneous leishmaniasis reported in
Khartoum province between September 1986 and March 1987, 61% were
males and 39% females (El-Safi and Peters, 1991). To what extent these
differences were due to exposure, or to failure of females to report for
treatment, cannot be assessed.
B. IMMUNOLOGICAL OBSERVATIONS
1. Helminthic infections
this concept is available. For example, the prevalence and levels of immuno-
globulin (Ig) G antibodies to the sheath of B. rnalayi and W . bancrofti micro-
filariae are much higher in sera from amicrofilaraemic than from microfilar-
aemic donors (Piessens et al., 1980). It has been observed that immigrants
developed IgM antibodies to microfilariae soon after arrival in a filariasis
endemic area but did not develop IgG antibodies to the same antigen
preparation until they had lived continuously in the area for many months
(Piessens et al., 1987). In a study by Kurniawan et al. (l990), it was assumed
that those with low levels of ( < 1 : 100) IgG antimicrofilarial antibodies were
“underexposed”. In this study, “exposed” individuals were divided into
three categories: those in whom no objective evidence of infection could be
detected; those with filarial antigens present in the sera; and those who were
microfilaraemic. The purpose of the study was to compare antigen recog-
nition patterns of defined groups of amicrofilaraemic persons with similar
degrees of exposure, and it was conducted in Indonesia with 81 adult
immigrant volunteers. Comparison of the immunological response by sex in
a representative population is undocumented, but it would be of interest (i)
to determine the proportion and age distribution of amicrofilaraemic
females, (ii) to define the characteristics of females with active infection, and
(iii) to ascertain the proportion of those individuals who are pregnant.
2 . Protozoal infections
I I I I I I I I I #
TABLE
3 Proportional risk ratios by age and sex for visceral leishmaniasis in Voo,
Kenya (1957-1961) (Southgate, 1964)
s(r59 10.9 -
> 60 6.3 ~
TABLE4 Skin test reactions and total and specific IgE antibody levels by sex in
Venezuelan patients with cutaneous leishmaniasis (Lynch et al., 1987)
both specific and total IgE were significantly higher in males. The authors
suggested that elevated total IgE levels may be associated with deficiencies in
T cell function. The presence of detectable levels of specific IgE antibody
seemed to depend on the cellular immune reactivity of the individual rather
than on clinical status.
Recent studies using the S . rnansoni-mouse system indicate that the schisto-
somicidal compound, praziquantel, may depend for its efficacy upon the
humoral immune status of the host (Brindley and Sher, 1987; Flisser et af.,
1989; Piper et al., 1990), although it is not yet known whether immune-
facilitated drug action is also a feature of schistosomiasis in humans
(Mitchell, 1990) nor how far it is directed against a limited subset of
antigens. Immunosuppression is known to reduce the efficacy of experi-
mental chemotherapy for several parasitic diseases, including malaria (Lwin
et af., 1987), trypanosomiasis (Frommel, 1988), onchocerciasis (Bianco et af.,
1986) and visceral leishmaniasis (Iwobi et af., 1991). Clinical studies have
suggested a link between poor efficacy of chemotherapy against severe
cerebral malaria and low levels of antimalarial antibody (Doenhoff et af.,
1991). Difficulties in treating diseases like diffuse cutaneous leishmaniasis
may reflect poor T cell immune responsiveness to parasite antigens (Doen-
hoff et al., 1991).
TABLE
5 Seroprevalence (indirectjuorescent antibody test) by age and sex in three areas of Gambian sleeping sickness in Congo ( F r k d .
1981)
Ngabe
Males 178 2.8 252 8.3 376 8.0 40 1 12.7 1207 8.9
Females 216 2.8 26 1 6.1 382 11.8 463 20.7b 322 12.3b
Niari
Males 809 0.7 969 0.9 121 1 4.3 I687 5.9 4676 3.5
Females 805 0.1 933 1.4 I189 2.2b 2232 5. I 5159 3.0
The sex of the host may affect the immune response to chemotherapy and
this may be expressed both in relation to toxicity (or adverse reactions) and
efficacy. This issue is difficult to address on the basis of current knowledge
since drug trials systematically exclude female subjects because of pregnancy
risks. There are also few experimental data available and, although Goble
and Konopka (1973) demonstrated the influence of sex on chemotherapy in
several systems, the finding that some drugs were more effective in males and
others in females could not be explained. Some observations on Indian kala-
azar are suggestive of a poorer treatment response in males. Post kala-azar
dermal leishmaniasis (PKDL) is more commonly observed in males (Acton
and Napier, 1927-1928; Napier and Das Gupta, 1931-1932). In 1000
consecutive cases of PKDL seen in Calcutta, 80% were males in the second
and third decade of life (Sen Gupta, 1956). In one study of the Bihar
epidemic (India) a large number of cases had previously received inadequate
treatment and all but two responded when managed on controlled thera-
peutic regimens (Aikat et al., 1979). Table 6 shows that the male:female
ratio of cases presenting without previous treatment was similar. Of patients
giving a history of treatment, most were males. This might suggest either
that males were more likely to discontinue treatment or that females
responded better to therapy. From Table 7 it is seen that the number of
untreated males and females was similar in all age groups, indicating that the
discrepancy in treated patients was not due to any one age category of
females failing to present for treatment.
TABLE
6 Male :female ratio of kala-azar cases in Bihar, India, grouped in relation to
previous treatment and method of diagnosis (Aikat et al., 1979)
TABLE
7 Age and sex distribution of treated and untreated kala-azar cases in Bihar,
India (Aikat et al., 1979)
Untreated 33 31 12 11 9 6 4 5
M , male; F. female.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 23
Iv. EVIDENCE
FOR SEX DIFFERENCES
ATTRIBUTED
TO HORMONAL
AND
GENETICFACTORS
1. Hormonal factors
increases male susceptibility, available evidence does not suggest that oestro-
gen increases resistance in females. A study by Wesley (1973) found that
resistance in females could be attributed to lack of androgen rather than
presence of oestrogen. In another study, older female jirds (retired breeders)
were found to be more susceptible than young female jirds (Devereux and
Ash, 1978). Declining oestrogen levels in old female jirds would be unlikely
to increase susceptibility. One interpretation of the data would be that
hormonal changes during pregnancy have an additional effect on adult
worms or depress parasitaemia. This would explain the higher parasitaemias
seen in post-reproductive jirds and does not conflict with the conclusion that
testosterone increases susceptibility. Although increased oestrogen levels
during pregnancy are immunosuppressive, the role of progesterone, levels of
which are also increased during pregnancy, is not fully understood.
cantly higher liver parasite burdens (Mock and Nacy, 1988) than females. In
the latter study testosterone treatment of female BALB/c mice resulted in an
88% increase in the number of liver amastigotes. It was suggested that
testosterone modulation of L. major infection could be due to a direct effect
of the hormone or to an indirect effect on cell-mediated immunity-over and
above susceptibility due to genetic factors. Exacerbation of infection with
Leishmania mexicana amazonensis in hamsters treated with testosterone was
also observed by Arcay ( 1 985). In one set of experiments using gonadecto-
mized mice with some males having silastic tubing implants containing
oestrogen, it seemed that oestrogen may determine the comparatively
greater resistance of female DBA/2 mice to L . mexicana (Alexander, 1988).
Other experiments, by contrast, showed relatively greater resistance to
infection with L. major in male mice (De Tolla et al., 1981; Giannini, 1986;
Alexander, 1988). Conflicting results may be the result of variations in
experimental procedure. Gonadectomy, for example, reduces sex hormone
levels, but does not totally deplete sex hormones due to compensation by
extra-gonadal tissue (Ansar Ahmed et al., 1985).
3. Genetic factors
the same segregation as males, but the range of parasitaemia was always
about 2 log,, values lower in females, except when the F1 generation was
back-crossed to BALB/c, when parasitaemias in both male and female
progeny were indistinguishable. Greenblatt and Rosenstreich (1 984) also
showed that the longer any strain of mice survived, the greater was the
difference in survival time between male and female mice exposed to
infection with the rhodesiense form of T. brucei. Their investigations
suggested that an X-linked gene could not account for the differences
observed between resistant (C57BI/6) and susceptible (BALB/c) mice, but
the mechanism responsible was not identified.
that cells from male and androgen-treated female mice presented a soluble
antigen (KLH) less efficiently than cells from normal female or castrated
mice (Weinstein et al., 1984). In conjunction with evidence that lymphocytes
from female mice are more reactive than those of male mice to cell-
associated allo-antigens, it seems that there are major sex-associated differ-
ences linked to functions known to be regulated by MHC-encoded glyco-
proteins.
v. EVIDENCE
FOR SEX DIFFERENCES
RESULTING
FROM IMMUNE
STIMULATIONDURING HUMANPREGNANCY
VI. PARASITIC
INFECTION
AND PREGNANCY
OUTCOME
TABLE 8 Mean age specific indirect fluorescent antibody titres (log,,) by age and
parity in pregnant women at jirst ante-natal visit in western Kenya (Nangina)
Parity group
Age group (years) P O P 1-3 P 4-6 >P 6
< 18 3.31 f 0.08" 3.54 f 0.90 - -
(Wb (7)
19-20 3.49 f 0.50 3.60 f 0.50 - -
(27) (27)
21-22 3.1 1 3.42 f 0.57 4.01 -
(2) (22) (1)
23-24 2.80 3.77 f 0.62 3.81 -
(3) (12) (3)
25-26 - 3.24 f 0.70 3.01 -
(7) (3)
> 26 - 3.33 f 0.40 3.70 & 0.62 3.68 0.58
(6) (14) (12)
Standard deviation.
Numbers in parentheses are number of patients screened.
A. MATERNAL MORBIDITY
1. Helminthic infections
2. Protozoal infections
Chronic infections with T. cruzi are not associated with severe morbidity.
Polyhydramnios and varicose veins were complications observed in sero-
positive pregnant women in Argentina (Hernandez-Matteson et al., 1983).
African trypanosomiasis is traditionally regarded as a severe and acute
infection in pregnancy, although it is now apparent that asymptomatic
seropositive women may have uncomplicated pregnancies (Lapierre and
Coste, 1963). African trypanosomiasis is frequently associated with anaemia
as a result of immunological mechanisms or splenomegaly, as is visceral
leishmaniasis.
Severe anaemia in pregnant women living in malarious areas is associated,
in several reports, with increased risk of pre-term delivery and perinatal
mortality. These reports have been summarized by B. J. Brabin (1991a).
Chronic splenomegaly is an important complication of malaria in women
living under holoendemic conditions (Topley, 1968; Brabin, B. J. et al.,
1988). Spleen rates of 25% are not uncommon in these areas and in coastal
Papua New Guinea spleen rates in women of over 50% have been reported
by several investigators over the past 40 years (Metselaar, 1956; Brabin, L.,
1988). Table 9 shows the relation between haematological indices and spleen
size in pregnant women from rural Madang, Papua New Guinea. Anaemia
increases with increasing spleen size, as does red cell folacin concentration.
The high red cell folate concentration may be related to the chronic
reticulocytosis of malaria, as reticulocytes have high concentrations of
folate, although this may not be the only mechanism to explain this
observation (Brabin, B. J., in press). The higher free erythrocyte protopor-
phyrin and lower mean corpuscular haemoglobin concentration with
increasing spleen size suggest that iron deficiency increases with the develop-
ment of splenomegaly. Iron deficiency is almost universally present in
malaria-endemic areas and may be causally associated with malaria
(McGregor, I. A., 1988).
During pregnancy in malaria-endemic areas, spleen rates and size increase
(Brabin, B. J. et al., 1988). This has important consequences for maternal
and foetal health. In view of the increased requirements for iron and folate
during pregnancy, the additional burden of splenomegaly can result in the
progressive development of iron deficiency and haemolysis with advancing
gestation. In Madang, Papua New Guinea, the prevalence of severe anaemia
( < 8 g/dl) in the last trimester was 44% in primigravidae and 29% in
multigravidae for those who had no antenatal care (Brabin, B. J. et al.,
1990b). Such women with severe anaemia have an increased mortality risk
during pregnancy, especially if associated with post parturn haemorrhage or
puerperal sepsis. Other complications of malaria in pregnancy include
hypoglycaemia and cerebral malaria, although under holoendemic con-
ditions these are very uncommon. Renal insufficiency is a rare complication.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 31
The risks and severity of malaria in pregnant women are discussed in detail
by B. J. Brabin (1991a).
TABLE9 Haematological indices and spleen size in pregnant women in rural Madang,
Papua New Guinea
Haemoglobin (g dl-') 8.9 f 1.5 8.6 f 1.5 8.4 f 1.4 8.3 f 1.8
(1 26) (55) (35) (30)
Haematocrit (YO) 29.6 f 4.3 29.4 f 4.7 28.3 & 5.3 28.3 f 4.7
(120) (52) (27) (27)
Free erythrocyte proto- 34.2 f 15.4 31.5 f 12.2 37.2 f 16.4 40.3 f 21.2
porphyrin ( l g dl- ') (1 20) (51) (35) (28)
Mean corpuscular haemo- 30.5 f 3.5 29.4 f 2.2 30.2 5.0 28.9 f 3.1
globin concentration (g dl) (1 19) (52) (27) (27)
Red cell folacin 1641 f 744 1821 f 1067 2340 f 842 2245 f 685
(nmol 1- l ) (63) (36) (17) (14)
Reticulocytes (YO) 2.1 f 2.0 1.8 f 2.5 1.9 f 1.7 3.1 f 4.0
(52) (22) (16) (16)
~~
Spleen size, distance palpable below costal margin. Numbers in parentheses are numbers of
a
patients examined (women examined at first antenatal visit).
1. Helminthic infections
parasite was present, from 20.5% in uninfected women to 23.2% with two or
more species. Short, presumably malnourished, women had a significantly
greater risk of foetal growth retardation. As there is suggestive evidence that
malnutrition influences the pathogenicity of intestinal parasites in humans
(Crouch, 1982; Crompton, 1989, these findings support the hypothesis
that foetal growth retardation is related both to parasitic burden and to
nutritional status.'
2 . Protozoal infections
In this study, 65% of babies were low birth weight (<25OOg) and born to
primigravidae anaemic at booking ( < 8 g/dl). Of these, three-quarters were
growth retarded and the rest were premature. This association of maternal
malaria anaemia and low birth weight will be influenced by the prevalence of
iron deficiency. There is very little information on the effect of maternal iron
deficiency on outcome of pregnancy in these anaemic populations. Maternal
iron deficiency has been associated with an increased risk of low birth weight
in a non-malarious area (Whiteside et a!., 1968).
Table 10 shows data from the Papua New Guinea study (Brabin, B. J. et
al., 1990b) which illustrate how the risk ratio for low birth weight increases
in primigravidae with decreasing levels of maternal haemoglobin. In this
same population, women with evidence of iron deficiency at booking (free
erythrocyte protoporphyrin (FEP) > 35 pg/dl) had a significantly increased
risk of delivering a low birth weight baby (relative risk 4.1, 95% confidence
limits 1.2-14.0). Maternal anaemia and iron deficiency were not associated
with increased risk in multigravidae. The population attributable risk of low
birth weight in primiparae associated with anaemia at booking in this
population would be 43.5% (haemoglobin < 9 g/dl) and 50.0% (haemo-
globin < 8 g/dl). The population attributable risk of low birthweight associ-
ated with iron deficiency at booking in primigravidae (FEP >35pg/dl)
would be 55.9%.
TABLE10 Relative risk for low birth weight at Alexishafen, Papua New Guinea, in
primiparae according to maternal haemoglobin at booking and delivery
Relative risk'
At booking At delivery
Haemoglobin ( g dl- ') (n = 48) (n = 71)
1, Helminthic infections
2. Protozoal infections
a An infection is termed congenital if the infected infant of an infected mother had never been in an endemic area, or if the parasite was found in the
7 Sina et al.
( 1979)
gam biense Cameroun - 30 Both treated and well 7 months later
Fever at 5 months gestation, but normal pro-
gression of pregnancy. Diagnosed and-treated
at 8 months. Twins born with cords and peri-
pheral blood films positive. Treated and
apparently well 4 months later
8 Darre et al. gambiense Chad/Cameroun -
b
Infection in expatriate mother at about 6 months
( 1 937) France gestation, with a febrile event. Febrile epi-
sodes increased in frequency post-natally.
Child severely mentally retarded from birth
9 Triolo et al. gambiense Cameroun 25 Febrile episodes during pregnancy associated
(1985) with trypanosomiasis and malaria. Child born
with hepatomegaly. Mother and child treated
with good response
10 Kalanda gambiense Zaire 25 Mother diagnosed with first stage infection at 8
(1991) months gestation. Twins born, apparently
well. Thick smear of twin I positive at birth,
but serological results in twin 2 not positive
till 6 weeks. Mother and twins were treated
and yielded normal results at 6 months
TABLE14 Gambiense sleeping sickness in children aged 0-4 years assessed by indirect
fluorescent antibody test in the Congo and parasitologically in Zaire (Frbzil, 1981;
Henry et al., 1982)
proportion of patients originated from rural areas but had lived for a
number of years in the city. The migration history of the mother of one
congenital case is probably similar to that of many urban women: she moved
to an urban area at 13 years of age, lived for a few years in an area of low
transmission, and finally moved to a non-endemic area where, 9 years later,
aged 33 years, she gave birth to congenitally-infected twins in her seventh
pregnancy (Hoff et al., 1978). The occurrence of congenital infection in
urban areas is of concern because transmission can occur by vertical
infection even in areas where vector control has interrupted direct trans-
mission through triatomine bugs although, if congenital infection occurs
only sporadically, this may not represent a serious threat to control. The
incidence of congenital infection has been estimated to vary between 2% and
10% (Bittencourt et al., 1985a). There is little information on its frequency
in endemic areas, but confirmed cases appear to be rare (Mott et al., 1976).
Although there is little evidence to question the assumption that immunity
to T. cruzi infection is lifelong, relevant information on how frequently
persons exposed to infective metacyclic forms of the parasite under natural
conditions are infected is not available. Nor is it known whether immune
response mechanisms in infected individuals living in non-endemic areas
differ from those living in places where exposure to reduviid vectors is
common, and whether this would alter maternal immunity during preg-
nancy.
The factors leading to congenital infection have never been clearly
delineated. Bittencourt et al. (1988) have suggested that parasitaemic
episodes can occur repeatedly during pregnancy. Of 77 women chronically
infected with Chagas disease, on whom xenodiagnosis was performed during
pregnancy, 10 ( 1 1 YO)had three or more positive diagnoses, and one of these
mothers gave birth to a congenitally-infected baby. It was considered that
the more frequent or persistent the parasitaemia, the more probable was
congenital infection. Whether parasitaemic episodes occur more frequently
in pregnant than in non-pregnant women requires further investigation. In
this study the same patients were taken as a control group after delivery, and
no significant difference in incidence rates of parasitaemia was observed.
These women were urban residents and ideally the results of the study need
to be compared with those of pregnant and postnatal women delivering in
an endemic area.
Besides frequency of parasitaemia, virulence of the infective strain,
duration of parasitaemia (a characteristic of different strains), gestational
stage when parasitaemia occurs, maternal age, and parity are also likely to
influence the timing or severity of congenital infection. Azogue et al. (1989,
in Bolivia, found that most congenital cases occurred in newborn infants
weighing 100&2500g, with a gestational age of 26-37 weeks. In a series
48 L. BRABIN A N D B. J. BRABIN
(Le Van Hung, 1951; Kortmann, 1972; Reinhardt et al., 1978; Marshall,
1983; Ezeoke, 1985). Infants born to these mothers are at higher risk of low
birth weight (Kortmann, 1972), although this may be because a higher
proportion were primigravidae. In such cases, parasitaemia appears to clear
rapidly or within a few weeks of birth (Kortmann, 1972; Diallo et al., 1983)
and symptomatic malaria does not occur. Several studies have shown a zero
cord infection rate despite high malaria prevalence in mothers. The reasons
for this are unknown.
I. Helminthic infections
( a ) Schistosomiasis
( i ) Passive immunity. Maternal transfer of antibody, resulting in the
capacity to resist challenge by a primary infection with cercariae, has been
demonstrated in newborn rats (Knopf and Coghlan, 1989). In human
studies, only a proportion of children born to mothers with detectable
schistosomal antibodies are antibody positive. In the West Indies, no
antibodies to S. mansoni were found in half of the newborn children of
seropositive mothers (Lees and Jordan, 1968). Of 31 seropositive newborn
infants, antibodies had waned by the age of 6 months. In Burundi, the
presence of schistosomal circulating antigens (CSA) in 24 of 26 cord sera
was associated with a lack of significant antibody titres in the newborn
infants (Carlier et al., 1980). It is not known how children with or without
detectable antibodies at birth differ in their subsequent immune response to
schistosomiasis.
newborn infants and it is possible that the placenta acts as a site for
dissociation of antigen-antibody complexes (Loke, 1982). Foetal exposure
to free or bound schistosomal antigens would influence the degree of antigen
exposure and might influence the subsequent development of either toler-
ance or sensitization. Both these effects have been reported for schisto-
somiasis in human (Lewert and Mandolitz, 1969) and animal studies (Uhr et
al., 1957; Hang et al., 1974).
Children (aged 7-36 months) of infected mothers were studied in Brazil
for delayed hypersensitivity reactions to S. mansoni antigens (Camus et af.,
1976). Delayed hypersensitivity was detected in about half the children. In a
later study sensitization was demonstrated in 17 of 25 newborn children of
mothers with positive delayed skin reactions to S. mansoni (Tachon and
Borojevic, 1978). Camus et al. (1976) considered that delayed hypersensiti-
vity reactions indicated sensitization to CSA. Neither of these studies
defined maternal characteristics or the epidemiological conditions under
which sensitization occurred, although the second study took place in an
area where transmission of S. mansoni had been interrupted 4 years pre-
viously. Sensitization may also have occurred postnatally from antigens in
breast milk, although this was not investigated.
(6) Filariasis
2. Protozoal infections
( a ) Malaria
( h ) T . cruzi
( c ) African trypanosomiasis
those of Whitelaw and Urquhart (1989, who demonstrated that young mice
suckled by mothers infected with T. brucei were immune to homologous
trypanosome challenge. The immunity was considered to be transmitted in
colostrum or milk, since mice born of infected mothers and transferred at
birth to normal foster mothers were susceptible to challenge. It is difficult to
relate these observations to human infection because placentation in rodents
is of the haemoendothelial type and the majority of immune transfer from
mother to offspring occurs after birth (Brambell, 1970).
In humans the amount of maternal antibody available to the neonate
depends on the time available for production and placental transfer and,
therefore, on the gestational time at onset of primary or recurrent infections
in pregnancy (Brabin, B. J., 1985a). Transplacental immunity is thought to
be less protective against infections caused by organisms eliciting maternal
antibodies that are predominantly of the IgM class (Miller and Stiehm,
1983), and Loke (1978) has observed that maternal protective antibodies
may not be able to penetrate the blood-brain barrier and prevent invasion of
the cerebrospinal fluid. IgM antibodies may be more effective in the control
of peripheral parasitaemia than IgG (Campbell et al., 1978), but less
effective than IgG class antibodies in reaching tissue parasites (Sacks et al.,
1980). It has been suggested that there are, in human sera, natural trypano-
cidal IgM antibodies which account, at least in part, for the occurrence of
natural immune defence mechanisms in the refractory host. Trypanocidal
activity and agglutinins to T. equiperdum could not be detected in cord blood
or newborn sera, but were present in the sera of individuals from 6 months
of age till late in life (Verducci et al., 1989). This was noted to be in
accordance with the significant appearance of IgM at 4 6 months after birth.
These observations suggest that the neonate may be relatively unprotected
from infection during early infancy.
( d ) Leishmaniases
VII. CONCLUSIONS
Loke (1982) noted that, before his review of the transplacental transmission
of parasites, the approach to this subject was concerned with the frequency
with which maternal infections affected the foetus and their pathological
consequences for the child. His own viewpoint was that of a reproductive
immunobiologist interested in the immune competence of the placenta and
the immunomodulation of the foetal response. The present review has
concentrated primarily on factors affecting immunity in both non-pregnant
and pregnant women and on the chronicity of several infections which are
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 57
A. MATERNAL-CHILD HEALTH
B. VACCINE DEVELOPMENT
C. DRUG TREATMENT
primary health care facilities. For example, at present the World Health
Organization recommends that patients serologically diagnosed as infected
with African trypanosomiasis should be followed up without treatment in
endemic situations, but in epidemic conditions treatment should be given if
at least two different serological test systems give positive results (WHO,
1986). The advisability of presumptive treatment of asymptomatic non-
pregnant women seropositive for sleeping sickness needs to be assessed. This
would involve establishing the relative risk of serologically positive, but
parasitologically negative, mothers infecting their children, and the risk of
recrudescence of infection during pregnancy.
Achieving optimal iron supplementation to reduce anaemia during preg-
nancy is difficult due to a high level of non-compliance in many areas
(WHO, 1990). Programmes should aim to improve haemoglobin levels
through iron supplementation and treatment of infectious diseases which
contribute to anaemia in non-pregnant women, especially girls aged 5-1 5
years. Cultural proscriptions against drug usage during pregnancy would
not apply to this group.
nation, to find schedules which optimally protect mother and child. They
should seek implementation of programmes understood by women and
operable within the framework of cultural constraints on them (Holland,
1989).
ACKNOWLEDGEMENTS
Part of the research for this review was funded by a grant from the UNDP/
World Bank/WHO Special Programme for Research and Training in Tropi-
cal Diseases, World Health Organization, Geneva, to Dr L. Brabin. We
thank Dr Uche Amazigo, University of Nigeria, Nsukka, for the photo-
graph of onchodermatitis in a pregnant woman, and Dr H. J. Van der Kaay
for permission to reproduce Fig. 1.
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PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 81
NICHOLAS J. WHITE
and
MAY HO
I. Introduction ............................................ 84
11. Animal Models ...................................... 85
111. Human Malaria ........................ ............... 86
A. Clinical features ........................................ 86
B. Causes of death and permanent sequelae .............................. 88
IV. Pathogenesis .......................... 93
A. Sequestration ...................................................... 93
B. Reduced red cell deformability 95
C. Cytoadherence .................................. 96
D. Putative endothelial cytoadherence receptors .......................... 98
E. Parasite cytoadherence ligands . . . . . . . . . . ............... 101
.................................. I02
V. Parasite Virulence Factors ................................... 105
A. Multiplication ........................... ............. 105
B. Synchronicity .................................... I08
110
110
A. Animal studies ................................................ 110
................. 112
C. Human studies ............................................. 112
I. INTRODUCTION
11. ANIMAL
MODELS
111. HUMAN
MALARIA
A. CLINICAL FEATURES
Factor Reference
Genetic
Protective erythrocyte abnormalities (surface receptors, Miller (1988)
cytoskeleton, haemoglobin type, enzymes, availability
of reticulocytes), HLA type Hill et al. (199
Age
Haemoglobin F Pasvol et al. (1 976)
Transfer of maternal immunity McGregor and
Pregnancy Wilson (1988)
WHO (1990)
Nutrition
Protective effects
Malnutrition Edington ( 1967)
Low iron Murray et al. (1975)
Deficiencies of riboflavin, a-tocopherol, Thurnham et al.
essential amino acids (1983)
Host defence
Humoral immunity Tharavanij et al. ( 1984)
Cellular immunity Druilhe et al. (1983)
Phagocytic function Ward et al. (1984)
Failure to augment splenic function rapidly?
Other diseases/conditions
Concomitant or previous infection impairing host defence?
Anaemia
Drug addiction
Parasite factors
Cytoadherence Ho et al. (1991a)
Rosetting Carlson et al.
Multiplication capacity? (1990b)
Antigenic variation?
Sporozoite inoculum/viability
Environmental/social factors
Location
Availability and use of antimalarial drugs
Family respanse to illness
Competence and resources of primary health care worker
Competence and resources of referral centre
88 N. J. WHITE AND M. HO
P . vivax, P . malariae and P . ovule kill very rarely during acute infections.
Occasional patients debilitated from other diseases may die, and rupture of
the enlarged spleen may cause death from haemorrhage if immediate surgery
is not available (Covell, 1955). In most adults who die from severe falci-
parum malaria there is multiple vital organ dysfunction and an adequate
explanation for the fatal outcome is apparent to the attending clinician. The
principal causes of death are pulmonary oedema, acute renal failure, and
metabolic acidosis with circulatory failure (WHO, 1986, 1990). Although
THE PATHOPHYSIOLOGY OF MALARIA 89
children, whereas renal impairment contributes to over half the adult deaths
(White et al., 1987a; Molyneux et al., 1989b; WHO, 1990). Pulmonary
oedema is also unusual. In contrast, hypoglycaemia and lactic acidosis with
terminal circulatory failure and aspiration pneumonia are more common in
young children. Many children die suddenly in the acute phase of severe
malaria without a clear explanation (unpublished observations). There is
primary respiratory arrest without circulatory failure. Of the 26 deaths in a
series of 180 Gambian children with severe malaria, no clear explanation
was evident in 14 children, four of whom died with primary respiratory
arrest (N. J. White and D. Waller, unpublished observation). The possibility
that this phenomenon results from brainstem compression- secondary to
raised intracranial pressure has been suggested by recent findings that
opening pressures at lumbar puncture are usually raised in children with
cerebral malaria (Newton et al., 1991; Waller et al., 1991). This is in contrast
to the findings in adults where opening pressures are normal in 80% of cases
(Fig. 11) and are significantly lower in fatal cases than in survivors (Warrell
et al., 1988). Some of the neurological findings in children with cerebral
malaria have been attributed to the development of a tentorial pressure
cone, but the specificity of these interpretations needs to be confirmed
(Section VII.F.2). Nevertheless, the observations are important because of
the possibility of providing specific treatment (e.g. with mannitol) which
would reduce intracranial pressure.
In areas of stable intense transmission (hyper- or holoendemicity),
anaemia becomes more common than cerebral malaria as a presentation of
severe malaria in children. The case specific mortality rate is lower. In a
recent large series of over 600 severely ill children reported from The
Gambia, overall mortality was approximately 7% in children with severe
anaemia compared with 16% in cerebral malaria (Brewster et al., 1990). The
causes of death in children with severe malarial anaemia are also different.
Some children have relatively low parasite counts, and in these the patho-
physiological processes relate largely to severe anaemia, whereas in others
both severe malaria and severe anaemia coexist. High output heart failure is
an important and potentially lethal manifestation of severe anaemia. Many
children die suddenly before or during blood transfusion. Twenty-four hour
electrocardiographic recordings suggest that cardiac arrhythmias are prob-
ably not a cause of “unexplained deaths” in acute cerebral malaria (F.
Nosten and N. J. White, unpublished observations), but this has not been
studied in severe malarial anaemia.
3. Permanent sequelae
(Molyneux et al., 1989b; Brewster et al., 1990). In over half the cases there is
hemiparesis (Fig. 2), but cortical blindness and clinical evidence of more
diffuse brain damage are also common. In half the children discharged from
hospital with a neurological deficit, there is full recovery within 6 months
(Brewster et al., 1990). The possibility that cerebral malaria causes more
subtle permanent defects such as slight motor impairment or mild intellec-
tual retardation in survivors has not been explored.
(a)
IV. PATHOGENESIS
A. SEQUESTRATION
All stages of parasite development are seen in blood smears taken during
infections with P. vivax, P . malariae and P . ovale, but the peripheral blood in
P. fakiparum malaria rarely contains pigmented trophozoites or meronts
(schizonts).* The intravascular sequestration of erythrocytes containing
these mature forms of the parasite is an essential pathophysiological feature
of falciparum malaria (Luse and Miller, 1971). The degree of vascular
sequestration varies between organs, being greatest in the brain in patients
with cerebral malaria and least in the skin (Li et al., 1983; MacPherson et al.,
1985). In patients who die without developing cerebral malaria, sequest-
ration is significantly less in the brain (Pongponratn et al., 1991). These
findings suggest a relationship between the organ distribution of sequest-
ration and pathology.
* Throughout this review, the etymologically more consistent terms meront and merogony have
been used instead of schizont and schizogony (eds).
94 N. J. WHITE AND M. HO
FIG.3. Cerebral venule packed with parasitized erythrocytes and pigment in a fatal
case of cerebral malaria (courtesy of Professor M.Aikawa).
Within a few years of Laveran’s discovery of the malaria parasite in the
blood of a febrile patient in Algeria, pathological observations from Italy
(Marchiafava and Bignami, 1894) recorded the extraordinary discrepancy
between the microscopical appearance of the peripheral blood and that in
the cerebral vessels of patients dying from cerebral malaria. The capillaries
and venules in the brain were packed with erythrocytes containing mature
forms of the parasite and abundant brown-black pigment, which were not
seen in ante-mortem blood samples (Fig. 3). These findings were confirmed
in pathological reports on the soldiers dying from falciparum malaria in
Macedonia during the First World War of 1914-1918 (Dudgeon and Clarke,
1917, 1918; Gaskell and Miller, 1920). It was suggested that the parasitized
erythrocytes had difficulty traversing the capillary bed and, as a result, blood
flow was obstructed. Initially it was thought that thrombus formation
occurred (Dudgeon and Clarke, 1917), altho.ugh the authors also conceded
that microvascular obstruction by parasitized erythrocytes might be revers-
ible (Dudgeon and Clarke, 1918). Later pathological studies have concluded
that widespread thrombus does not occur in fatal cerebral malaria, and have
favoured the concept of “plugging” (i.e. obstruction) of small vessels by
masses of parasitized erythrocytes (Gaskell and Miller, 1920; Spitz, 1946)
(Section VI1.T).
The mechanism of microvascular obstruction was investigated in a series
THE PATHOPHYSIOLOGY OF MALARIA 95
Red cells containing malaria parasites do not pass through micropore filters
as easily as unparasitized erythrocytes. This suggests that these infected
erythrocytes are less deformable than normal cells, and might therefore not
pass as easily through capillary beds (Miller, L. H. et af., 1971, 1972; Lee, M.
V. et al., 1982). In normal microcirculatory flow, red cells (diameter 7-8 pm)
must undergo considerable deformation in their passage through the capil-
lary (diameter 3 4 p m ) . Capillary blockage does occur when red cells are
unusually rigid, as in sickle cell crisis, but the clinical features and organ
distribution of vascular obstruction in this condition are most unlike those
of severe malaria. The reduced deformability of red cells infected with P.
falciparum is directly proportional to the maturity of the parasite (Cranston
et al., 1984); the older, and larger, the parasite, the more rigid is the infected
cell. There is increased expression of phosphatidylserine and phosphatidyl-
ethanolamine and reduced phosphatidylcholine on the outer leaflet of the
trophozoite-infected erythrocyte membrane. Cells containing meronts also
have reduced sphingomyelin in the outer leaflet (Maguire et al., 1991). These
abnormalities of phospholipid distribution, which may result from depletion
of adenosine triphosphate, oxidative stress and alterations in the cytoskele-
ton, influence the surface properties of the infected erythrocyte. They are
associated with increased phagocytic clearance and adherence to monocytes
and endothelial cells (Section VII.P.4). Several other factors contribute to
reduced red cell deformability: increased membrane stiffness, increased
cytoplasmic viscosity resulting from changes in membrane permeability
96 N. J. WHITE AND M. HO
C. CYTOADHERENCE
The stage and host cell specificity of cytoadherence suggest that the inter-
action between PRBC and endothelial cells involves specific parasite ligands
and host receptors. The quest for these proteins has proved more difficult
and confusing than expected originally, and has relied heavily on the use of
models in vitro. The search began with the observation that PRBC adhere to
cultured human umbilical vein endothelial cells (HUVEC) in vitro (Udeinya
et al., 1981) with the same stage and host cell specificity as observed with
sequestration in vivo. This model proved difficult and unpredictable. A
number of normal cell types and continuous cell lines have subsequently
been shown to have cytoadherent properties, of which the human amelano-
tic melanoma cell line C32 (American Type Culture Collection, no.
CRL1585) (Schmidt et a/., 1982) has been the most extensively employed. In
addition, putative receptor proteins have been purified and studied either
immobilized on plastic or in their soluble forms. Although such studies
provide definitive information regarding cytoadherence to a particular
molecule, it has been argued that the immobilized or free receptor molecule
in vitro may not have the same tertiary structure as that expressed on the cell
surface in vivo. One approach to circumvent this problem has been to
transfect COS cells with plasmids carrying the complementary deoxyribo-
nucleic acid (cDNA) for the receptors. The receptor under investigation is
then expressed on the otherwise antigen-free surface of the COS cell.
1. Thrombospondin
The first molecule identified as a potential receptor for cytoadherence was
thrombospondin (TSP), an adhesive glycoprotein produced by activated
platelets and involved ubiquitously in cell-to-cell interactions (Tandon et al.,
1989). Using purified TSP, Roberts et al. (1985) showed that PRBC adhered
selectively to TSP in a dose-dependent manner, but not to other adhesive
proteins such as fibronectin and von Willebrand’s factor. Cytoadherence
was specifically inhibited by anti-TSP monoclonal antibodies and soluble
TSP, and occurred under both static and shear-flow conditions (Rock et al.,
1988). However, further work has revealed that while TSP may contribute to
cytoadherence, it is not sufficient to mediate the process alone. PRBC do not
adhere to every melanoma cell line which secretes TSP (Panton et al., 1987)
and anti-TSP antibodies neither bind, nor inhibit cytoadherence, to C32
melanoma cells (Ockenhouse et al., 1989a).
2. CD36
3. ICAM-I
4. Clinical correlates
have lower melanoma cell binding rates than other patients with severe
disease. These findings support the hypothesis that CD36 mediates sequest-
ration in vital organs other than the brain but question the role of CD36 in
mediating cerebral sequestration. In immunohistochemical studies of human
tissues using the monoclonal antibody OKM5, CD36 can be demonstrated
on vascular endothelium in sections of lung, kidneys and liver (Knowles et
af., 1984), but not in the brain (A. Berendt, personal communication).
However, using a different monoclonal antibody, CD36 was detected on
cerebral vascular endothelium (Barnwell et af., 1989). This suggests that a
different receptor epitope of CD36 may be expressed in the cerebral
microvasculature.
In contrast to the results with CD36, cytoadherence of freshly isolated P .
,fakiparum to purified ICAM- I , and to a sub-clone of the C32 melanoma cell
line bearing ICAM- I but not CD36, was generally low in one study and bore
no quantitative relationship to any clinical manifestations of malaria (Ock-
enhouse et af., 1991b). When both CD36 and ICAM-I were expressed
together, as on the surface of the C32 melanoma cells, there was preferential
binding to CD36.
The weight of evidence suggests that CD36 is the most important of the
candidate receptor molecules thus far identified. However, the true role of
these molecules, and others perhaps yet undiscovered, in P . fakiparum
sequestration will undoubtedly require more investigation. Further studies
must also take into account the distribution and density of the endothelial
ligands on different tissues, in order to reconcile the variability in end organ
damage seen in patient populations of differing age and background immu-
nity.
1991a). This finding further strengthens the hypothesis that CD36 is the
receptor for the parasite ligand on vascular endothelium. The definitive
proof of the role of these parasitized red cell surface proteins in cyto-
adherence awaits the production of specific isotypic antibodies and/or the
cloning of the genes encoding these antigens.
Two other molecules have been proposed as the cytoadherence parasite
ligand, although the evidence supporting them is far less convincing. The
155 kDa ring-infected erythrocyte antigen (RESA) antigen, which is trans-
ferred from the merozoite to the erythrocyte membrane during invasion,
was thought initially to be entirely submembranous, but recent evidence
suggests that part of the molecule is exposed on the exterior of the red cell as
the parasite matures. RESA could therefore have a role in cytoadherence.
The RESA antigen has been shown to have cross-reactive epitopes with
band 3 protein (Holmquist et al., 1988), the human erythrocyte anion
transporter, and this too has been implicated as a ligand for cytoadherence
(Winograd and Sherman, 1989). Presumably, changes in the erythrocyte
cytoskeleton which occur as a result of parasitization expose previously
hidden host molecules (neoantigens) on the cell surface. At present there is
considerably less evidence to support a role for RESA or modified band 3 in
cytoadherence than for PfEMPl, but the situation is far from resolved.
Regardless of the eventual identity of the cytoadherent ligand, a conserved
component must be present since all P. falciparum parasites causing natural
infections cytoadhere. In addition, there must be a strain-variable compo-
nent since inhibition or reversal of cytoadherence by immune sera occurs in
a strain-specific manner (Udeinya et al., 1983; Singh et al., 1988). Indeed, the
cytoadherence surface proteins show antigenic variation within cloned
parasite lines in a manner analogous to the schizont-infected cell antigen
(SICA) of P. knowlesi. The constant and variant components could be either
closely associated molecules or different epitopes on the same molecule. This
ability of the parasite to vary the surface antigenicity of the cytoadherent
protein is obviously important for its survival as it helps to evade host
recognition and thus parasite removal.
F. ROSETTING
Non-parasitized erythrocytes will agglutinate around red cells containing
mature forms of the parasite in vitro (David et al., 1988; Udomsangpetch et
al., 1989b). This phenomenon is termed rosetting and may sometimes be
seen in fresh blood samples (Ho et al., 1991). It shares many characteristics
with the properties of cytoadherence. Rosetting occurs only with species of
Plasmodium which also exhibit cytoadherence (Handunnetti et al., 1989).
Both phenomena occur with mature stages of P . falciparum and begin after
approximately 26 h of intra-erythrocytic development (David et al., 1988).
THE PATHOPHYSIOLOGY OF MALARIA 103
* Ethyleneglycol-bis-(P-aminoethylether)-~,,N,llr,K-tetraacetic
acid.
104 N. J. WHITE AND M. HO
(a)
VIRULENCEFACTORS
V. PARASITE
A. MULTIPLICATION
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1. Stabilization of parasitaemia
Most untreated episodes of falciparum malaria are not fatal. The log-linear
rise in parasite numbers is checked, and the host reaches an equilibrium with
the parasite burden, and later effects its removal from the body. With
repeated infections in areas of intense transmission, the level at which
parasitaemia stabilizes falls; also, the threshold for symptoms rises (Kitchen,
1949). Eventually parasitaemias become asymptomatic, a state known as
premunition. Concurrently the risks of unrestrained parasite multiplication
to lethal burdens declines. Several factors converge to limit parasite multi-
plication. These include increased splenic clearance function, the inhibition
of merozoite invasion by antibodies, the humoral immune response with
antibody opsonization of red cells and merozoites, various antibody-inde-
pendent cellular effector mechanisms, exhaustion of more receptive erythro-
cytes (certain red cells are more susceptible to invasion than others), and
non-specific activation of host defences leading to release of cytokines and
other toxic species that directly or indirectly damage parasites (White and
Krishna, 1989). However, equilibration of the parasite population can occur
in the absence of these factors. For example, a simple feedback mathemat-
ical model in which fever (temperature >40°C) inhibits merogony (Kwiat-
kowski, 1989) illustrates this tendency to equilibrate (Kwiatkowski and
Nowak, 1991)-but also, as in other biological systems, shows fundamental
instability (chaotic dynamics) of the parasite population when multiplication
rates are high. Such a system has a tendency to “overshoot”-i.e. the
number of parasites might become very large indeed before the feedback
factor reduces the population. The overshoot may prove fatal.
The benign human malarias have an inbuilt brake on their capacity to
multiply; P . vivax and P . ovule have a predilection for young erythrocytes,
whereas P . malariae is thought to invade mature cells only (Garnham, 1988).
P . falciparum also preferentially invades younger erythrocytes (Pasvol et al.,
108 N. J. WHITE AND M. HO
1980), but can invade cells of all ages-hence the propensity for untram-
melled multiplication to reach lethal parasite burdens (Field, 1949). P .
falciparum does not develop well in erythrocytes containing haemoglobin F
(Pasvol et al., 1976, 1977), which partly explains why malaria is rarely severe
in the first few months of life.
B. SYNCHRONICITY
1. Fever patterns
2 . Rigors
VI. CYTOKINES
IN MALARIA
PATHOLOGY
A. ANIMAL STUDIES
(Grau et al., 1987b,c). The treatment, however, does not influence the
parasitaemia, and protected mice eventually die of anaemia. Furthermore,
monoclonal antibodies to certain other cytokines, e.g. the combination of
anti-interleukin 3 and anti-granulocyte macrophage-CSF (anti-GM-CSF)
(Grau et al., 1988b), as well as anti-y interferon (IFN-y) (Grau et al., 1989d),
can also prevent cerebral symptoms through a reduction in TNF produc-
tion. The administration of recombinant TNF to mice infected with P .
berghei, and which are resistant to cerebral symptoms, apparently induces
the lethal neurological syndrome (Grau et al., 1989a,b,c). Interleukin (IL) 6
levels are also elevated in this model (Grau et al., 1990), but this cytokine
seems less directly involved in pathology; high IL-6 levels also occur in the
absence of cerebral pathology in animals infected with non-lethal P . yoelii
parasites, and anti-IL-6 antibody does not protect against the development
of cerebral symptoms.
The following sequence of events is proposed. In genetically susceptible
strains of mice, CD4+ T cell activation during acute malaria leads to the
production of cytokines which “upregulate” a variety of macrophage
functions, one of which is the release of TNF. Elevated TNF levels in the
circulation alter the surface properties of endothelial cells and cause the local
accumulation of leucocytes. These sequestered leucocytes in turn release
more TNF, thus amplifying the cytotoxic effects on endothelial cells, with
resultant vascular wall damage and haemorrhagic necrosis. In this murine
model, sequestration of leucocytes and monocytes is considered to be more
important than that of infected erythrocytes.
Cytokines have also been implicated in the pathogenesis of anaemia,
hypoglycaemia and pulmonary oedema in rodent models. Bone marrow
dysfunction results from TNF-induced dyserythropoiesis and erythrophago-
cytosis (Clark, I. A. and Chaudhri, 1988a; Miller, K. L. et al., 1989). If
recombinant TNF is infused into mice infected with P . vinckei, hypo-
glycaemia, midzonal liver necrosis and neutrophil adhesion in pulmonary
vessels occur (Clark, I. A. et al., 1990). These features are commonly seen in
terminal P . vinckei infection in association with high levels of circulating
TNF. Administration of TNF has also been shown to cause foetal death in
pregnant mice infected with P . vinckei (Clark, I. A. and Chaudhri, 1988b).
In contrast to the deleterious effect of high levels of TNF on the host, small
doses of parenteral IFN-y and TNF have been shown to reduce parasitae-
mia in P . chabaudi adami (Clark, I. A. et al., 1987) and P . yoelii (Taverne et
al., 1987) infections. More recently, low doses of IL-I were found to protect
C57BL/6J mice against cerebral pathology induced by P . berghei and also to
reduce parasitaemia, although the two effects were separate (Curfs et al.,
112 N. J. WHITE AND M. HO
1990). These observations have led to the concept that low levels of
cytokines could be beneficial by exerting an indirect antiparasitic effect on
blood-stage parasites, but high concentrations of the same cytokines may act
in concert to produce toxic damage in the host. The antiparasitic effect is
known to require other serum components since recombinant T N F and
IFN-y are not directly cytotoxic to intra-erythrocytic parasites in vitro
(Taverne et al., 1987), and IL-1 has no effect when given to T cell-deficient
animals (Curfs et al., 1990). The additional toxic serum components remain
to be identified, although the killing of blood-stage P. falciparum in vitro by
serum containing T N F results mainly (> 70%) from the lipid peroxide
content (Rockett et al., 1988). These peroxides are formed by the interaction
of lipoproteins with reactive oxygen intermediates and are unaffected by
antioxidants. Malaria parasites are readily killed by free radicals (Malhotra
et al., 1988). Lipid peroxidation has the effect of stabilizing the reactive
oxygen groups, and thus creating a more stable cytotoxic molecule than
other oxygen radicals.
C. HUMAN STUDIES
There are several problems with the cytokine story in human malaria.
First, cytokine levels are high in malaria due to P.vivax as well as that due to
P.fakiparum-but P. vivax does not kill. Second, there are differences
between the clinical manifestations of severe malaria and lethal septic
shock-a condition in which there is good evidence for cytokine-mediated
pathology (Parillo et al., 1990). Third, the clinical and pathological features
of so-called “cerebral malaria” in the P. berghei-infected mouse model
(where much of the evidence supporting a role for cytokines in malaria
pathology has been obtained) are quite different from those seen in human
disease. In humans, there are no haemorrhagic foci or focal intravascular
accumulations of large mononuclear cells, and endothelial cell damage is
114 N. J. WHITE AND M. HO
OF VITALORGAN
VII. PATHOPHYSIOLOGY DYSFUNCTION
A. CARDIOVASCULAR ABNORMALITIES
C. PULMONARY OEDEMA
1. Cerebral bloodflow
Although the cardiac index is usually high, and systemic vascular resistance
is low in severe malaria (White, 1985), flow in some vital organs may be
reduced. In health, autoregulation ensures that blood flow to the brain
provides sufficient oxygen and substrates for metabolic demands. In 12
patients with cerebral malaria, cerebral blood flows were within the range
considered normal in healthy adults, but were considered inappropriately
low for the arterial oxygen content (i.e. oxygen supply) and the augmented
metabolic demands associated with fever and infection (Warrell et al., 1988).
The cerebral metabolic production of lactate was increased during coma, but
fell to normal on recovery of consciousness. In a separate study, CSF
concentrations of lactate were found to be elevated consistently in cerebral
malaria (White et af., 1985), and were significantly higher in fatal cases than
in survivors (Fig. 9). CSF lactate was inversely correlated with CSF
concentrations of glucose, and values also returned to normal with recovery
of consciousness. Similar findings have been reported in children (White et
al., 1987b; Molyneux et al., 1989b). These observations all indicate anaero-
bic glycolysis within the brain in cerebral malaria. This results presumably
from interference with microcirculatory flow (i.e. ischaemia causing
hypoxia), but in addition could reflect a flow-independent shift to anaerobic
respiration (e.g. inhibition of citric acid cycle activity by toxic moieties such
as cytokines or other secondary products). Obligatory anaerobic glycolysis
by the sequestered parasites also contributes to local lactate accumulation.
But coma in cerebral malaria cannot be explained simply by hypoxia;
118 N. J. WHITE AND M. HO
I4 0
.
a
12
a
10
a
8
a
CSF
Lactate
m moll1
# 6
4 a
a
--
2
NO R M A L RANGE
_c.
FATAL SURVIVORS CONVALESCENT
’
10.7 ml kg- min- in six fatal cases of falciparum malaria compared with
’
15.6 ml kg- min-’ in survivors. There was also a significant inverse corre-
lation between venous lactate concentrations and LBF at flows less than
’ ’.
15 ml kg- min- This suggests that reduced LBF could contribute towards
liver dysfunction and lactic acidosis in life-threatening infections (Pukrit-
tayakamee et al., in press). Recent studies of forearm blood flow and
metabolism in severe malaria indicate that a shift to anaerobic glycolysis
also occurs in skeletal muscle. However, there is no evidence that muscle
blood flow is reduced sufficiently to impair or retard intramuscular drug
absorption in severe malaria (White, 1985; Waller et al., 1991).
The increase in lactate production in these various organs cannot be
explained simply by reduction in measured total organ blood flows, and it
is unlikely to be explained solely by parasite glycolytic metabolism. This
suggests that either microcirculatory obstruction is variable or “patchy” (i.e.
low flow in some capillaries, high flow in adjacent vessels) or that the
cytoadherent parasitized erythrocytes interfere with gas and substrate
exchange and host metabolic functions but do not cause a large increase in
the resistance of the circuit. In either case it is likely that normal erythrocytes
can squeeze past the adherent parasitized red cells (White, 1986). Otherwise,
permanent ischaemic neuronal damage would be the rule rather than the
exception following cerebral malaria.
E. CEREBRAL MALARIA
throughout the white matter of the brain (Spitz, 1946). Retinal haemor-
rhages are also commonly seen in cerebral malaria (Looareesuwan et al.,
1983a), particularly with the use of indirect ophthalmoscopy and fluorescein
angiography (E. Schulenberg, T. M. E. Davis and N. J. White, unpublished
observations) (Fig. 10). The microvascular damage in the connecting path-
ways appears to be related to sequestration rather than thrombus formation
and may contribute to nervous system dysfunction, seizures and possibly
residual sequelae.
2. Irnrnunopathology
FIG. 10. (a) Retinal haemorrhages and exudates in cerebral malaria (courtesy of S.
Ward and Dr E. Schulenberg). (b) Fluorescein angiography of the retina; disruption
of the macular microvasculature in cerebral malaria (courtesy of S. Ward and
Dr E. Schulenberg).
122 N. J. WHITE A N D M. HO
F. CAPILLARY PERMEABILITY
On the basis of his autopsy observations, Rigdon (1944) concluded that the
primary pathological process in cerebral malaria was anoxia, that hypoxia
damaged the cerebral capillary endothelium and that this led to an increase
in capillary permeability. Maegraith and Fletcher (1972) later concluded,
from a series of studies on rhesus monkeys infected with P . knowlesi, that
increased capillary permeability. was the primary pathological process in
cerebral malaria. In terminally infected animals there was increased per-
meability to albumin labelled with ‘’’1 and fluorescein isothiocyanate, and
increased penetration of the brain by water-soluble dyes. This was rapidly
reversed by corticosteroids and the antimalarial drugs chloroquine and
mepacrine (Migasena and Maegraith, 1967; Maegraith and Fletcher, 1972).
Extravasation of plasma into the cerebral interstitium was considered to
account for cerebral oedema-a common post-mortem finding in cerebral
malaria. Reduced microcirculatory flow leading to cerebral hypoxia was
considered a secondary phenomenon attributed to local haemoconcen-
tration. It was proposed that the increase in cerebral capillary permeability
resulted from the local release of inflammatory mediators, originally thought
to be kinins (Onabanjo and Maegraith 1970a,b; Maegraith and Fletcher,
1972; Desowitz, 1987), but more recently suggested to be free oxygen
radicals (Clark, I. A. et al., 1986; Migasena and Areekul, 1987). As an
extension of this suggestion, the beneficial effects of the quinoline anti-
malarial drugs in cerebral malaria were thought to result from their anti-
inflammatory, rather than their antiparasitic, activity (Migasena and
Maegraith, 1967). The “permeability theory” was widely accepted, and
provided the theoretical basis for the use of corticosteroids and osmotic
agents to reduce cerebral oedema in human cerebral malaria.
The hypothesis that increased capillary permeability causes cerebral
malaria is no longer generally accepted. There are several reasons for this.
First, there are criticisms of the experiments on which the theory was based;
P . knowlesi infection in the rhesus monkey is clinically and pathologically
most unlike cerebral malaria in man. The identification of the vasoactive
kinins and their precursors relied on non-specific bioassays and, although a
permeability defect was identified, the blood/CSF albumin partition was
unchanged. This latter finding necessitated postulating increased bi-direc-
tional flux of albumin (i.e. exporting albumin from the CSF compartment
against a 100 : 1 concentration gradient), which is most unlikely. Second,
there is now considerable evidence from studies of human adults with
cerebral malaria (Looareesuwan et al., 1983b; Warrell el al., 1986) that
THE PATHOPHYSIOLOGY OF MALARIA 123
2. Intracranial pressure
.
..
300-
....
. .
.
200 -
..
... ...
.
.....
om
...
-
.. .
0..
100
..
0..
.. .. . .
0.
.
0- I III
Survivors Fatal cases Survivors Fatalcases
LAdUWS 1 L Chlldnn 1
FIG. 11. Opening pressures of cerebrospinal fluid (CSF) at lumbar puncture in
adults and children with cerebral malaria.
adequate cerebral perfusion pressure will reduce cerebral blood flow and
may cause ischaemic damage. RICP and subsequent tentorial or foramen
magnum coning could explain the sudden respiratory arrests which appear
to be an important cause of death in childhood cerebral malaria. On the
other hand, the observations that lumbar puncture opening pressures are no
higher in fatal cases than in survivors (Waller et al., 1991), the frequency
with which neurological signs attributed to coning occur in survivors
(Newton et af., 1991), the lack of convincing evidence that lumbar puncture
causes neurological deterioration, and the excellent recovery in children with
very high opening pressures all argue against an important role of RICP in
the pathophysiology of cerebral malaria. These questions are unresolved at
the present time, but as RICP is a potentially treatable condition (with
osmotic agents such as mannitol), further studies are clearly needed.
Patients with severe malaria, particularly those who have been comatose
with high fever for more than 24h, may be significantly dehydrated on
126 N. J. WHITE AND M. HO
H. ELECTROLYTE CHANGES
active haemolysis and possible tissue injury which tend to raise plasma
potassium, and activation of the renin-angiotensin system, and quinine-
induced insulin release, which tend to reduce it.
1. ENDOCRINE DYSFUNCTION
J. RENAL IMPAIRMENT
Acute renal failure is a major cause of death in adults with severe falciparum
128 N. J. WHITE AND M. HO
cell casts, proteinuria is mild or absent, children are unaffected and in most
cases renal impairment is transient. It is possible that occasional patients
develop significant glomerular disease following acute falciparum malaria,
but this appears to be rare.
The relative roles of haemoglobin (Brant et al., 1951) and the large
amounts of erythrocyte and parasite debris released at merogony in the
pathogenesis of malarial renal failure are unresolved. Massive haemolysis
causing “black water” certainly causes renal failure both in acute malaria
associated with quinine treatment (Blackie, 1944), and also in some patients
with glucose-6-phosphate dehydrogenase deficiency who receive oxidant
antimalarial (or other) drugs (Section VI1.R). Originally, it was thought that
renal failure resulted from blockage of the renal tubules by haemoglobin and
related pigments, but studies of renal histology in humans argue against this
hypothesis (Maegraith and Findlay, 1944; Dukes et al., 1968). How quinine
and severe Malaria consort to induce massive haernolysis remains to be
discovered-but the end result is acute tubular necrosis. The natural history
of renal failure is determined initially by the overall severity of the infection
(approximately two-thirds of deaths associated with malaria renal failure
occur rapidly from multiple organ dysfunction), and then by the ability of
the medical facilities to conduct peritoneal or haemodialysis. Provided no
complications on dialysis ensue, most patients’ renal function will return to
normal over a period of several weeks (T. T. Hien, personal communi-
cation).
K. GASTROINTESTINAL DYSFUNCTION
L. LIVER DYSFUNCTION
M. HYPOGLYCAEMIA
100
80
Mortality
('A)
60
40
20
0
I I I I I
<1 1-2 2-3 A
Lactate I ~iucomratio
FIG. 12. Relationship between the ratio of admission venous plasma lactate to
glucose in 200 adults and children with severe malaria (mean and 95% confidence
interval).
132 N. J. WHITE AND M. HO
1. Iatrogenic hypoglycaemia
2. Impaired gluconeogenesis
malaria (White et al., 1987b; Taylor et al., 1988; Molyneux et al., 1989b).
Fasting rapidly depletes hepatic glycogen in children (who have an average
12 h worth of stores) even if they are well nourished, and glycogen depletion
certainly contributes to hypoglycaemia in some cases, but not all
children are ketotic, and other factors are undoubtedly responsible.
Counter-regulatory hormone concentrations (i.e. growth hormone and cor-
tisol) are elevated (Taylor et al., 1988) and there is often concomitant lactic
acidosis. These biochemical features, together with hypoglycaemia, have
been described in other severe childhood infections (Phillips et al., 1988;
Kawo et al., 1990), but they are particularly common in severe P. falciparum
infections, occurring in approximately one-third of all children with cerebral
malaria.
In humans, clearance of the monosaccharide galactose is normal in severe
malaria (Pukrittayakamee et al., in press). This suggests that there may be a
specific defect in glycolysis (rather than a generalized reduction in glycolytic
enzyme activity) and that the biochemical locus of impaired hepatic gluco-
neogenesis may reside outside the small segment of the glycolytic pathway
between galactose and glucose.
Biochemical studies on isolated liver slices from rats with severe P.berghei
infections also suggest that there may be a specific defect in the glycolytic
pathway (P. A. H. Holloway and D. H. Williamson, personal communi-
cation). Hepatic gluconeogenesis from lactate is particularly impaired, and
cannot be explained entirely by the reduction in hepatic adenosine triphos-
phate concentrations. Hepatic ketogenesis is also reduced, but ketogenesis
from endogenous substrates shows a relative increase in hydroxybutyrate
synthesis.
Elevated plasma concentrations of the cytokine T N F are correlated with
hypoglycaemia and death in severe falciparum malaria (Grau et al., 1989a-d;
Kwiatkowski et al., 1990) (Section V1.C). T N F is a potent inhibitor of
hepatic gluconeogenesis (Evans et al., 1989), inducing many of the metabolic
derangements observed in malaria, and this cytokine could well be impli-
cated in the pathogenesis of hypoglycaemia in severe malaria.
Taken together, these findings point principally to an impairment of
hepatic gluconeogenesis which is proportional to the severity of malaria and
might be localized to specific biochemical steps in carbohydrate metabolism.
The counter-regulatory hormone response is appropriate and proteolysis
and lipolysis are unimpaired. There appears to be primary hepatocyte
dysfunction. This is compounded by reduced hepatic perfusion, increased
peripheral glucose consumption and lactic acid production, reduced or
absent glycogen stores, and-in some patients-uinine-stimulated hyper-
insulinaemia. The liver cannot clear the increased quantities of lactate and
alanine produced peripherally; hypoglycaemia and lactic acidosis result.
134 N. J. WHITE A N D M. HO
3. Glucose consumption
N. LACTIC ACIDOSIS
The muscles are not painful in severe malaria, but there is biochemical
evidence of muscle damage (elevations in serum concentrations of myo-
globin and creatine kinase) which parallel disease severity (Miller, K. D. et
al., 1989). On histopathological examination, skeletal muscle is a site of vas-
cular sequestration, but muscle cell abnormalities are usually mild. Rarely,
rhabdomyolysis occurs (De Silva et al.,. 1988) and acute myoglobinuric
renal failure may develop. The quantitative contribution of skeletal muscle
(the largest tissue mass in most bodies) to the development of lactic
acidosis remains to be determined.
136 N. J. WHITE AND M. HO
P. ANAEMIA
1. Oxygen delivery
3. Role of antibody
4. Membrane abnormalities
response is often inadequate. Bone marrow, white cell and platelet produc-
tion is normal or increased. Preliminary data suggest that the erythropoietin
response in patients with normal renal function is usually appropriate (P. M.
Cotes, personal communication). Morphologically the bone marrow is
dyserythropoietic in both falciparum and vivax malaria (Srichaikul et al.,
1967; Abdallah et al., 1980; Knuttgen, 1987; Wickramasinghe et al., 1987,
1989). Iron is plentiful in the marrow, but serum iron is low and serum
ferritin is very high (Phillips et al., 1986a). In rodent malarias, cytokines,
particularly TNF, appear to contribute to bone marrow dysfunction (Clark,
I. A. and Chaudhri, 1988a; Miller, K. L. et al., 1989). In human malaria,
local cytokine release could be responsible for dyserythropoiesis, but-as in
the other situations where cytokine induced pathology is hypothesized-
definitive proof is lacking. In P.fakiparum infections there is some sequest-
ration of parasitized erythrocytes in the bone marrow, and it has been
suggested that this could cause local hypoxia (Wickramasinghe et al., 1987).
R. BLACKWATER FEVER
S. THROMBOCYTOPENIA
The platelet count is usually low in malaria. A moderately reduced count (ca.
100 000 PI-') is observed in symptomatic infections with all four species
of human Plasmodium (Horstmann et al., 1981; Hill et al., 1964), but much
lower counts may be recorded, particularly in severe falciparum malaria.
Several hypotheses have been proposed to account for thrombocytopenia;
disseminated intravascular coagulation (Devakul er al., 1966; Dennis et al.,
1967), antibody-mediated clearance (Kelton et al., 1983; Grau et al., 1988a;
Srichaikul et al., 1988), and enhanced aggregation (Essien, 1989).
Bone marrow megakaryocyte numbers are normal in malaria, suggesting
adequate production of platelets. Increased turnover is suggested by obser-
vations of platelet enlargement (Fajardo and Rao, 1971), and studies of the
disposition of platelets labelled with 'Cr have confirmed accelerated peri-
pheral platelet destruction and splenic pooling (Sheagren et al., 1970;
Skudowitz et al., 1973). There is usually increased coagulation cascade
activity (Pukrittayakamee et al., 1989), but full blown disseminated intravas-
cular coagulation is rare and occurs in only 5% of cerebral malaria cases
(White and Looareesuwan, 1987; WHO, 1990); it is therefore unlikely to be
the sole cause of the reduced platelet count. Increased platelet-associated
IgG has been demonstrated in both vivax and falciparum malaria (Kelton et
al., 1983; Mohanty et al., 1988). Platelets were shown to have saturable
binding sites for malarial antigens, and it was suggested that malaria-specific
IgG bound to platelet-absorbed antigen via the Fab portion of the molecule.
However, in Thailand no relationship was evident between either free or
bound platelet-directed antibodies and either the absolute platelet count or
changes in the count (S. Looareesuwan and N. J. White, unpublished
observations). Platelets taken from patients with acute malaria have shown
increased ability to aggregate (Mohanty et a[., 1988). Some workers have
reported increased plasma concentrations of the platelet-specific proteins, p-
thromboglobulin and platelet factor 4, in uncomplicated malaria (Essien,
1989), but in other studies of severe malaria there was no evidence of platelet
activation (Supanaranond et al., in press). Thus there is contradictory
evidence on both hypotheses, and no firm conclusion can be drawn on the
relative importance of immune mechanisms or activation. Platelet antibody
is unlikely to play a significant role as the platelet count rises coincident with
the resolution of symptoms in malaria, but the other proposed mechanisms
require further study.
T. COAGULATION
and Nkrumah, 1972; Sucharit et al., 1975; Goodall, 1981; Horstmann and
Dietrich, 1985), but the clinical importance of these laboratory findings has
been overemphasized. The coagulation cascade is certainly activated in acute
malaria (Pukrittayakamee el al., 1989). Erythrocytes containing mature
falciparum malaria parasites promote coagulation (Udeinya and Miller,
1987). There is accelerated fibrinogen catabolism (Devakul et al., 1966) with
elevated serum fibrin degradation products, but fibrinogen concentrations
are usually raised even in severe malaria as part of the acute phase response.
Thus, in most patients, synthesis exceeds consumption (Jaroonvesama, 1972;
Looareesuwan et al., 1983b; Pukrittayakamee et al., 1989). Hypofibrino-
genaemia indicates significant consumptive coagulopathy and may be associ-
ated with a bleeding diathesis. In most patients antigen related to factor VIII
and von Willebrand factor concentrations are raised, and are correlated
directly with parasitaemia and inversely with the platelet count (Horstmann
and Dietrich, 1985). Plasma concentrations of antithrombin I11 (the natural
inhibitor of thrombin) are reduced in malaria (Pukrittayakamee et al., 1989),
and antithrombin 111-thrombin complexes are increased even in uncompli-
cated malaria. These changes closely parallel disease activity. Thus acceler-
ation of coagulation cascade activity is proportional to disease severity, but
only in a relatively few patients with severe disease does disseminated
intravascular coagulopathy with consumptive coagulopathy cause signifi-
cant bleeding (Borochovitz et al., 1970). An association between dissemi-
nated intravascular coagulopathy and the development of acute pulmonary
oedema has been suggested (Punyagupta et al., 1974), but the high incidence
of bleeding in the patients studied may have been more related to coexistent
uraemia and treatment with heparin, low molecular weight dextran and
steroids.
The importance of thrombus formation in the pathology of fatal malaria
is uncertain. The pathological interpretations of autopsy findings have been
contradictory. Several authors have reported finding microthromboses with
fibrin deposition, particularly in the cerebral vessels of patients who died
with cerebral malaria (Dudgeon and Clarke, 1917; Rigdon, 1944; Thomas,
1971; Schmid, 1974; Toro and Roman, 1978; Boonpucknavig et al., 1990),
but others have considered true thrombus formation to be relatively unusual
(Spitz, 1946; MacPherson et al., 1985) or not to occur at all (Gaskell and
Miller, 1920; Edington, 1967; Janota and Doshi, 1979). These latter authors
considered that the microvascular obstruction was not caused by thrombus,
but by “plugging” with a compressed or agglutinated mass of parasitized
cells and pigment (Dhayagude and Puranare, 1943; Kean and Smith, 1944;
Arieti, 1946; Spitz, 1946; 00et al., 1987). Occasional fibrin strands may be
seen, but electron microscopy reveals a “striking absence of platelets” in the
obstructed vessels (MacPherson et al., 1985). Overall these results suggest
THE PATHOPHYSIOLOGY OF MALARIA 141
FIG. 13. (a) Ultrastructure of the spleen: a parasitized erythrocyte (PE) is adherent
to the littoral cells (LC) of the splenic sinusoid. E, uninfected erythrocyte; K, knob; M,
mitochondrion of littoral cell; P, parasite. (b) Trapping of a parasitized erythrocyte
(PE) between two splenic littoral cells (LC). E, uninfected erythrocyte; P, parasite.
(Reproduced with kind permission from Pongponratn et al., 1989.)
142 N. J. WHITE AND M. HO
U. SPLENIC FUNCTION
1. Filtration
2. Immune mechanisms
and host neoantigens are exposed on the surface of the red cell, and more
antibody is bound (Luzzi et al., 1991). Once the cell has sequestered, it
cannot be removed by the spleen. Thus, there is a balance between the ability
of the spleen to remove the increasingly opsonized and rigid erythrocyte, and
the parasite's capacity to export cytoadherence proteins to the red cell
surface and effectively remove itself from the circulation. Although phago-
cytic activity is generally increased in uncomplicated malaria, there is
evidence that it is compromised in severe malaria (Ward et al., 1984). In this
.
situation, it is difficult to distinguish cause from effect.
50 -
0.
.
0
.
* .
40 -
.
0.
Hematocrlt 0 .
(%) *.
.
30 - 0
R=0.791
pco.001
20 , I I ,, I
10 20 30 40 " >40
tIn (hours)
FIG. 14. Relationship between haematocrit and clearance half-time (t,,J of erythro-
cytes coated with immunoglobulin G in acute falciparum malaria. (Reproduced from
Ho et al., 1990a.)
W. IMMUNE DYSFUNCTION
1. Immune suppression
2. A ct ivation
X. COMPLEMENT
1. Activation
the time of merogony) (Neva et al., 1974). In both falciparum and vivax
malaria, concentrations of the later components of the complement pathway
were not reduced (Neva et al., 1974; Petchclai et al., 1977). Despite these
associations, there is no convincing evidence that complement activation or
immune complexes have a pathological role in acute malaria. In murine
malaria, immune complexes inhibit Fc receptor-mediated phagocytosis, but
there is no evidence for this in humans (Ho et al., 1990a).
2. Quartan nephropathy
Y. PREGNANCY
VIII. CONCLUSIQN
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The Interaction of Leishmania Species with
Macrophages
JAMES ALEXANDER
AND
DAVID G. RUSSELL
I. Introduction . . . . . . . . . . . . . . . 176
178
179
B. Entry into the vertebrate . . . . . . . . . . . ................... 181
111. Outer Membrane Molecules of Leishmania . . . . . . . . . . . . . . . . . . . . . 181
A. Promastigotes . . . . . . . . . ............................. 181
188
191
191
B. Characterization of the parasitophor 196
C. Intracellular trafficking and the para vacuole ................ 199
200
200
V. Macrophage Heterogeneity . . . . . . . . . . . . . ............... 202
A. Genetic control of Leishmania infection ............. 202
207
VI. Antigen Presentation and the Induction of Immunity 207
A. Parasite interference with antigen-presenting cell 208
B. Antigen complexity and antigen-presenting cell heterogeneity . . . . . . . . . . . . 209
C. Processing of parasite antigen and activation of T cells 214
VII. Lymphocyte Control of Macrophage Anti-Leishmania Activity . . . . . . . . . . . . . . . 215
A. B and T cell involvement . . . . . . . . . . . . .............. 215
B. Lymphokines and macrophage activation . . . . . . . . . . . . . . . . . . . . 220
C. Macrophage activation and parasite killing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1
I. INTRODUCTION
11. LIFECYCLESTAGES
OUTSIDE
THE MACROPHAGE
The parasites found within the sandfly are particularly important as these, or
at least a subpopulation of them, initiate natural infection, and it is against
these forms that humans must be protected. Not only does the sandfly
midgut present the parasite with a totally different environment from that
within the macrophage, but throughout the length of the gut differing
conditions induce well-characterized morphological changes (reviewed by
Killick-Kendrick, 1979, 1990).
Following an infected blood meal, amastigotes enter the gut of the female
sandfly. Macrophages have never been reported in the sandfly midgut. Not
all amastigotes survive (Franke et al., 1987) and those that do seem to go
through at least one division before developing a flagellum (Warburg et al.,
1986). Whether a subpopulation of amastigotes is pre-adapted for life in the
invertebrate is a matter for speculation. As well as amastigotes, several
promastigote types and morphologically distinct paramastigotes have been
described as the parasites migrate forward in the sandfly gut (reviewed by
Killick-Kendrick, 1979). Within the midgut, two morphologically distinct
types of promastigote have been described; the long, thin nectomonad (body
INTERACTION OF LEISHMANIA WITH MACROPHAGES 179
gotes have low infectitivy for host cells and animals, while stationary phase
promastigotes have comparatively high infectivity. This has been demon-
strated for L. major (Sacks and Perkins, 1984, 1985; Sacks et al., 1985;
Mallinson and Coombs, 1989a,b), L. donovani (Giannini, 1974; Doran and
Herman, 1981; Howard et al., 1987), L. mexicana (Mallinson and Coombs,
1989a,b) and L. braziliensis (Kweider et al., 1987). Changes in infectivity
have been associated with differential gene expression (Meade et al., 1989),
changes in cell surface characteristics (Sacks et al., 1985; Howard et al.,
1987; Sacks and da Silva, 1987; Cooper et al., 1988; Pimenta et al., 1989) and
biochemical properties (Doran and Herman, 1981; Mallinson and Coombs,
1986, 1989a,b). Infective stationary phase promastigotes of L. major (Sacks
et al., 1985; Sacks and da Silva, 1987) and L. donovani (Howard et al., 1987;
Cooper et al., 1988) lose the ability to bind the lectin peanut agglutin (PNA).
This is associated with a developmental modification of the surface lipo-
phosphoglycan (LPG) (Sacks and da Silva, 1987), which results in a
thickening of the glycocalyx from 7 nm in log phase promastigotes to 17 nm
in infective stationary phase promastigotes (Pimenta et al., 1989). Increased
promastigote infectivity in L. braziliensis and L. mexicana may also be
associated with an upgrading of the expression of the major surface
glycoprotein gp63 (Kweider et al., 1987; Russell and Alexander, 1988).
There are also quantitative and qualitative changes in enzyme content
between log and stationary phase promastigotes of L. donovani (Doran and
Herman, 1981), L. major and L. mexicana (Mallinson and Coombs, 1986,
1989a,b). In L. mexicana, isoenzymes and the content of certain amino acids
found in stationary phase promastigotes are normally associated with
amastigotes, suggesting that these forms are already partially pre-adapted
for life in the macrophage (Mallinson and Coombs, 1986, 1989a,b).
These stationary phase infective promastigotes, metacyclic forms as they
are invariably called, have a characteristic morphology (reviewed by Killick-
Kendrick, 1990), and promastigotes of similar morphology are found in the
thoracic midgut (Sacks and Perkins, 1984, 1985) and proboscis (Fig. 1) of
sandflies (Killick-Kendrick, 1986; Killick-Kendrick et al., 1988). While
proboscis forms have always been likely candidates to initiate infections
because of their close proximity to wounds (Adler and Theodor, 1931),
numerous authors have postulated that regurgitation during feeding from
more posterior parts of the gut is equally likely to facilitate transmission
(Davies et al., 1990; Sacks and Perkins, 1984, 1985; Warburg et al., 1989).
Using a monoclonal antibody (3F12) specific for metacyclic epitopes of L.
major LPG (Sacks and da Silva, 1987), Davies and coworkers (1990) have
identified metacyclic parasites in the pharynx, oesophagus and thoracic
midgut of P. papatasi 7-10 days after infection. No parasites were ever
found in the mouthparts, although infections could be transmitted to mice at
INTERACTION OF LEISHMANIA WITH MACROPHAGES 181
the second blood meal 6 days after infection. Expression of the metacyclic
marker recognized by monoclonal antibody 3F12 was, however, strongest in
the “short-stumpy” pharyngeal forms, indicating that paramastigotes could
also infect the vertebrate host, as suggested by Walters and coworkers
(1989a,b) studying L. chagasi and L. panamensis.
3. Lipophosphoglycan
workers (Orlandi and Turco, 1987; Turco et al., 1987, 1989) revealed that the
molecule was tripartite, comprising repeated phosphorylated saccharide
units linked via a mannose-rich carbohydrate core to a lysoalkylphos-
phatidylinositol lipid anchor. More recent studies in other species (McCon-
ville et al., 1987; Sacks et al., 1990) have revealed that, although LPG is
represented on all species, its structure shows considerable polymorphism
primarily in the phosphorylated saccharide repeats. For example, the repeat-
ing units of L. donovani LPG are phosphorylated disaccharides of PO,-
6Gal( IH)Manl, whereas the repeats of L . major consist of tetra, tri- and
disaccharide units containing galactose, mannose, glucose and arabinose.
One of the most intriguing observations concerning LPG has come from
the work of Sacks and colleagues on the differentiation of the promastigote
form from a non-infective dividing cell in mid log phase, to a non-dividing
infective form in stationary phase culture (Sacks and Perkins, 1984). This
attainment of infectivity as a culture passes into stationary phase has been
shown to mirror the increase in infectivity detected in promastigotes within
the sandfly vector as they migrate out of the midgut and into the proboscis
(Sacks and Perkins, 1985). This differentiation is reflected in a profound
alteration in the structure of LPG in Leishmania spp. from the Old World
(Sacks, 1990; Sacks et al., 1990). Originally, this alteration was detected in L.
major promastigotes by the loss of binding of the lectin PNA, which
conveniently allowed direct isolation of the infected forms from culture
(Sacks et al., 1985). Sacks et al. (1990) have now characterized these
differences further, and shown that LPG from metacyclic forms has under-
gone changes in both the structure and number of phosphorylated repeat
units. The number of neutral hexoses per lipid molecule is virtually doubled,
indicating that the LPG from metacyclic forms is twice the size of its log
phase counterpart. The effect of such a change on the surface topography of
the promastigote has been graphically illustrated by Pimenta et al. (1989,
1991), showing the appearance of a glycocalyx on the surface of metacyclic
forms.
This change in the LPG structure has been shown to correlate with
important alterations in the parasite’s biology. It had already been noticed
that metacyclic promastigotes of L . major showed considerably more resist-
ance to lysis by complement than their log phase counterparts (Puentas et
al., 1988). Intriguingly, activation of complement by metacyclic forms
proceeds via the classical pathway even in the absence of antibody. Sacks
and colleagues (1990) have suggested that the elongation of LPG, which is
known to act as the predominant deposition site of C3b, effectively blocks
access of the later lytic components of the complement system to the
parasite’s membrane. Recent demonstration of released CS-9 complexes that
have fallen off the promastigote surface supports this proposal (Puentas et
186 J. ALEXANDER A N D D. G. RUSSELL
B. AMASTIGOTES
Due to the relatively inaccessible location of the amastigote form, and the
comparative difficulty of completely isolating it from contaminating host cell
proteins, study of the surface-exposed molecules on the amastigote has
lagged behind that of promastigotes. However, some recent studies have
addressed the expression of the two major promastigote surface glycoconju-
gates on the surface of the amastigotes.
Earlier studies on the expression of gp63 throughout the parasite’s life cycle
reported conflicting findings (Fong and Chang, 1982; Chang et al., 1986;
Colomer-Gould et al., 1985). A more recent study (Medina-Acosta et al.,
1989a) has resolved this issue. Monoclonal antibodies raised against L. m.
mexicana promastigote gp63 were screened against extract from metaboli-
cally labelled amastigotes. Two of the antibodies recognized one or more
proteins in the extract. Characterization of the recovered material by
enzymatic deglycosylation and chemical peptide mapping of immunopreci-
pitated material demonstrated that it shared a common peptide backbone
with promastigote-derived gp63 (Medina-Acosta et al., 1989a). Frommel et
al. (1990) recently demonstrated that antibody raised against recombinant
FIG. 3. Two-dimensional polyacrylamide gel electrophoretogram (2D-PAGE) of surface-labelled amastigotes; from
Medina-Acosta et al. (1989a). Autoradiographs from 2D-PAGE gels run with modified amastigotes isolated from infected
mice reveal that the major protein, of parasite origin, on the surface of lesion amastigotes is a gp63. (a) The amastigotes in this
gel were covalently modified with N-hydroxysuccinimide-sulpho-biotin (NHS-biotin), washed, fractionated by 2D-PAGE,
and transferred to nitrocellulose. The membrane was then probed with 1Z5[I]streptavidin,and autoradiographed. The most
abundant polypeptide is host actin (open arrow). The other arrowed protein is amastigote gp63, in the form present on the
surface. (b) Amastigotes metabolically labelled for 3 h with 3s[S]methionine,covalently modified with NHS-biotin and then
detergent extracted. The metabolically labelled modified polypeptides were recovered with streptavidin agarose, removed with
2-mercaptoethanol and separated by 2D-PAGE. By this procedure parasite surface proteins can be discriminated from those
of host origin. The most abundant amastigote surface protein (arrowed) has been shown immunologically to be amastigote
gp63.
190 J. ALEXANDER AND D. C. RUSSELL
gote LPG has fewer larger units that show a reduction in overall anionic
charge. This difference is supported by the description of promastigote LPG-
specific epitopes.
With respect to the biology of the amastigote LPG, while promastigote
LPG on infective L . major metacyclic forms develops a detectable glyco-
calyx, no such coat could be found on the amastigote surface (Pimenta et al.,
1991). Attempts to surface-label the LPG by ~eriodate-NaBr[~H] before
isolation yielded little recoverable label (Turco and Sacks, 1991). So,
although amastigotes must also survive exposure to serum components and
enter macrophages, the protection of the LPG glycocalyx does not appear to
have been retained. Properties of the amastigote LPG require further study.
IV. PARASITIZATION
OF THE MACROPHAGE
All Leishmania spp., regardless of the disease syndrome resulting from the
infection, parasitize members of the host’s mononuclear phagocyte system.
The problems inherent in exploiting the macrophage fall into three main
categories: first, identification and entry into the “chosen” cell type; second,
survival within a cell that has evolved to kill invading microbes; and third,
long-term survival within an antigen-presenting cell.
Rizvi and colleagues (1988) reported that gp63 exhibited some structural
similarity to the Arg-Gly-Asp cell-binding domain of fibronectin. Peptides
based on this sequence inhibited binding of promastigotes of L. chagasi to
macrophages. In another study published shortly after, Russell and Wright
(1988) demonstrated that isolated gp63, reconstituted on to the surface of
C18-derivatized silica beads, bound to macrophages as a function of the
density of incorporated gp63 (Fig. 6). Interaction of these gp63-beads with
macrophages was blocked if the macrophages were plated on to plastic
coated with anti-CR3 monoclonal antibodies. Binding was also inhibited by
synthetic peptides based on the Arg-Gly-Asp and Ala-Gly-Asp regions of C3
194 J. ALEXANDER AND D. G. RUSSELL
and fibrinogen. The original published sequence for gp63 from L. major
contained an Arg-Gly-Asp sequence (Button and McMaster, 1988). Synthe-
tic peptides based on this region were also inhibitory to gp63/CR3 binding.
Although at that time it was proposed that binding was mediated by this
Arg-Gly-Asp region, it is now known that this region of the deduced amino
acid sequence was incorrect (Button and McMaster, 1990; Russell, 1990).
So, although it is clear that gp63 binds to CR3, the nature of the interaction
remains unknown.
indicated that the vacuole was capable of fusing with compartments that
appeared to be secondary lysosomes (Alexander and Vickerman, 1975).
Early histochemical studies have also shown the presence of acid phospha-
tase in the vacuole (Antoine et al., 1987); however, as the parasite itself is
known to produce a soluble acid phosphatase, the relevance of this result to
the definition of the compartment is limited. A more definitive study on the
nature of the lysosomal enzyme in the parasitophorous vacuole and in entire
macrophages infected with Leishmania has been more revealing. Prina et al.
(1990) have shown that cathepsins D, B, H and L are delivered to vacuoles
containing parasites. The studies were all carried out on relatively new
infections, initiated by L. amazonensis promastigotes, because older estab-
lished infections resulted in larger vacuoles which were technically difficult to
label. Interestingly, the total hydrolytic activity of a range of lysosomal
enzymes was unaltered in infected macrophages.
Although circumstantial evidence indicates that the parasitophorous
vacuole is lysosomal in derivation, a careful description in terms of defined
membrane protein markers and lysosomal hydrolases is lacking and is
fundamental to further analysis of the vacuole as a dynamic cellular
compartment. Employing immunofluorescence and immunoelectron mi-
croscopy, Russell et al. (1991b) have reported preliminary analysis of the
distribution of various membrane proteins and hydrolases in an established
L. mexicana infection of murine macrophages.
The markers of particular relevance for the endocytic pathway employed
were the lipid-associated membrane proteins LAMPl and LAMP2 (Chen et
al., 1985), present in both late endosomal and lysosomal compartments, and
the mannose-6-phosphate receptor which is present in the membranes of late
endosomes, but absent from lysosomal compartments (Kornfeld and Mell-
man, 1988). In addition, antibodies against the hydrolases cathepsin D and
P-glucuronidase were used. The parasitophorous vacuole was positive for
both cathepsin D and P-glucuronidase. The density of gold label for both
hydrolases within the lumen of the vacuole was similar to that of small
lysosomes around the periphery of the vacuole, indicating that a constant
supply of lysosomal enzymes is released into the vacuole. The parasito-
phorous vacuole membrane was shown by both immunofluorescence and
immunoelectron microscopy to contain the lysosomal glycoproteins LAMPl
(seen in Fig. 7) and LAMP2, and Lgp 120. In contrast, labelling with anti-
mannose-6-phosphate receptor antibody reveals an extensive concentration
of vesicles in the perinuclear space, but label appeared at low density within
the parasitophorous vacuole, which was also LAMP-positive. By accepted
convention (Kornfeld and Mellman, 1988), the compartment is late endo-
soma1 to lysosomal in nature.
198 J. ALEXANDER AND D. G. RUSSELL
(a)
INTERACTION OF LEISHMANIA WITH MACROPHAGES 199
Whatever the cellular basis, the results of these studies raise several
interesting questions concerning the accessibility of the parasitophorous
vacuole to endocytosed material and its relevance to the possible potentia-
tion of leishmanicidal drugs by targeting them to the parasitophorous
vacuole of infected macrophages. With respect to the latter point, Mukho-
padhyay et al. (1989) indicated that methotrexate coupled to maleylated
BSA, which it was presumed was being internalized via scavenger receptors,
was two orders of magnitude more effective than free drug in eliminating
Leishmania from an infection in vivo.
cated that nitrous oxide intermediates were responsible for much of the
parasite killing (Green et a/., 1990). It is not yet known if Leishmania
possesses any ability to combat this response in the way that LPG acts as a
scavenger of oxygen radicals. However, the effect of both LPG and acid
phosphatase on the generation of superoxide intermediates should extend to
other expressions of macrophage activation.
V. HETEROGENEITY
MACROPHAGE
L. mexicana
Gene Chromosome L. donovani L. major Primary cutaneous Metastaticlvisceral
Lsh 1 ++ - - ++
H-2 17 ++ + - ++
H-11 NK ++ ++ + ++
Scl-1 8( 1 1) - ++ + -
Scl-2 4 - - ++ + +/NK
+ +, Major influence; +, minor influence; -, no influence; NK, not known.
1. Lsh
2. H-2
curing mice (Rezai et al., 1980). Non-cure in B10.D2 mice is also associated
with the generation of CD4’ T cells (Blackwell and Ulczak, 1984). Low
infecting inocula in this non-cure mouse strain result in a cure profile, while
large doses, > lo7 amastigotes, given to the cure H-2b haplotypes result in a
variable phenotype response. Thus, for L . donovani infection in B10 H-2
congenic mice, there appear to be two determinants for the development of a
non-curing disease: a sufficiently high antigenic load and the type of class I1
molecule presented in association with the parasite antigen by the antigen-
presenting cell. The generation of T cells able to determine a non-healing
response occurs later, > 30 days after infection (Ulczak and Blackwell,
1983). Control of this response, as indicated above, has been mapped to IE
at the K end of H-2 (Blackwell, 1983). However, some non-cure haplotypes,
as well as cure haplotypes, fail to express IE molecules, suggesting that both
non-cure and cure phenotypes controlled by these haplotypes must also map
to K end genes other than IE. As both curing and non-curing responses are
mediated by CD4’ T cells, which recognize antigen in association with class
I1 molecules, IA seems to be the likely candidate.
Treating B10.D2 mice, bearing the H-2“ non-curing haplotype, with
specific monoclonal antibodies against IA or IE molecules has clarified the
involvement of these molecules in disease outcome (Blackwell and Roberts,
-=
1987). Early treatment, at 30 days, with either anti-A or anti-E resulted in
an increased liver and spleen parasite burden. After day 50, anti-A treatment
resulted in exacerbated infection, while anti-E treatment led to the resolution
of disease. These results reaffirm that L . donovani antigens presented in
association with IE class I1 molecules on the macrophage are invariably
associated with non-curing disease.
H-2, by contrast, has only a limited effect on subcutaneous L . major
infections (Howard et al., 1980a). However, despite a failure to observe any
H-2 controlled differences in primary cutaneous growth of L. mexicana, this
genetic locus did markedly influence the visceralization and metastatic
spread of this organism, with the L. donovani “non-curing” haplotypes
developing the heaviest infections with generalized metastasis (Roberts et al.,
1989).
3. H-11
4. Scl-1
5. Scl-2
PRESENTATION
VI. ANTIGEN AND THE INDUCTIONOF IMMUNITY
1987; Unanue and Allen, 1987; Weaver and Unanue, 1990; Kaye et al.,
199I ) . The generation of antigen-specific lymphokine-producing T cell
populations, particularly those producing IFN-y, promote in their turn
macrophage leishmanicidal activity (Section VI1.B). Although a role for
CD8 T cells cannot be discounted in influencing leishmanial infections, the
+
T,I and T,2 subsets of CD4' T cells, which are dependent on class I1
molecule-associated antigen presentation, appear to control healing and
disease exacerbation, respectively (Section VI1.A).
Mouse Cvtokine
Cytokine Species Cell type strain Healing Cell stimulation activity Reference
Murine
IL-l L. donovani RPM BALB/c No -
1 Reiner ( 1 987)
IL-l L. major RPM BALB/c No -
T Cillari et al. (1989)
IL-2 L. donovani splenic BALB/c No Leishmanial lysate Ag 1 Murray, H. W. et
al. (1987)
IL-2 L. donovani splenic BALB/c No PHA 1 Reiner and Finke
( 1983)
IL-2 L. major splenic BALB/c No PHA 1 Cillari et al. (1986)
IL-2 L. major LNC BALB/c No Leishmanial crude Ag 1 Solbach et al.
(l987b)
L. major LNC C57BL/6 Yes Leishmanial crude Ag t Solbach et al.
(l987b)
I L-2 L. major splenic BALB/c No Fraction 1 Ag 1 Scott et al. (1988)
vaccine
fraction 1
I L-2 L. major splenic BALB/c Yes Leishmanial soluble Ag t Scott et al. (1988)
vaccine
fraction 9
IL-3 L. major splenic BALB/c No Leishmanial soluble Ag t Lelchuk et a[.
(1988)
I L-3 L. major splenic CBA Yes Leishmanial soluble Ag 1 Lelchuk et al.
( I 988)
IL-3 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Lelchuk et al.
irradiated ( 1 988)
IL-3 L. major splenic BALB/c No Leishmania1 soluble Ag t Lelchuk et al.
vaccine sc (1989)
IL-3 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Lelchuk et al.
vaccine iv (1989)
I L-4 L. major splenic BALB/c No Fraction 1 Ag t Scott et al. (1988)
vaccine
fraction 1
IL-4 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Scott et al. (1988)
vaccine
fraction 9
IL-5 L. major splenic BALB/c No Fraction 1 Ag Scott et al. (1988)
vaccine
fraction 1
IL-5 L. major splenic BALB/c Yes Leishmanial soluble Ag Scott et al. (1988)
vaccine
fraction 9
TNF-a L. donovani BMDM BALB/c No LPS Descoteaux and
Matlashewski
( 1989)
TNF-a L . donovani BMDM BIOLsh' Yes LPS Blackwell et al.
(1989)
TNF-a L. major LNC C3H Yes Leishmanial whole Ag t Titus et al. (1989)
TNF-a L. major LNC BALB/c No Leishmanial whole Ag 1 Titus et al. (1989)
IFN-y L. major LNC C57BL 16 Yes Leishmanial crude Ag Sadick et al. (1986)
IFN-y L. major LNC BALB/c No Leishmanial crude Ag Sadick et al. (1986)
IFN-./ L. major LNC BALB/c Yes Leishmanial crude Ag Sadick et al. (1 986)
IFN-7 L. donovani splenic BALB/c No Leishmanial lysate Ag Murray, H. W. et
al. (1 987)
IFN-y L. major splenic BALB/c No Fraction 1 Ag 1 Scott et al. (1988)
vaccine
fraction 1
IFN-y L. major splenic BALB/c Yes Leishmanial soluble Ag r Scott et al. (1988)
vaccine
fraction 9
IL-4 L. major splenic C57BL16 Yes In vivo 1 Heinzel et al.
(1989)
TABLE
3 (Continued)
Mouse Cytokine
Cytokine Species Cell type strain Healing Cell stimulation activity Reference
Human
IL-2 L.d. chagasi Lymphocytes - No Leishmanial soluble Ag 1 Carvalho et af.
(19854
IL-2 L.d. chagasi Lymphocytes - Yes Leishmanial soluble Ag t Carvalho et al.
(1985a)
IL-2 L. donovani PBMC - No PHA 1 Cillari et al. (1988)
IL-2 L . donovani PBMC - Yes PHA 1 Cillari et al. (1988)
IL-2 L . major PBMC - Yes Leishmanial lysate Ag Passwell et al.
(1 987)
IFN-y L. mexicana PBMC - No Leishmanial lysate Ag 1 Murray, H. W. et
al. (1984)
IFN-y L. mexicana PBMC Yes Leishmanial lysate Ag Murray, H . W. ct
u1. ( 1984)
IFN-y L. bra:iliensis PBMC - No L. amazonensis r Carvalho et ul.
soluble Ag (1985b)
IFN-y L. donovani PBMC - No Leishmanial soluble Ag 1 Sacks et al. (1987)
IFN-y L. donovani PBMC - Yes Leishmanial soluble Ag f Sacks er al. (1987)
IFN-y L. braziliensis PBMC ~ No L. rnexicana pijianoi 1 Rada er al. (1987)
diffuse whole Ag
cutaneous
IFN-y L. braziliensis PBMC - No L. mexicana pijanoi t Rada et al. (1987)
muco- whole Ag
cutaneous
IFN-y L. major PBMC - Yes Leishmanial lysate Ag Passwell et al.
(1987)
Ag, Antigen; BMDM, bone marrow derived macrophage; IFN, interferon; IL, interleukin; iv, intravenous; LNC, lymph node cells; LPS, lipopolysacchar-
ide; PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin; RPM, resident peritoneal macrophages; TNF, tumour necrosis factor; t. up-
graded; 1, down-graded.
214 J. ALEXANDER AND D. G. RUSSELL
VII. LYMPHOCYTE
CONTROL
OF MACROPHAGE
ANTI-LEISHMANIA
ACTIVITY
2. CD8' T cells
While the studies reported above indicate that the contribution of the
humoral response to disease progress cannot be ignored, an enormous
weight of evidence continues to identify T cell immunity as the controlling
factor (Bryceson et al., 1972; Preston et al., 1972; Skov and Twohy, 1974b;
Liew et al., 1982; Sheppard et al., 1983). Moreover, those T cells conferring
protection or counterprotection primarily belong to the CD4' T cell subset
(Mitchell et al., 1981; Liew et al., 1982; Louis et al., 1982; Gorczynski, 1985).
Some evidence does, however, suggest a protective, though not a suppressor,
role for CD8' T cells. The contribution of CD8' T cells to protective
immunity was first suspected because higher numbers of antigen-specific
CD8' T cells were generated in mice resistant to L. major than were
generated in susceptible mice (Milon et al., 1986). Furthermore, adminis-
tration in vivo of anti-CD4' monoclonal antibodies increased resistance
(Titus et al., 1985; Hill et al., 1989) to L. major, while administration in vivo
of anti-CD8' monoclonal antibodies exacerbated infection (Farrell et al.,
1989). Further evidence 'suggesting a role for the CD8' T cell subset in
protection came from studies on L. donovani. An influx of CD8' T cells was
associated with the inhibition of parasite growth in hepatic nodules (McEI-
rath et al., 1988), and both CD4' and CD8' T cells had to be adoptively
transferred from euthymic litter mates to reconstitute resistance against L.
donovani in athymic BALB/c mice (Stern et al., 1988). The mode of action of
these cells in protective immunity remains to be clarified. Early indications
that infected macrophages could be destroyed by specific cytotoxic cells
(Bray and Bryceson, 1968) have yet to be convincingly substantiated (Mauel
and Behin, 1987). However, CD8' cells can produce INF-y upon specific
stimulation (Kaufmann, I988), and this product has been repeatedly shown
INTERACTION OF LEISHMANIA WITH MACROPHAGES 217
3. CD4' T cells
Protective
Cytokine Species Host cell Mouse strain function Reference
Murine
IL-3 and L. major Macrophages BALB/c No Louis et al. (1987)
GM-CSF
GM-CSF L. major RP macrophages CBA/H Yes Handman and Burgess (1 979)
BALB/c
C3H/He
CSF (M-CSF L. major RP macrophages C3H/HeN Yes Ralph et al. (1983)
and
GM-CSF)
GM-CSF L. major PE macrophages CBA No Titus et al. (1984)
M-CSF
GM-CSF L. major S macrophages BALB/c No Greil et al. (1988)
IFN-y L. major PE macrophages CBA Yes Titus et al. (1984)
L. major BM macrophages C57BL/6 Yes Titus et al. (1984)
L. enriettii PE macrophages C57BL/6 Yes Titus et al. (1984)
L. enriettii PE macrophages CBA Yes Titus et al. (1984)
IFN-7 L. donovani RP macrophages BALB/c Yes Murray, H. W. et al. (1985)
IFN-y L. major P macrophages C57BL/6 Yes Wyler et al. (1987)
IFN-y L. major RP macrophages C3H/HeN No Davis et al. (1988)
IFN-y L. major S macrophages BALB/c Yes Greil et al. (1988)
IFN-y L. major RP macrophages C3H/HeN No Belosevic et al. (1988)
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et af. (1 988)
+ IL-2
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et al. (1988)
+IL-4
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et al. (1988)
+ GM-CSF
IFN-7 L. major PE macrophages CBA Yes Liew et al. (1989)
IFN-1 L. major PE macrophages CBA No Liew et al. (1989)
+ IL-3
IFN-y L. major PE macrophages CBA No Liew et al. (1989)
+ IL-4
TNF-a L. major PE macrophages BALB/c No Bogdan et al. (1990)
TNF-a L. major PE macrophages BALB/c No Bogdan et al. (1 990)
+ IL-4
TNF-a L. major PE macrophages BALB/c Yes Bogdan et al. (1 990)
+ IFN-y
TNF-a L. major PE macrophages CBA Yes Liew et al. (1990b)
TNF-a L. major PE macrophages CBA Yes Liew et al. (l990a)
+ IFN-y
Human
GM-CSF L. donovani M D macrophages - Yes Weiser et al. (1987)
IFN-y L. donovani MD macrophages - Yes Murray et al. (1983)
IFN-y L. donovani Monocytes - Yes Hoover et al. (1986)
IFN-7 L. donovani Monocytes - Yes Hoover et al. (1985b)
L. major Monocytes - No Hoover et al. (1985b)
IFN-7 L. mexicana Monocytes Yes Murray, H. W. et al. (1984)
+
~
BM, Bone marrow; CSF,colony stimulating factor; GM, granulocyte-macrophage;IFN,interferon; IL,interleukin; M, macrophage; MD, monocyte-
derived: PE. Deritoneal exudate: RP. resident Deritoneal: S. sdeen; TNF.tumour necrosis factor.
220 J. ALEXANDER AND D. G. RUSSELL
Until recently the toxic metabolites of oxygen, superoxide (0, -), singlet
oxygen (lo,), the hydroxyl radical (OH) and most especially hydrogen
peroxide (H,O,), have been thought to be responsible for macrophage
leishmanicidal activity (Murray, H. W. 1981; Pearson et al., 1982). Evidence
for this viewpoint arose because of studies that demonstrated that amasti-
gotes of L. donovani and metacyclic promastigotes of L. major (Da Silva et
al., 1989) survived better than, and triggered the macrophage respiratory
burst only weakly compared with, log phase promastigotes; this ability
was also attributable to amastigotes having higher levels of glutathione
peroxidase, superoxide and catalase than promastigotes (Murray, H. W.,
1982; Pearson et al., 1983). Intracellular amastigotes are also capable of
down-regulating the macrophage oxygen-dependent microbicidal potential
(Buchmuller-Rouiller and Mauel, 1987). Nevertheless, there has been
increasing evidence from several studies that oxygen-independent mechan-
222 J. ALEXANDER AND D. G. RUSSELL
isms are capable of killing Leishmania; not only are L. donovani, L. mexicana
and L. major resistant to oxygen radicals (Pearson et al., 1982; Mallinson
and Coombs, 1989b), but they can be killed by macrophages deficient in the
production of oxygen metabolites (Murray, H.W. and Cartelli, 1983; Scott
et al., 1985). Recently a new mechanism of macrophage anti-leishmania1
killing has been characterized as a novel metabolic pathway synthesizing
nitric oxides [nitric oxide (NO), nitrite (NO,-) and nitrate (NO,-)] from
L-arginine (Green et al., 1990; Liew et al., 1990a,b) with L-citrulline as a co-
product. Macrophage NO-mediated killing of L. major has been shown to be
induced by TNF-a acting synergistically with IFN-y (Liew et al., 1990a).
This biochemical pathway, as well as anti-Leishmania activity, is inhibited in
the presence of D-arginine and N-monomethyl-L-arginine (James and Hibbs,
1990; Liew et al., 1990a,b). Although the exact mechanism of NO-mediated
killing is not known, it is suspected that NO reacts covalently with intracel-
lular iron leading to the inhibition of enzymes with Fe-S prosthetic groups.
Such enzymes include some involved in DNA synthesis as well as
the proximal two oxidoreductases of the mitochondria1 electron transport
system (James and Hibbs, 1990).
AND VACCINATION
VIII. THERAPY
A. THERAPY
1. Chemotherapy
Protective1
Route of Mouse therapeutic
Cvtokine Species administration strain function Exacerbative Reference
Murine
IL-2 L. donovani ip + iv BALBIc No No Murray, H. W. et al.
(1987)
IL-2 L. donovani ip + Pentostam nu/nu/BALB/c Yes No Murray, H. W. et al.
(1989)
IL-2 L.m. amazonensis sc local BALBIc No Yes Mazingue et al. (1989)
IL-3 L. major iP BALBIc No Yes Feng et al. (1988)
CBA No No
IL-3 L. major ? BALBIc No Yes Louis et al. (1 987)
I L-4 L. major sc local BALBIc Yes No Carter et al. (1989)
TNF-a L. major iv C3H Yes No Titus et al. (1989)
BALBIc Yes No
GM-CSF L. major iP BALB/c No Yes Solbach et al. (1987a)
GM-CSF L. major iP BALBIc No No Corcoran et al. (1988)
GM-CSF L. major iP BALBIc No Yes Greil et al. (1988)
IFN-y L. donovani ip, iv + im BALBIc Yes No Murray, H . W. et al.
( I 985)
IFN-7 L. major sc local BALB/c Yes No Kiderlen and Lohmann-
iv C57BL/6 Yes No Matthes (1988)
IFN-.( L. donovani ip + iv BALB/c Yes No Murray, H. W. et al.
(1987)
IFN-.( L. donovani ip + Pentostam BALB/c Yes No Murray, H. W. et a/.
(1988)
IFN-y L. donovani ip + Pentostam nu/nu BALB/c Yes No Murray, H. W. et al.
(1989)
IFN-)I L. donovani sc osmotic minipump BALB/c Yes No Murray, H. W. (1990)
TNF-a L. major sc local CBA Yes No Liew et al. (1990~)
Human
IL-2 L. aethiopica Intranodular - Yes No Akuffo et al. (1 990)
IFN-.I L . braziliensis sc local - Yes No Harms et al. (1989)
L. tropica sc local Yes No
IFN-.I L. d. chagasi +
im Pentostam - Yes No Badaro et al. (1990)
IFN-7 L. tropica sc + pentavalent - Yes No Kurkcuoglu and Tandogdu
antimony ( 1990)
CSF, Colony stimulating factor; G, granulocyte; IFN, interferon; IL, interleukin; ip, intraperitoneal; iv, intravenous; M, macrophage; sc, subcutaneous;
TNF. tumour necrosis factor.
226 J. ALEXANDER AND D. G. RUSSELL
3. Immunotherapy
genetic background of the host, the route and dosage of cytokine adminis-
tration and the state of disease progression at the time of treatment. Of the
two CD4' T cell subsets shown to be involved in controlling Leishmania
infections, T,1 cells (which produce IFN-y, IL-2 and IL-3) have been
associated with protection, while T,2 cells (which produce IL-3, IL-4 and
IL-5) have been associated with disease exacerbation (Section VII.A.3).
Logically, one would expect that treatment with IFN-y and/or IL-2 should
induce a protective response against this organism, while IL-4 and/or IL-5
would promote disease exacerbation. Similarly, it would be expected that
cytokines which are known to promote macrophage leishmanicidal activity,
such as GM-CSF (Handman and Burgess, 1979; Weiser et al., 1987; HO et
al., 1990) and TNF-a (Liew et al., 1990a,b) would be protective in vivo, while
those generally associated with inhibiting microbicidal activity such as IL-3
(Liew et al., 1989) would exacerbate disease.
Although, as one would expect, IFN-y has been shown in numerous
studies to control the growth of Leishmania in vivo (Table 5 ) , an effect
reversed by treating mice with antibodies neutralizing IFN-y (Belosevic et
al., 1989; Squires et al., 1989), the few reports that have been published on
administration of IL-2 in vivo suggest that its role is open to question. Where-
as, for example, local administration of IL-2 to patients infected with L.
aethiopica did induce a protective response (Akuffo et al., 1990), similar
treatment of mice infected with L. amazonensis exacerbated lesion and
parasite growth (Mazingue et al., 1989). Systemic injection of IL-2, on the
other hand, had no effect on the growth of L . donovani in BALB/c mice
(Murray, H. W. et al., 1987). All studies in vivo have so far confirmed the
expected disease-exacerbating capacity of injected IL-3 (Louis et al., 1987;
Feng et al., 1988) and the protection-inducing capacity of TNF-a (Titus el
al., 1989; Liew et al., 1990c), the TNF-a effect being reversible with
neutralizing antibodies (Liew et al., 1990~).Surprisingly, when one considers
the ability of GM-CSF to activate macrophage leishmanicidal activity (Hand-
man and Burgess, 1979; Ralph et al., 1983; Ho et al., 1990), therapeutic
studies to date indicate a counter-protective role for this cytokine (Solbach
et al., 1987a; Greil et al., 1988). The exacerbative effects of GM-CSF and
IL-3 have been related to their increasing the pool of circulating monocytes
and providing additional host cells for the parasites. The role of IL-4 in
macrophage activation remains unclear and somewhat controversial (Sec-
tion'VI1.B). Its role in vivo in inducing protection or counter-protection also
awaits further clarification. Whereas, for example, treating mice with anti-
bodies neutralizing IL-4 allowed BALB/c mice to inhibit the growth of L.
major (Sadick et al., 1990), by distinct contrast treating developing lesions of
L. major locally with low concentrations of IL-4 in a slow-release matrix
promoted healing and protective immunity (Carter et al., 1989). The effects
228 J. ALEXANDER AND D. G . RUSSELL
of IL-4 injected directly into footpads infected with L. major have recently
been studied (Lezama-Davila et al., in press). Treatment at the onset of
infection promoted increased parasite growth while, by contrast, treatment
of established but still developing lesions inhibited further lesion growth and
parasite multiplication. Obviously T cellkytokine-macrophage interplay is
complex and awaits further detailed study, but the prospects for future
cytokine immunotherapy in leishmaniasis remain exciting.
B. VACCINATION
:s ca M ce. Sukutaneaua.
control.(6/6)
CBA/ca Mice. Intraperitoneal.
rude &.(2/6)
10
ICE. u u onwuo. W B / C Mice. Intmparitoneol.
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 20
TIME (weekm).
FIG. 8. A compilation of data from Russell and Alexander (1988) concerning the
protective properties of liposomes with gp63 reconstituted into the bilayer. Female
BALB/c or CBA mice were inoculated with either phosphatidyl choline liposomes
(control), phosphatidyl choline liposomes with crude membrane antigens (crude Ag),
or liposomes with gp63. Inoculations were delivered by the subcutaneous or intra-
peritoneal route twice, 4 and 8 weeks before challenge with 5 x lo4 stationary phase
L. rnexicunu promastigotes. The numbers in parentheses at the end of each curve
indicate (first) the number of infected individuals and (second) the group size.
Complete protection, determined by the inability to detect lesion development, was
found in CBA mice inoculated with gp63 liposomes by both subcutaneous and
intraperitoneal routes.
1988; Alexander and Russell, 1988). In CBA/Ca mice, under some experimen-
tal conditions, the level of protection appeared complete. Protection in
BALB/c mice, which are exquisitely sensitive to L. mexicana and show a low
T cell response to gp63, was less impressive. This result is reproducible in the
CBA/Ca system which, in contrast to the BALB/c, is a high responder for
gp63 (unpublished results). Protection can be transferred with T cells that
are predominantly of the CD4 phenotype. Since our results appeared,
several groups have published studies, mainly using the BALB/c-L. major
model, that were unable to document similar levels of protection or response
(Kahl et al., 1989; see also Handman et al., 1990). However, considerable
protection against L. major has been induced in CBA/Ca mice by means of
an oral Salmonella typhimurium vaccine expressing gp63 (Yang et al., 1990).
Furthermore, Jardim et al. (1990) have recently made considerable progress
in defining the “desirable” epitopes on gp63 by studying the protective
response induced by synthetic peptides selected on the basis of Rothbard’s T
cell epitope algorithm (Rothbard and Taylor, 1988). One peptide, PT3, from
amino acids 154-168, generated, when injected with adjuvant, a strong
protective response against L. major in BALB/c mice, L. mexicana in CBA/
Ca mice, and L . infantum in hamsters (unpublished results). Interestingly,
this peptide spans the zinc binding site of the metalloprotease and is 100%
conserved in theifour species of Leishmania sequenced to date (Button and
McMaster, 1988; Miller et al., 1990; E. Medina-Acosta and D. G. Russell,
unpublished results). The T cells responsible for immunity were CD4’ and
secreted IL-2, a characteristic ascribed to the TH1 functional T-helper cell
subset. Surprisingly, PT3 inoculated without adjuvant resulted in disease
exacerbation following challenge infection with L. major.
3. Macrophage involvement
response and protection rather than a TH2 cell response and counter-
protection can be induced against a single T cell epitope. Therefore,
adjuvants which are potent stimulators of TH1 cell activation, namely
Freund’s complete adjuvant (Grun and Maurer, 1989) and certain non-ionic
surfactants (Brewer and Alexander, in press), have transformed a potentially
counter-protective T cell epitope, PT3, into one that inhibits disease pro-
gression (Jardim et d., 1990).
IX. CONCLUDING
REMARKS
Despite the volume of literature cited, indicating how intensely the subject is
being studied at present, this review was not intended as an encyclopaedic
compendium of all that is known about Leishmania. It was heavily slanted
towards our current understanding of the biology of the host-parasite
interaction and, as such, avoided mention of basic molecular genetics and
also parasite metabolism. Parasite metabolism was left out because the line
had to be drawn somewhere, and neither of us felt we could do this field
justice. Basic molecular genetics was omitted because it does not, as yet,
contribute greatly to our understanding of host-parasite interplay, although,
through the development of transfection, transformation and gene deletion
techniques, this area will probably include the next generation of exper-
iments.
ACKNOWLEDGEMENTS
We thank Denise Williams for her help in compiling the tables and Ellen
Anne Quinn for her secretarial assistance.
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INTERACTION OF LEISHMANIA WITH MACROPHAGES 253
GUNTERA. SCHAUB
I. INTRODUCTION
IT. PARASITWENIC
ALTERATIONS
OF HOSTBEHAVIOUR
As this is the first review summarizing publications concerning the influence of trypano-
somatids on insects, it is possible that I have missed some publications. If any reader knows of
any such missing reports I should very much appreciate the information, so as to be able to in-
clude them in a later review.
258 G. A. SCHAUB
A. REDUCTION OF FITNESS
1. General
proceeded to the cibarium region of the foregut probed at least three times-
in most cases more often-and took only a little or no blood during a period
of 15 min or more (Beach e f af., 1985).
The mechanism of the action of the parasites is still unknown. The theory
of blockage of the foregut has been called in question by Killick-Kendrick et
a f .(1977b). They emphasized that “the blockage is probably more apparent
than real, since the powerful dilator muscles of the cibarium and pharynx
would easily widen the canal” and suggested that parasites might interfere
with sensilla in the cibarium. At that time such sensilla were known only
from other bloodsucking insects, but later they were indeed described in the
proboscises of uninfected sandflies (Killick-Kendrick and Molyneux, 198 1)
and their presence in the labrum and the cibarium was suggested by Lewis
( 1 984). In a detailed scanning electron microscopical investigation, Jefferies
(1987) described in the cibarium two to five trichoid sensilla with a tapering
hair that were not chemoreceptors, but were perhaps mechanoreceptors. In
addition to the blockage and the sensilla theory, calculations of the fluid
mechanisms of blood flow suggest a third possible mechanism, as they
indicate that the attached parasites, especially those in the pharynx, are
likely to impair flow (Jefferies et af.,1986). Perhaps this results in an indirect
feedback effect on receptor functions in the anterior foregut. In a later
publication Killick-Kendrick et a f . ( 1988) described a pharynx blocked by
Leish. major and indicated the importance of a gel around the parasites,
possibly an excreted factor. This report supports the blockage theory
(Killick-Kendrick and Molyneux, 1990).
colonization of the foregut and/or the interference with the sensilla seem to
be responsible for the altered feeding behaviour of infected flies (Livesey et
al., 1980). However, pathological effects on the salivary glands should also
be considered. Such effects occur in infected Gfossina(see Section 1II.E) and
it has been suggested that they are responsible for affecting the feeding
behaviour of mosquitoes infected with malaria (Rossignol et af., 1986).
Effects on feeding behaviour are also known to occur with triatomines after
infection with Tryp. cruzi or Tryp. rangeli (D’Alessandro and Mandel, 1969;
Aiiez and East, 1984). If uninfected larvae and adults and those which are
naturally infected with Tryp. rangeli and/or Tryp. cruzi were given an
opportunity to feed on mice, infected larvae fed less frequently (the
difference was statistically significant) than uninfected larvae (D’Alessan-
dro and Mandel, 1969). This phenomenon was also evident with infected
adults, but the difference was not statistically significant from those infected
with Tryp. cruzi. Probing behaviour of R. robustus and R . prolixus infected
with Tryp. rangefi was also affected (Aiiez and East, 1984): whereas unin-
fected bugs probed on average twice before engorging (range 1-5 probes),
infected bugs probed on average 13 times (range 2-28 probes) and for longer
periods than uninfected ones. Some of the infected bugs ingested only small
amounts or no blood at all, one of them even after 28 probes.
The mechanisms of these disturbances, e.g. the colonization of the
foregut, have not been elucidated. Effects on the salivary gland have to be
considered, because their cells can be damaged or destroyed (Schwarzen-
bach, 1987) (see Section 1II.E).
In addition, more features have to be included to elucidate the action of
the trypanosomatids on bugs. In a series of investigations of the effects of
starvation on trypanosomatid-triatomine interactions (Schaub and Boker,
1986b; Schaub, 1988b, 1990d, 1991; Schaub and Losch, 1989; Schaub et al.,
1989a), we had the impression that prolonged starvation affected the volume
of ingested blood. In our most recent study of the feeding behaviour of
FIG. 1. Sensilla (arrow heads) in the labrum of Glossina morsitans morsitans
associated with Trypanosoma parasites (P) (a, b: scanning electron micrographs; c:
transmission electron micrograph). (a) Trypanosoma (Trypanozoon) brucei. Bar =
5 pm. (b, c) Trypanosoma (Nannomonas) congolense. (b) Bar = 2 pm. (c) Hemides-
mosomal plaques (arrowheads) are present in the attachment zone of parasites to the
cuticle (C) of the labrum, and to the basal cup (B) and stalk (S) of a sensillum. Bar =
I pm. (Fig. la, b reproduced by permission from Molyneux and Jenni, 1981,
Transactions of the Royal Society of Tropical Medicine and Hygiene 75, 160-163, and
Fig. lc reproduced by permission from Thevenaz and Hecker, 1980, Acta Tropica 37,
163-175.)
264 G. A. SCHAUB
111. DISTURBANCES
IN ORGAN SYSTEMS
compared with the normal white, yellow or dark brown drops (Schaub,
1988a; Schaub and Breger, 1988; Schaub and Meiser, 1990; Jensen et al.,
1990). Whereas in uninfected bugs the onset of digestion of haemoglobin at
the anterior end of the small intestine coincides with a colour change to
brown, infected bugs regularly have red contents in the dilated small intestine
(Schaub and Meiser, 1990). Interestingly, none of the bugs which die of
starvation has red intestinal contents (Schaub and Losch, 1989). Occa-
sionally haemolymph of Tri. infestans infected with B. triatomae is a light red
colour (Schaub, 1988a, 1990a; Schaub and Meiser, 1990; Jensen et al., 1990).
Similar effects in sheep keds infected with Tryp. melophagium were later
shown to be caused by experimental conditions resulting in blockage of the
spiracles (Nelson, 1956, 1981; Hoare, 1972).
Whereas red rectal fluid is usually deposited by healthy Anopheles ste-
phensi (Briegel and Rezzonico, 1985), in triatomines both reddening
phenomena indicate a disturbance of the function of the intestine. By starch
gel electrophoresis, the posterior intestines and the haemolymph of these
bugs were shown to possess proteins with the same migration behaviour as
marker haemoglobin (Schaub and Meiser, 1990). In additional photometric
measurements of the contents of different regions of the intestine, absorption
spectra of red stomach contents of infected and uninfected Tri. infestans, and
also of the red contents of the posterior small intestine of infected bugs,
showed the two typical haemoglobin maxima, whereas brown contents
showed neither of these maxima (G. A. Schaub, unpublished observations).
These data support the interpretation that ingested blood is not fully
digested in bugs infected with B. triatomae.
What is the mechanism of these disturbances in bugs infected with B.
triatomae? Valuable indications are offered by an ultrastructural study in
which we detected sequential steps of the damage process to the functional
subunits of the midgut, which are the extracellular membrane layers (acting
like the peritrophic membranes in other insects), the microvilli and the
epithelial cells (Figs2a, 3). These subunits are also affected by other
trypanosomatids.
(4 (b)
FIG. 3. Transmission electron micrographs of sections of small intestine of Tria-
toma infestans infected with Blastocrithidia triatomae, showing different types of
pathology. Bar = 2 p.(a) Cell with reduced microvilli. (b) Lysed epithelial cell with
parasites. The cell on the basal lamina (arrowhead) is vacuolated. (Reproduced by
permission of Cambridge University Press from Jensen et al., 1990, Parasitology 100,
1-9.)
( c ) Intestinal cells. Not only the apical microvilli but also the body of the
intestin'al cells can be affected by the flagellates. One group of trypano-
somatids frequently destroys the cells if they are invaded for intracellular
multiplication. For example, only a mere membrane remains from the
stomach cells of the rat flea invaded by Tryp. lewisi after multiplication of
the trypanosome (Wenyon, 1926).
Members of a second group of trypanosomatids penetrate the midgut cellS
270 G . A. SCHAUB
may occur in the haemocoele, indicating that this trypanosomatid might also
be pathogenic to this host under certain conditions (Kramer, 1961).
Only insertion of the flagellum occurs in water striders infected with B.
gerridis (Tieszen et al., 1983) and in triatomines infected with B. triatomae
(Jensen et a[., 1990; Schaub et al., in press b). B. triatomae also inserts its
flagellum into the epithelial cells of the Malpighian tubules (Schaub and
Schnitker, 1988) and into host cells co-cultivated in vitro (Reduth et al.,
1989). Penetration of the intestinal wall of R . prolixus has been postulated by
Peng (1979), based on haemocoele infection in five of 16 bugs after
experimental rectal infection. However, artefactual damage to the intestinal
wall cannot be excluded and, in seven of 16 bugs, B. triatomae was found in
the haemocoele 2-1 6 weeks after inoculation into the haemocoele. After
infection by coprophagy or feeding in vitro through a membrane, we found
no flagellates in the haemocoele of about 50 Tri. infestans (G. A. Schaub and
C . Jensen, unpublished observations). Whereas the other trypanosomatids
of this group rarely affect the intestinal cells, the cells of midguts colonized
by B. triatomae are often vacuolated or lysed (Jensen et al., 1990) (Fig. 3).
Thus, the basal lamina is freely accessible to the intestinal contents, and
cannot be a barrier to the passage of haemoglobin into the haemolymph.
Perhaps the very rare penetration by Tryp. corvi and Leish. major, cited
above, is restricted to degenerating cells in weakened insect hosts, i.e. cells
which had perhaps been affected before invasion (Mungomba et al., 1989).
This could also be the explanation for the intracellular development of Tryp.
cruzi in cells of the bug’s intestinal wall (Gomes de Faria and Cruz, 1927) or
its penetration and infection of the coelomic cavity (Lacombe, 1980). Also
the phenomenon that bacteria were found only in G. m. morsitans infected
with Tryp. brucei (see Hecker and Moloo, 1981) might be due to parasito-
genic weakening of the insect. The importance of the fitness of the host is
also shown by Herpetomonas sp. in Drosophila melanogaster (Lushbaugh
et al., 1976): the trypanosome normally develops in the lumen of the
intestinal tract, but penetrates the cells of the intestinal wall and multiplies
intracellularly if the insect has a concomitant infection with a yeast-like
fungus.
3. EfSects in the hindgut
The German term “Schorf” [scab] indicates a reaction of bees to infection
with C . mellificae (syn. Lept. apis), occurring in the dorsal part of the pylorus
and only at its end. However, Lotmar (1946) and Fyg (1954) suggested that
this scab material was of flagellate origin. The low colonization density also
argues against pathological effects. Number and size of the scabs was
affected by the type of food but not by a concomitant infection with Nosema
apis (Bahrmann, 1967).
272 G. A. SCHAUB
nates Tryp. brucei and Leish. hertigi in vitro, and the agglutinin titres are
increased by a prior inoculation of either trypanosomatid into the haemo-
coele (Ingram et al., 1984).
After injection of Tryp. rangeli into the haemocoele of Tri. infestans or R .
prolixus, the number of phagocytic cells increases greatly (Zeledon and de
Monge, 1966). Uninfected Tri. infestans already possess more than twice as
many haemocytes than R. prolixus, and thereby Tri. infestans can overcome
the infection. Some strains can also be controlled by R. prolixus and nearly
all by Tri. infestans (Zeledon and Blanco, 1965; D’Alessandro, 1976).
Whereas Tryp. rangeli multiplies inside the phagocytic haemocytes after
inoculation into the haemocoele, Tryp. cruzi is killed by the haemocytes of
R. prolixus (Tobie, 1968, 1970). Initially, the number of haemocytes in-
creases in R. prolixus, but to differing extents for the various haemocytic
cells (Gomez, 1967). Since all types of haemocytes are parasitized by Tryp.
rangeli (Schwarzenbach, 1987), their number is considerably reduced in old
and heavy infections (Grewal, 1957).
The prophenoloxidase system is not activated by Tryp. rangeli in R .
prolixus or in Tri. infestans. Since the intensity of this immune response is
lowered if the parasites are incubated together with a microbially derived
molecule, which normally activates the prophenoloxidase system, it was
suggested that the susceptibility of R. prolixus might be explained, at least
in part, by immune suppression. In the tissues of the refractile Tri. infestans,
agglutinating and trypanolytic factors seem to be more widely distributed
than in those of R. prolixus (Gregorio and Ratcliffe, 1991a,b).
The haemolymph of bugs infected with Tryp. cruzi has a normal appear-
ance, but implantation experiments indicate a strongly reduced cellular
immune response of infected Tri. infestans (Bitkowska et al., 1982). Since the
fluid from cultures in vitro caused similar effects, the authors suggested that
some parasites may develop in the haemocoele after suppression of the
host’s immune reactions. However, in our Tryp. cruzi-Tri. infestans system
the cellular encapsulation of pieces of nylon thread seemed to be identical in
infected and uninfected larvae (G. A. Schaub, unpublished observations),
but in bugs infected with B. triatomae, the cellular encapsulation and
melanization reactions were almost totally inhibited (G. A. Schaub, unpub-
lished observations). The latter effect may be due to the decreased concen-
tration of amino acids used for melanization (see Section 1II.D).
Many parasites affect the colour of the cuticle of their hosts. In R. prolixus,
Pyr. apterus and Tri. infestans infected with trypanosomatids the cuticle is
often paler (Grewal, 1957; Lipa, 1963; Watkins, 1969, 1971a; Schaub, 1988a;
Schaub et al., 1990b). However, C . cimbexi, which develops in the haemo-
coele of the hymenopteran host larvae, causes no apparent alterations of
external appearance (or behaviour) of the host larvae (Lipa and Smirnoff,
1971).
The translucent and pale cuticle of R. prolixus infected with Tryp. rangeli
seems to be caused by the parasite’s multiplication in the epidermal cells
(Watkins, 1971a). The pigment granules disappear in heavy haemocoelic
infections, and periodically orange-coloured urine is excreted (Watkins,
1969). An effect on pigmentation seems also to be evident in the eyes of
infected R. prolixus. However, the fact that about 50% of infected adults
have white eyes, while only 0.2% of uninfected adults do so (Watkins, 1969),
might also be explained by a survival of tolerant or refractory bugs if the
presence of white eyes is a genetic marker.
280 G . A. SCHAUB
FIG. 6. Male Triatoma infestans infected with Blastocrithidia triatomae (left) and
uninfected (right) 2 weeks after ecdysis. (Reproduced by permission of Pergamon
Press from Schaub et al., 1990, Journal of Insect Physiology 36, 843-853.)
Infected bugs before moulting contain lower levels of the amino acids which
are incorporated into the cuticle.
Unfortunately, this effect can also be caused by retarded development,
which is seen normally in infected bugs. Including data from bugs which had
not changed their metabolism in preparation for the development of the new
cuticle presumably lowered the mean values obtained for infected bugs, e.g.
for tyrosine. However, three pieces of indirect evidence support the theory
that a lower concentration of tyrosine occurs and is responsible for the
reduced tanning. (i) Amino acid analysis of cultures of B. triatomae in vitro
indicates that the flagellates may compete with the bug for essential amino
acids in the food. (ii) The fat body, which presumably makes or stores most
of the amino acids needed for the new cuticle, is greatly reduced in bugs
infected with B. triatomae (see Section 1II.E). (iii) The most important
indication is that after the moult we could find detectable concentrations of
p-alanine and an accumulation of its precursor, aspartate, in infected bugs
only. There is increasing evidence that not only N-acetyl-dopamine but also
N-P-alanyl-dopamine plays an important role in sclerotization, tanning and
melanization. In mutants of Diptera, Lepidoptera and Coleoptera, inhibi-
tion of the incorporation of p-alanine prevents tanning and causes intense
melanization (discussed by Schaub et al., 1990b). Why did this melanization
not occur in our bugs? The failure of tanning seems to occur only if the
substrate for melanization is not available. This also is dopamine, which is
made from tyrosine. Together, these results strongly indicate that it is a
reduced concentration of tyrosine that is responsible for the reduced tanning
in infected bugs, and not a reduced oxygen supply due to the reduced
tracheal system (see Section 1II.B). However, possible actions on enzymes
and .hormones involved in sclerotization and tanning cannot at present be
ruled out.
The outer appearance, but not the tanning of the cuticle, of the hymen-
opteran Caliroa cerasi is affected by an infection (Carl, 1976; Lipa et al.,
1977). The yellow spots on so-called “slug larvae” infected with B. caliroa
are caused by an effect on the mucous coating, which dries up and peels off.
The cause of the change of the colour of the larvae to dark brown or
blackish brown was not identified, but the colour indicates a disruption of
the gut during penetration of the flagellate into the haemocoele.
An obvious effect on colouration also occurs in the salivary glands of R.
prolixus; they are normally pink and become whitish in bugs infected with
Tryp. rangeli (Grewal, 1956). This might be caused by the parasites pene-
trating the cells on their way from the haemocoele into the lumen of the
282 G. A. SCHAUB
gland. In cases of severe infection the tissue is damaged and the basal
lamina is detached from the gland cells (Schwarzenbach, 1987; Hecker et al.,
1990). An opposite effect on colouration occurs with salivary glands of
infected tsetse flies, which normally have a chalky appearance, but become
brown to black in flies with very old natural infections of Tryp. brucei (Burtt,
1942, 1950), a phenomenon reported only from the Amani region of
Tanzania. In experimentally infected flies, the host membrane of the micro-
villi in the salivary gland shows a clear reaction at the attachment site of
Tryp. brucei, a clustered arrangement of intermembranous particles (Vicker-
man et al., 1988). Salivary glands of uninfected G. m. morsitans when
dissected into saline display sinuous motility, which is not seen with glands
heavily infected by Tryp. brucei (Golder et al., 1987). These changes coincide
with considerable alterations of the composition of the secretion, e.g.
reduced cholinesterase activity (Patel et al., 1982; Golder et al., 1987), which
might be the cause of the reduced feeding behaviour of infected flies (see
Section II.B.3).
In three host-parasite systems in which the parasite is highly virulent, the
fat body is considerably reduced (Smirnoff and Lipa, 1970; Watkins, 1971a;
Schaub et al., 1990b). This might explain the retarded development of sawfly
larvae infected with H. swainei, bugs infected with B. triatomae and R .
prolixus infected with Tryp. rangeli. Because the concentration of metab-
olites concerned with moulting does not increase above the critical level, the
hormonal induction of moult is not initiated (see Section 1II.D).
Since Tryp. rangeli invades the haemocoele and develops intracellularly in
all organs, they are all affected by the flagellate. In addition to the gut
cells, Malpighian tubules, haemocytes, cuticle, tracheal and epidermal cells,
salivary glands and fat body, all discussed in earlier sections, Tryp.
rangeli damages the nervous system of R. prolixus (Watkins, 1969, 1971b;
D’Alessandro, 1976).
Iv. EFFECTS
ON PRE-ADULT DEVELOPMENT
AND MORTALITY
There is only one report of adverse effects of Tryp. cruzi on the larval
, developmental times of the pre-adult stages of triatomines (Reis dos Santos
and Lacombe, 1985). However, the retarded development of infected bugs
might be explained by their having been maintained in isolation (see Section
V1.C) or it might have been unique to the Tryp. cruzi-bug system used in
that study (reviewed by Schaub, 1989b). Such effects did not occur in my
system (Schaub, 1988c,d), and Juarez (1970) also reported no adverse effect
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 283
fected groups more females than males developed, but after infection of first
instar larvae the numbers of both sexes were identical. Sex also influences
salivary gland infections; they arise in a higher proportion of males than
females.
aMortality rate (YO)calculated for each instar from the number of dead larvae in the
respective instar and the number which entered that instar.
LI, L2, etc., first, second, etc. instar larvae.
'The same capital letter marks control and infected group data originating from one
investigation, as follows: A, Tobie (1965), R. prolixus; B, Gomez (1967), R. prolixus; C, Aiiez
(1984). CI, R. prolixus, C2, R. robustus; D, Aiiez er al. (1987), R. prolixus; X, Grewal (1957).
R. prolixus. X I , X2. X3, increasing infection rates.
Total mortality rate.
Observations discontinued.
L1 0 3 28 3 0 3 16 7 1 8 2 4 4 0 2 7 11
f0 f5 f 4 f 2 -+o f3 +
- 16 f 5 f2 f8 fll f4 f0 +3
- f3 f5
L2 1 3 5 3 1 8 5 0 4 9 4 5 2 1 8 1 4
f l f4 f 5 +2
- f2 f5 f 7 +O
- f4 f7 f8 f5 f26 f 13 f2 f5
L3 3 8 10 12 0 8 2 4 13 5 1 22 54 2 3 1
f3 f7 f 9 f 4 fO f4 f 3 f 3 f7 +4 f2 f12 *37 +5 f2 f2
L4 1 3 5 11 2 2 1 1 25 8 4 36 30 1 0 1
f2 f5 f 9 f 9 +3
- f2 f 2 f2 f14 f7 f7 f20 f16 f l fO 5-2
L5 5 6 6 20 21 6 0 3 75 21 38 68 83 4 1 1
+2
- f5 fll f 16 f12 f6 fO f2 f17 f l l f21 f24 f15 f 3 f2 f2
Ll-LSd 10 22 46 40 24 25 22 14 85 41 59 85 94 16 11 17
f4 f14 f 9 f 18 flO f3 f 17 f3 flO f20 f10 f13 f7 f 15 f4 f11
Infection 93 49 11 69 48 34 5 2
rate f6 f27 57 f16 f15 f 6 f7 f3
a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered that instar;
mean and standard deviation of three to five groups, initially each consisting of 20-45 first instar larvae.
LI, L2, etc., first, second, etc. instar larvae.
'The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1988a) Triafoma
infesfans;B, Schaub and Breger (1988) Tri. sordida; C, Schaub and Breger (1988), Tri. pallidipennis; D, G . A. Schaub (unpublished data), Tri.
spinolai; E, Schaub and Breger (1988), D. maxima; F, Schaub (1988a), R.prolixus; G, G. A. Schaub (unpublished data), R. robustus; H, G . A. Schaub
(unpublished data), R . neglectus.
Total mortality rate.
286 G. A. SCHAUB
100
-s-
-3
c
v)
U
50
c
-
VI
c
3 Bugs in control group:
r" Bugs in coprophagy groups
uninfected: o
infected: 0
10
0
15 20 25 29 34
Weeks after first feeding
FIG.7. Cumulative percentage of moults to adults plotted against age for Triatoma
infestans in uninfected populations and those infected with Blastocrithidia triatomae.
(Reproduced from Schaub and Jensen, 1990, Journal of Invertebrate Pathology 55,
17-27.)
Instar-specific mortality rate varies in the different species (Table 2). The
infection rate of Tri. infestuns and--clearly correlated therewith-the mor-
tality rate, is higher in groups given more infected bugs (Schaub and Jensen,
1990). In all sensitive species the final larval instar shows the highest
mortality rates (Table 2). This is similar to the situation with Rhodnius spp.
infected with Tryp. rangeli. Bugs often die during ecdysis in both systems.
However, this increase of mortality during ecdysis is not specific to B.
triatomae, but is correlated with the higher mortality (Schaub and Jensen,
1990), an aspect which has not been considered in the Tryp. rungeli
infections.
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 287
a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered the respective
instar; mean and standard deviation of three to five groups, initially each consisting of 2 M 5 first instar larvae.
LI, L2, etc., first, second, etc. instar larvae.
The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1990a), Tri. infestans;
E H , G . A. Schaub (unpublished data): B, Tri. sordida; C , Tri. pallidipennis; D,Tri. spinolai; E, D . maxima; F, R. prolixus; G , R. robustus; H,
R. neglectus.
Total mortality rate.
290 G. A. SCHAUB
teran Caliroa cerasi (Carl, 1976; Lipa et al., 1977). N o laboratory study has
been performed with this species, but of 2500 collected larvae, 53% died
during rearing, usually in late larval instars, and 92% of these larvae
contained heavy flagellate infections in the haemocoele. At another locality a
mortality rate of about 40% was associated with the infection. First the
colour of the larvae changed (see Section III.E), and then they eventually
stopped feeding and died.
V. EFFECTS
ON ADULTLIFESPANAND REPRODUCTION
RATE
B. HOMOXENOUS TRYPANOSOMATIDS
V I. EFFECTS
SYNERGISTIC OF TRYPANOSOMATIDS STRESSORS
AND OTHER
So far the effect of the most important abiotic stressors, temperature and
relative humidity, on insects infected with trypanosomatids have been
investigated only in the B. triatomae-Tri. infestans system (see section IV.B),
but the temperature steps used were too large to recognize synergistic effects.
Laboratory studies are necessary to clarify whether the significantly lower
prevalence of C. bombi in spring queens of bumble bees than in previous
summer workers is caused by reduced hibernation success of infected queens
or by loss of the parasites during the winter (Shykoff and Schmid-Hempel,
I99 1b).
Observations by Gorla's group indicate that a synergistic stress of infec-
tion and abiotic factors might act on natural populations of Tri. infestans.
The percentage of bugs infected with Tryp. cruzi is statistically significantly
lower in winter and early spring, with mean daily minimum and maximum
environmental temperatures of about 6" and 17"C, respectively, than it is in
mid spring and autumn (15" and 27°C) (Giojalas et al., 1990). However,
other possibilities, such as temperature dependency of the development of
Tryp. cruzi and the age structure of the population, could be excluded only
by a detailed study, e.g. using populations in chicken houses (Gorla and
Schofield, 1989).
Another stress factor for specimens from naturally infected populations
could be capture and transport to the laboratory. This was evident with
sandflies infected with different trypanosomatids of toads and lizards. The
highest rates of infection occurred in flies that did not survive the transport
(Ayala, 1973).
In other systems synergistic effects of the infection and a second stressor
are evident.
A. SENSITIVITY TO INSECTICIDES
The sensitivity of tsetse flies infected with Tryp. brucei to a low dose (50%
lethal dose or less) of different insecticides was tested by Golder et al. (1982,
1984). Within 48 h after topical application of endosulfan, about 50% more
infected flies than uninfected ones were dead. The increased sensitivity of
infected flies was also evident after application of a natural pyrethrum
extract, and in both studies males reacted more sensitively than females. In
addition, G. m. morsitans infected with Tryp. congolense had reduced
resistance to deltamethrine (Nitcheman, 1988). Whereas the results con-
cerning the effect of infection on longevity of flies are contradictory, these
insecticide data indicate at least a subpathological effect of the trypano-
somes.
In our Tryp. cruzi-Tri. infestans system we used different insecticides. The
effective dose which killed 50% of the populations did not differ between
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 297
B. STARVATION RESISTANCE
triatomae. Thus, flagellates and bug either seem to compete for essential
metabolites whose depletion results in death, or else the flagellates excrete
toxic substances (Schaub and Losch, 1989).
Similarly to Tryp. cruzi, which does not affect survival time of regularly
fed bugs reared under optimum conditions, B. gerridis and/or C .flexonema
infections reduced starvation resistance of male water striders (Arnqvist and
Maki, 1990). Surviving males showed a statistically significant lower parasite
load than those dying of starvation during the first days. Since water striders
do not survive such long starvation periods as triatomines (mean survival
time of starved males was 5 days), their natural populations are probably
more affected than those of triatomines.
eventually developed had moulted to adults. By that time 20% more bugs
had emerged in all the other infected groups (Schaub, 1990b).
Mean total mortality rates of about 10% in uninfected groups were
unaffected by group size. In most groups of infected bugs the mean mortality
rates were about 50%, but in the most crowded groups, consisting of 50
larvae, a higher mean mortality rate of 75% (a statistically significant
difference) was observed. This indicates a subpathological overcrowding
stress, increased by the synergistic action of the flagellate (Schaub, 1990b).
In natural populations, crowding of Tri. infestans reduces blood intake
(Schofield, 1982), but isolation is likely to be a less important stress factor
for triatomines. The more sensitive reaction of infected bugs to these
stressors implies that other stress factors may well also act synergistically
with B. triatomae and perhaps also with Tryp. cruzi (see Section VIII).
VII. MECHANISMS
OF PATHOGENICITY
VIII. CONCLUSIONS
TABLE
4 Summary of effects of trypanosomatids on insectsa
Feature assessedb
Trypanosomatid Host insect A B C D E F G H I K
Endotrypanum Sandfly +
schaudinni
Leishmania Sandfly + + +
donovani
L. major Sandfly + + +
L. braziliensis Sandfly +
L. amazonensis Sandfly + +
L. aethiopica Sandfly +
Trypanosoma sp. Sandfly + +
(of toads,
lizards)
Trypanosoma sp. Sandfly + +
(of bats)
T. congolense Tsetse fly f + f
T. brucei Tsetse fly f + + f +
T. lewisi Rat flea + +
T. avium Mosquito + +
Tabanid +
T. corvi Mosquito +
T. cruzi Triatomine + f + f +
T. rangeli Triatomine + + + + + + + + +
Bed bug +
Blastocrithidia Triatomine + + + + + + + + +
triatomae
B. gerridis Water strider +' + +' -c
B. pessoai Mosquito +
B. caliroa Hymenoptera + +
Herpetomonas Hymenoptera - + + +
swainei
H.muscarum Diptera + + +
Crithidia bombi Bumble bee + -
+ +
C. cimbexi Hymenoptera
Leptomonas Bug + + + +
pyrrhocorris
L. oncopelti Bug +
L. pyraustae Corn borer +
a References are given in the text.
bA, fitness; B, probing and engorgement; C, intestine; D, Malpighian tubules; E, haemo-
lymph; F, cuticle; G, further organs; H, larval development and mortality rate; 1, adult
-.
longevity and fecundity; K, survival, usually subpathogenically affected (synergistic stressors).
+, Affected (sometimes only slight indications for the respective effect); not affected; k,
contradictory results.
' Double infections with Crithidiujlexonemu.
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 303
methods commonly available, production costs are low, and it can be stored
easily.
Since the effects of B. triatomae vary in groups infected with the same dose
and maintained under identical conditions, the intensity of the effects may be
caused in part by secondary infections. A weakening of the insects has to be
considered (see Section III.A.2.c). Thus, there might have been an increase in
either the sensitivity to B. triatomae infection or the susceptibility to other
pathogens which cannot develop in uninfected bugs, or can do so only to a
harmless level. Under natural conditions even a weakening of the insect host
would probably be sufficient to permit an adjustment of the insect popu-
lation at a lower equilibrium level.
The correlation of infection rate and mortality rate indicates that the use
of B. triatomae as a biological control agent against triatomines is possible
only if high infection rates can be achieved. Since large numbers of cysts can
be readily obtained from experimentally infected bugs or from cultures in
vitro, it is feasible to spray the resting places of the bugs with a suspension of
cysts (Schaub and Jensen, 1990; Schaub et al., 1990a). A further advantage
of the use of B. triatomae is the suppression of the development of Tryp.
cruzi in double infections (G. A. Schaub and M. Mehl, unpublished
observations). However, initial exploratory field tests are required to estab-
lish whether high infection rates can be achieved and whether B. triatomae
will be the first trypanosomatid used for biological or integrated control of
insects.
ACKNOWLEDGEMENTS
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Echinococcus multiloculuris Infection: Immunology and
Immunodiagnosis
B. GOTTSTEIN
I. Introduction 321
A. The parasite, its habitat and life cycle 321
B. The prevalence, distribution and speciation of the parasite 323
C. The disease: alveolar echinococcosis (AE) in humans 325
11. Immunology 327
A. Definitive hosts 327
B. Intermediate hosts 334
111. Immunodiagnosis 339
A. Immunodiagnosis in definitive hosts 339
B. Antibody detection in human AE 343
C. Immune-complexed and circulating antigens in AE 352
D. Cellular immune response in human AE 354
IV. New developments 355
A. Recombinant E. multilocularis antigens 355
B. Diagnosis by the polymerase chain reaction 359
C. Vaccination against infection with E. multilocularis 363
Acknowledgements 366
References 361
I. INTRODUCTION
The adult tapeworms attach to the mucosa of the small intestine. The
strobila of the fully developed parasite ranges between 1.2 and 4.5 mm long
(Thompson, 1986), and usually consists of two to six (mean five) proglottids.
The rostellum of the scolex may be extended into crypts of Lieberkiihn with
rostellar hooks lightly penetrating the epithelium (Thompson and Eckert,
1983) and the four suckers of the scolex adhering to the base of the villi.
Some of the worms may occasionally break through into the lamina propria
at the site of the anterior scolex. Such induced microlesions may become of
special interest in the context of immunobiological events discussed later in
this chapter. The intimate contact between parasite scolex and host tissue is
reflected by dense microtriches covering the scolex region, which shows a
structure different from that of the strobila and which may be responsible
for absorbing nutrients directly from the mucosal wall (McManus, 1981).
Rostellar glands may be indirectly involved in such mechanisms by the
release of bioactive molecules involved in processing nutritive host compo-
nents for subsequent uptake. Excreted/secreted parasite molecules may,
thus, be of potential antigenicity.
The hermaphroditic adults reach sexual maturity in about 4 weeks (Vogel,
1957; Yamashita et al., 1958). Egg production starts as early as 28 days after
infection of definitive hosts (Thompson and Eckert, 1983); some degree of
variation may depend on parasite isolates and definitive host species. Gravid
proglottid uteri contain round to ovoid eggs (3CL36 pm in diameter), with a
single fully differentiated oncosphere embedded in an oncospheral mem-
brane and surrounded by a thick embyrophore made of closely fitting
keratin blocks (Lethbridge, 1980). Such proglottids, and the free eggs
released on their rupture, are shed in the faeces of infected definitive hosts.
Eggs released into the environment show a high degree of longevity and
resistance to degradation, due to the thickened embryophore described
above.
When ingested by a suitable intermediate host (small mammals such as
microtine and arvicolid rodents, occasionally muskrats and others), digestive
processes and other factors in the host gut result in hatching and release of
the oncospheres. These become subsequently ‘activated’, most likely by the
surface-active properties of bile, an event which can be observed by the
release and disintegration of the enveloping oncospheral membrane. The
activated oncospheres penetrate the epithelial border of the intestinal villi
within 30-120 min (Lethbridge, 1980). Assisted by hook movements and
histolytic enzymes, the oncospheres then enter venous and lymphatic vessels,
and are distributed to other anatomical sites. Most of the oncospheres
develop in the liver (although some may reach the lungs or other organs).
Maturation to the asexually proliferating metacestode involves degeneration
of the oncospheral tissue, cellular proliferation, vesicularization and forma-
ECHINOCOCCUS MUL TILOCULARIS INFECTION 323
outcome: for cases without radical surgery, the mortality rate was found to
be 92% within 10 years after primary diagnosis (Schicker, 1976). The
mortality rate has significantly decreased to 1&14% within the last decade
(Ammann et al., 1988), probably due to marked improvements in diagnosis
and therapy.
11. IMMUNOLOGY
A. DEFINITIVE HOSTS
1. Intestinal immunity
way, and the elucidation of this pathway will help to explain the basic
differences between a persistence of infection and the elimination or expul-
sion of intestinal helminths.
As it was previously thought that adult cestodes were either poorly or
non-immunogenic (It0 and Smyth, 1987), there is little detailed information
on the specific immunology of adult cestode infection in definitive host
animals. The potential risks of handling mature and gravid adult E.
multilocularis may have been the main reason hampering the immunological
investigation of intestinal adult E. multilocularis infections. Most research
with cestodes has been carried out on the Hymenolepididae in laboratory
animals, and it has been shown that destrobilation and expulsion of
immature (10 to 14 day-old) Hymenolepis diminuta in mice after primary
infection is immunologically mediated, apparently involving thymus-depen-
dent immune mechanisms (It0 and Smyth, 1987). H . nana infections in mice
result in immunity against adult infection, but do not prevent egg develop-
ment to larval cysticercoids. Immunology of Hymenolepis has been reviewed
by Rickard (1983) and by Ito and Smyth (1987) and will not be further
considered in the present review.
So far, no investigations have been undertaken to demonstrate a local
intestinal immune response (at the specific humoral and cellular level) to
adult stage E. multilocularis; thus any discussion of the specific host-parasite
interface and interactions in the parasite-harbouring intestine of definitive
hosts is speculative. The structures of adult E. multilocularis predisposed for
interaction with the intestinal immune system are the scolex, the integument
and all molecules excreted or secreted by the tapeworms. The presence of
scolex/rostellar gland cells has been described in E. multilocularis (Thomp-
son and Eckert, 1983). Secretory substances originating from such cells may
be delivered directly into areas where the rostellum is deeply embedded in
the crypts of Lieberkiihn. Host receptor cells in this area and IEL, dendritic
cells and macrophages at the base of the villi are likely to contact and to take
up antigenic parasite components for further processing. This may happen
especially if these parasite products are secreted in large quantities and the
surrounding host tissue exhibits slight damage by hook penetration (Thomp-
son, 1986). For E. granulosus there is experimental evidence for induction of
an adult stage-specific humoral immune response (see Section 1I.A). The
induction of a local immune response, however, does not necessarily imply
functionally protective interactions. The demonstration of such mechanisms
has not been undertaken at the experimental immunological level in vivo
until recently. One theoretical approach was adopted by Kamiya, H. et al.
(1980a) who observed adult E. multilocularis lysed in vitro in the presence of
serum, with the subsequent activation of the alternative pathway via
complement factor C3. Other reports lacking either substantial pathology or
host cellular reaction have been based on single infections of dogs with
330 B. GOTTSTEIN
nity to infection with 50% of the dogs showing immunity by the sixth
infection. Little or no protective immunity against reinfection has been
found against Taenia spp. in dogs and cats (Rickard et al., 1977; Williams
and Shearer, 198I). Parenteral administration of various kinds of antigens
(somatic adult or metacestode antigens, living oncospheres, secretory/excre-
tory antigens produced in vitro, and others) demonstrated controversial
results (either success or failure in inducing protective immunity), variation
depending on different research groups, experimenh and parasite species
(Gemmell, 1962; Herd et al., 1975; Rickard et al., 1975; Herd, 1977). All of
these empirical experimental approaches lacked a specific immunological
foundation, as none respected the generation, demonstration and investi-
gation of immunological mechanisms at the appropriate site of interaction.
A realistic target would be to induce a specific homing of immune cells to the
potential site of action, i.e. the epithelium of the small intestine. Neverthe-
less, some interesting immunologically related information can be deduced
from some of these experiments; for example, the application of adjuvant
(Bordetella pertussis emulsified in Freund’s complete adjuvant) non-specifi-
cally potentiated the immune status of control dogs, and thus induced a
certain degree of protection to the challenge of infection with E. granulosus
through macrophage activation (Herd, 1977). Similar observations will be
discussed later concerning metacestode infection (see Section 1I.B). Irra-
diated protoscoleces could be used as a primary approach to target the
gastrointestinal immune system. Movsesijan et al. (1968) found that oral
immunization of dogs with 1000-2500 irradiated E. granulosus protoscoleces
per animal induced protective immunity to the challenge of infection;
unfortunately the authors failed to demonstrate any immune mechanisms
responsible for this effect. Accumulating evidence suggests that new strat-
egies aimed at the vaccination of definitive hosts will not only have to
elucidate accurately and specifically the potential immunological modes of
protective responses, but will also have to develop new technologies for
vaccine antigen production, administration and presentation to the intesti-
nal immune system. Recombinant DNA techniques may be used for antigen
synthesis. This synthesis could be achieved by expression of candidate E.
multilocularis genes in biocarriers such as live attenuated Salmonella spp.,
which may prove ideal to deliver the recombinant parasite antigens to the
correct anatomical site of the definitive host (see Section 1V.C). Additional
facilities to undertake immunological studies with adult stage E. multilocu-
laris were developed by Kamiya nd Sato (1990), who showed that adult stage
E. multilocularis survived, strobilated and matured sexually in the small
intestine of young male golden hamsters and Mongolian gerbils (Meriones
unguiculatus). Immunosuppression (prednisolone tertiary-butylacetate treat-
ment) of the animals amplified susceptibility, as shown by increased survival
periods and worm numbers. Such work would be greatly advantageous for
332 B. GOTTSTEIN
southern Germany (Barutzki et al., 1990), proving the capacity for complete
metacestode development in definitive hosts.
B. INTERMEDIATE HOSTS
(1980a) showed that host resistance was related to the extent of lysis of the
protoscoleces in fresh serum in vivo. However, although they were unable to
detect host immunoglobulins on the parasite tegument, the presence of C3
was demonstrated, thereby indicating that host resistance was correlated
with serum complement levels.
T lymphocytes probably play the most important role in the immunologi-
cal control of E. multilocularis infection. Baron and Tanner (1976) reported
that depletion of T cells enhanced metastasis formation of E. multilocularis.
In congenitally athymic nude mice, E. multilucularis developed very rapidly,
and the host tissue reaction was minor compared to that of heterozygote
mice (Kamiya, H. et al., 1980b). Baron and Tanner (1977) concluded that
activated macrophages may be included as key participants as they could
be seen to adhere to the metacestode and this adhesion was enhanced by
opsonization. Ali-Khan and Siboo ( 1980) suggested that neutrophils also
could attack E. multilocularis metacestode cells coated with antibody.
Alkarmi and Behbehani (1989) suggested that the parasite survives by
actively impairing cellular mechanisms of recognition and neutrophil
chemotaxis in experimentally infected mice. This effect was attributed to
inflammatory and chemotactic properties of E. multilocularis antigens,
which may also modulate the intense inflammatory response and amyloido-
genesis in AE (Alkarmi and Ali-Khan, 1989).
The effector functions of different populations or subsets of lymphocytes
(such as THelperl or THelper2),as well as regulatory lymphokine mechanisms,
have not been studied in experimental murine AE. Linked to criteria of
susceptibility and resistance, such investigations may provide key findings
for the understanding of the different forms of progression and development
of the disease.
to normal after 83 days. The same author observed no evident sex difference
in resistance or susceptibility of AKR mice. Yamashita et al. (1963) con-
cluded from infection experiments that some mouse strains showed resist-
ance in the females to E. multilocularis infection.
In general, the determination of progressive or restrictive metacestode
growth forms, or even inability to establish infection, is markedly dependent
upon host (inbred) strains (Ohbayashi et al., 1971) and their genetically
encoded diversity or peculiarity of immune responsiveness. Strains of mice
that have been found to be particularly susceptible include AKR (Liance et
af., 1984~).CBA (Lukashenko, 1966), Balb/c and C57BL/6J (Alkarmi and
Ali-Khan, 1984); C57L/J was found by several authors to be the most
susceptible mouse strain (Kroeze and Tanner, 1987). Relatively resistant
inbred mouse strains include A/J (Lubinsky and Desser, 1963), C57BL/10
(Liance et al., 1984~)and C3H/HEJ (Yamashita et al., 1958). However,
many contradictory reports exist concerning the degree of susceptibility or
resistance of several mouse strains. These contradictions may reflect a more
complex situation concerning potential strain or isolate variations of the
parasite (Thompson and Lymbery, 1988).
Since mice of the same H-2 haplotype may differ significantly in their
susceptibility to E. multilocularis, it is probable that the control of suscepti-
bility genetically maps outside of the H-2 complex (Kroeze and Tanner,
1985). The specific immunological features that modulate metacestode
proliferation have not yet been delineated sufficiently to explain the various
courses in progressive or restrictive infection types. There is only fragmen-
tary information on cell-mediated immunity restricted to more general
aspects concerning different host cell populations. Detailed analysis of
parasite-specific T lymphocyte responses, particularly subsets of lympho-
cytes with their characteristic production of lymphokines as well as cytokine
interaction with other populations of immunologically competent cells, has
not yet been fully undertaken. Work in this direction was performed by
Kamiya, H. et al. (1980a,b), who reported that athymic nude Balb/cA (nu/
nu) mice were more susceptible to E. multilocularis than their heterozygote
+
nu/ littermates. However, these differences, obviously related to thymus-
dependent T lymphocytes, have never been substantiated by the transfer of
appropriate cell populations for sequential analysis of this primary phenom-
enon. Similar effects were observed after thymectomy, lethal irradiation
(followed by reconstituting with syngeneic bone marrow cells) or treatment
with anti-thymocyte serum of infected susceptible mice. These experiments
all resulted in enhanced E. multilocularis metastasis formation (Baron and
Tanner, 1976).
Immune suppression phenomena may also play some role in murine AE at
a more general level. Hinz and Domm (1980) showed that progeny of
ECHINOCOCCUS MUL TILOCULARIS INFECTION 339
infected NMRI female mice have a reduced humoral immune response and
are more susceptible to proliferation of E. multilocularis metacestodes than
the offspring of uninfected mothers. Malignant sarcomas are more likely to
develop in A/J mice infected with E. multilocularis, indicating a potential
depressive regulation of anti-tumour mechanisms by the parasite (Lubinsky
and Desser, 1963). A decline of peritoneal lymphocyte, monocyte and
eosinophil cell number replaced subsequently by neutrophils was observed
during the phase of E. multilocularis proliferation (Devouge and Ali-Khan,
1983). This included splenomegaly, involution of the thymus and depletion
of T cells in lymph nodes draining the metacestode lesion (Ali-Khan and
Siboo, 1980), although mechanisms responsible for these effects could not be
elucidated. From the non-specific immunological point of view, Liance et al.
( 1 990) observed the delayed-type hypersensitivity (DTH) response in vivo,
after antigen challenge of infected resistant mice, to be significantly higher
than in susceptible mice. The same research group (Bresson-Hadni et al.,
1990~)analysed the phenotypic patterns of cells within the periparasitic
granuloma in susceptible and resistant mice. Susceptibility was associated
with a persistence of numerous LY4(CD4+) lymphocytes and low macro-
phage number, whereas the periparasitic granuloma of resistant animals
showed elevated numbers of LY2(CD8+) T cells. The authors concluded
that the cell composition of this periparasitic granuloma might be of crucial
importance in controlling metacestode proliferation. Functional regulatory
interactions between immune cells causing the differences responsible for
susceptibility or resistance require investigation, including analyses of cyto-
kine secretions and their influence on interacting host and parasite cells. As
well as systematic analysis of the patterns of host cellular and humoral
immune responses, more information is sorely needed on the biological
activity in vivo of metacestode parasite cells themselves, especially the
interference of released parasite molecules which may modulate the estab-
lishment of immune responses.
111. IMMUNODIAGNOSIS
problematical for infections with low worm numbers. More recent reports
indicated that animal hosts infected with adult cestodes respond to the
infection with the formation of parasite-specific immunoglobulins (Rickard,
1983). Antibody detection, therefore, has been experimentally investigated
for diagnosis of E. granulosus infection in dogs (Jenkins and Rickard, 1985,
1986a,b). The antigens used initially in these experiments were those con-
sidered most likely to be accessible to the immune system of the host. They
were derived either from the scolex region (scolex E/S antigens), which is
intimately associated with the intestinal mucosa, or from hatched onco-
spheres, which might penetrate the intestinal wall. Serum antibodies to scolex
E/S antigens were detected by ELISA 2-3 weeks after experimental in-
fection with E. granulosus; anti-oncospheral antibodies were found 1 week
after eggs were seen in the faeces of the infected dogs. No cross-reactions
were observed with serum antibodies from dogs experimentally infected with
T. hydatigena and T. pisformis. Gasser et al. (1988) used E. granulosus
protoscolex somatic antigens to detect parasite-specific serum antibodies in
16 of 22 (73%) feral dogs with naturally acquired E. granulosus infection.
The same test was evaluated under field conditions for the assessment of E.
granulosus infections in dogs shot in the hyperendemic area of north-western
Turkana (Jenkins et al., 1990). Unfortunately, this study demonstrated that
the use of E. granulosus protoscolex antigen did not result in a reliable
diagnosis of currently infected dogs, in contrast to the Australian study
(Gasser et al., 1988). By radiolabelling and immunoprecipitation of E.
granulosus protoscolex E/S products, Gasser et al. (1989) identified two
major components of M , 27 000 and M , 94 000, both with a high diagnostic
specificity, but a lesser degree of diagnostic sensitivity. Stage-specific anti-
bodies against E. granulosus oncospheral antigens were observed in 11 of 21
dogs (52%) naturally infected with E. granulosus. The stage specificity of the
anti-oncospheral humoral immune response strongly suggested that onco-
spheres from Echinococcus eggs actually hatch in the intestine of the specific
definitive hosts. This may happen immediately after the egg-release from
ruptured terminal gravid proglottids shed from mature tapeworms, or it may
happen after peroral ingestion of E. granulosus eggs followed by hatching
induced during gastrointestinal passage. Similar features have been
suggested for E. multilocularis (Gottstein et al., 1991a).
As discussed in Section ILA, the metacestode stage specificity of the Em2-
antigen indicated that its synthesis started at day 12 after oncospheral
development after hatching. The Em2-antigen has been evaluated by Em2-
ELISA for assessing fox populations with E. multilocularis infection. The
species specificity of the test was also proven for adult stage infections, as no
cross-reactions occurred with antibodies from animals infected with intesti-
nal or tissue-dwelling non-Echinococcus cestodes or nematode species. Inves-
ECHINOCOCCUS MUL TILOCULARIS INFECTION 34 1
A
N
-
405nm
O
-
( D
e
I
o
D *
o
N
o
1 I
SPF-dogs
1. Clinical immunodiagnosis
and 1972. Mortality was much lower (190/,) between 1979 and 1983 (Gloor,
1988), and more recent data indicate a reduction of mortality to 10-14% for
the last few years (Ammann et al., 1988). These reports and similar reports
from France and Japan suggest that new diagnostic techniques and strat-
egies, including large-scale serological screening of human populations at
risk or living in endemic areas, may, in addition to new improved surgical
techniques and new measures of chemotherapy, be responsible for this
reduction in the mortality rate.
For both support of clinical diagnosis of AE and primary serological
diagnosis, the selection of a particular immunodiagnostic test involves
consideration of the diagnostic sensitivity and specificity of the technique
and the purpose for which it will be used. The operating characteristics of
most tests vary according to the method used. These include (i) the nature,
purity and quality of the antigen, (ii) the nature of patients’ immunoglobu-
lins (isotypes, etc.) specified in the test and (iii) the methodical sensitivity
of the test procedure selected. A comparison of the diagnostic quality of
different test systems then depends very much upon the characteristics of the
groups of AE patients and non-AE control persons used to carry out the
comparative study. For these reasons, judging the merits of tests is relatively
difficult, except when the various tests are evaluated in the same groups of
cases and controls. The results of such studies indicated that problems of
sensitivity fortunately appear less important (at least when employing
methodically sensitive assays such a ELISA) in immunodiagnosis of E.
multilocularis than of E. granulosus infections (Schantz and Gottstein, 1986).
Most persons infected with E. multilocularis appear to have developed a
detectable humoral immune response.
Until recently, most serological tests for immunodiagnosis of human AE
employed heterologous E. granulosus antigens. This was partly because E.
granulosus antigens could be obtained easily from many sources world-wide,
and in a very early study E. granulosus hydatid fluid appeared to be a better
diagnostic reagent for AE than antigens prepared from homologous parasite
material (Norman et al., 1966). In addition, many diagnostic laboratories
primarily investigated cystic echinococcosis, as it is a more frequent problem
than AE. The use of heterologous E. granulosus hydatid fluid antigen was
subsequently reported to be diagnostically relatively sensitive (75%-94%)
for the indirect haemagglutination test (IHA) (Hess et al., 1974; Schantz et
al., 1983; Auer et al., 1988b), or to be only slightly inferior to crude E.
multilocularis antigens in the same test procedure (Liance et al., 1984a).
Similarly, protoscoleces of both species used in IFAT yielded adequate
diagnostic sensitivities (Liance et al., 1984b).
The most specific diagnosis of cystic echinococcosis ( E .granulosus) to date
relies on the demonstration of serum antibodies reacting with an antigen
ECHINOCOCCUS MUL TILOCULARIS INFECTION 345
called “antigen 5”, which was initially demonstrated by Capron et al. (1967)
in immunoelectrophoresis with the respective precipitation of arc-5. Anti-
bodies precipitating antigen 5 also occur in serum of human patients with
AE (Varela-Diaz et al., 1977), and comparative studies showed that 58% of
AE patients from Switzerland were arc 5-positive compared to 74% of
patients with cystic echinococcosis (Gottstein et al., 1986b). Antigens shared
by E. granulosus and E. multilocularis (called the Em1 fraction) have been
isolated from crude extracts of E. multilocularis metacestode tissue by
affinity chromatography, and used as reagent for immunodiagnosis of both
cystic echinococcosis and AE (Gottstein et al., 1983). The Em1 fraction
significantly improved specificity for nematode and trematode cross-reac-
tions, compared to E. granulosus hydatid fluid antigen (Gottstein, 1985). In
general, the investigation of homologous E. multilocularis metacestode
antigens repeatedly proved to be superior to heterologous E. granulosus
antigens. Knobloch et al. (1985), for instance, evaluated crude soluble E.
multilocularis antigens by ELISA and reported antibody-binding activity in
96% of human cases of AE, without however investigating the specificity of
the crude antigen. Similar findings were described by various other authors
and have been reviewed by Schantz and Gottstein (1986). Using crude E.
multilocularis antigens, however, non-specific reactions and cross-reactions
created similar difficulties to those well known for E. granulosus antigens. An
analytical study has shown that the degree of cross-reactivity in crude E.
multilocularis antigens is markedly variable for different parasite isolates
(Gottstein, 1991). Thus, recent research has concentrated on purification of
highly specific antigens from E. multilocularis. The first documented attempt
was done by our group and employed affinity chromatographic procedures
to immunosorb cross-reactive antigenic components from a crude E. multilo-
cularis metacestode antigen solution (Gottstein et al., 1983). The resulting
fractions (Em 1- and Em2-antigen) were simultaneously applied (in ELISA)
to correctly differentiate 95% of human cases with cystic echinococcosis
from patients with AE. Such discrimination rates are potentially dependent
upon strain variations and implicated variation within the spectrum and
nature of antigens (Gottstein, 1991). Presumed variation may reflect various
sensitized B lymphoblast populations and antibody profiles associated with
different E. multilocularis epitopes. Thus, it was necessary to demonstrate
conservation of Em2 expression by examining the ubiquity of anti-Em2
antibodies in serum from patients originating from geographically disparate
endemic areas (Gottstein et al., 1986a). This study confirmed the previously
observed discrimination rate (E. multilocularis versus E. granulosus) by
differentiating 95% of 82 patients with either cystic echinococcosis or AE,
indicating that potential inter- and intraspecific strain differences do not
influence antibody response to the Em2-antigen. In conclusive studies
346 B. GOTTSTEIN
A B C M B1 M 82 83 M
2. Sero-epidemiology
Blood donors
Serological results
No. Percentage (anti-Em2-IgG detection) Clinical findingsb
17 160 99.97 Negative No investigation performed
4 0.02 Positive No liver lesion detected by
US/CT
2 0.01 Positive Liver AE confirmed clinically
Total
17166 100.00
3. Post-treatmept control
Meben-
Group/ dazole IgG IgE IgA IgM
patient concentration'
- - - -Parasite
No. (Pmolll) rb rib r rr r rr r rr localization
E. multilocularis, partial removal
1 0.25 63 18 0 0 15 0 0 0 Liver
2 0.35 10 14 0 0 0 0 0 0 Liver
3 0.90 33 3 4 0 0 0 0 0 Liver
E. granulosus, inoperable
17 0.26 91 40 6 0 48 0 0 0 Liver/pancreas
18 0.31 92 42 81 0 37 0 18 0 Liver/lungs
19' 0.10' 64'67' 15' 0' 7' 7' 0' 0' Liverlkidney
Intraperitoneal
1. Clinical diagnosis
2. Post-treatment control
IV. NEWDEVELOPMENTS
M r i 2 3 1 2 3 1 2 3
92-
66-
45-
31 -
21 -
14 -
A B C
FIG.3. SDS-PAGE and immunoblot analysis of the recombinant Echinococcus
multilocularis antigen 11/3-10, for different purification steps from bacterial cell
extracts. Immunoblots contain antigen 11/3-10 incubated with (A) a pool of sera
from healthy blood donors and (B) a pool of sera from E. multilocularis-patients; (C)
shows the corresponding silver-stained protein patterns. Lanes 1 were loaded with
crude protein extracts from bacterial cells, lanes 2 with a peak fraction from DEAE-
Sephacel chromatography and lanes 3 with the pure antigen 11/3-10 fraction from a
subsequent phenyl-Sepharose CL-4B chromatography. (After Muller et al., 1989a.)
358 B. GOTTSTEIN
Specificityb
Infecting parasites
E. granulosus 108 1 107 (99) 5 103 (95)
T. solium 15 2 13 (87) 0 15 (100)
Trematodes 26 0 26 (100) 0 26 (100)
Nematodes 71 0 71 (100) 0 71 (100)
synthesized in high yield and was quantitatively exported into the peri-
plasmic space. The recombinant (GBP-) E. multilocularis antigen could be
conveniently purified in soluble form from bacterial cell culture supernatants
following an osmotic shock procedure. The purified recombinant antigen
comprised greater than 50% of total cellular protein and could be applied
directly in ELISA.
New alternative systems for recombinant protein synthesis have been
proposed and developed using yeast, plant cells and mammalian cells
(reviewed by zu Putlitz et al., 1990). One of the most promising high-level
expressions of foreign genes has been achieved in Spodoptera frugiperda (fall
armyworm) cells infected with recombinant baculovirus. The expression in
this system is under the control of the strong polyhedrin promoter of
Autographa californica nuclear polyhedrosis virus (Luckow and Summers,
1988). This allows expression of prokaryotic and eukaryotic genes to
produce fused or non-fused recombinant proteins. One of the main advan-
tages of this invertebrate virus expression vector over bacterial expression
systems is the abundant expression of recombinant proteins, which are often
immunologically and functionally similar to their authentic counterparts (zu
Putlitz et al., 1990). In addition, baculoviruses are not pathogenic to
vertebrates or plants and do not employ transformed cells or transforming
elements as do other expression systems. The baculovirus vector also uses
many of the protein modification, processing and transport systems that
occur in higher eukaryotic cells, which may be essential for the complete
biological function of a recombinant antigen (Luckow and Summers, 1988).
As an alternative to individual E. multilocularis gene expression in host
cells, the synthesis of E. multilocularis antigens was investigated by inducing
continuous growth of E. multilocularis cells in vitro by fusing them to a
murine tumour cell line (Dieckmann-Schuppert et al., 1989). Such hybrido-
mas secreted parasite antigens as demonstrated by indirect immunofluores-
cence analysis. The hybrid E. multilocularis antigens were investigated by
ELISA for the potential immunodiagnostic value. Results indicated a genus-
specific operating level with potential for discriminating E. multilocularis
from E. granulosus.
In conclusion, the outlook for cloning of E. multilocularis genes encoding
epitopes with immunodiagnostic potential is optimistic. Molecular biology
procedures facilitate the production of serological reagents (antigens) in
large amounts and of standardizable quality, thus contributing to the
development of accurate and inexpensive immunodiagnostic tests for AE.
adult stage parasites or eggs in samples derived from the faeces or the small
intestines of definitive hosts.
5.0 5.0
4.0k d4.0
3.0 - - 3.0
2.0L -2.0
1.6 - - 1.6
1.0 - - 1.0
0.51- - 0.51
5.0 5.0
4.0k d4.0
3.0 -
2.0- - 3.0
1.6 -
-2.0
- 1.6
1.0 - - 1.0
0.51- - 0.51
30- - 3.0
20- - 2.0
16- - 16
10- - 10
0 51-
- 0.51
0.39-
- 0.39
030- - 0.30
0.22 -
- 0.22
3.0 - - 3.0
2.0 - - 2.0
1.6 - - 1.6
1.0 - - 1.0
0 51- - 051
0 39- - 0.39
0 30- - 0.30
0 22- - 022
w l i
-
G
other active cells to interact with the parasite. It is important to realize that
the two B and T lymphocyte populations recognize different kinds of
parasite epitopes. The presumed identification of E. multilocularis antigens,
or more importantly their respective epitopes, which may be related to host-
protective immunity, will most probably be achieved through a combination
of B and T cell epitopes applied and presented as a bifunctional hybrid
vaccine. A model system has been optimized for generating specific T and B
cell proliferation to E. multilocularis antigens (Gottstein et al., 1990a). Live
attenuated Salmonella had shown promise as live oral vaccines (Germanier
and Furer, 1975) and as carriers of recombinant heterologous antigens to
host immune systems (Dougan et al., 1987). Salmonella typhimurium and S.
typhi offer the great advantage that one of their main host cell (macro-
phages) exhibits the potential to perform MHC class I and MHC class I1
restricted antigen presentation. The gene fragment from E. multilocularis,
coding for a species-specific antigen 11/3-10 (Muller et al., 1989a) was
expressed in the live attenuated Salmonella typhimurium vaccine strain
LT2 MlC; an analysis of the respective antigen is shown in Fig. 7. The
recombinant vaccine was assessed for its potential to induce both a humoral
and cellular immune response in potential intermediate (mice) and definitive
(dogs) hosts. Both subcutaneous and peroral administration of the vaccine
resulted in the generation of murine antibody synthesis and the priming of
murine T lymphocytes against S. typhimurium antigen, as well as against the
recombinant E. multilocularis antigen. Significant serum antibody levels
against Salmonella and recombinant parasite antigen were found in immu-
nized dogs, whereas the proliferation of peripheral blood lymphocytes
stimulated with S . ryphimurium as well as with recombinant E. multilocularis
antigen was only borderline. This may have been related to an incompati-
bility between S. typhimurium as bacterial carrier species and the canine
cellular immune system as host species. Also, the use of peripheral blood
cells may not have been the optimal source for potentially antigen-primed
cells or the corresponding lymphocyte subset has not the potential to
proliferate in vitro. Translocation of Salmonella from the intestinal lumen
occurs mainly via PP to the lamina propria and mesenteric lymph nodes,
followed by a locally restricted dissemination to the reticulo-endothelium
system of liver and spleen. The specific cellular immune response thus may
only remain localized in the gastrointestinal area, as described also for
experimental murine Giardia lamblia infection (Gottstein et al., 1990b). A
specific local gut response may become of marked importance for a potential
vaccine against intestinal adult stage E. multilocularis infection.
In conclusion, the outlook for the development of a vaccine against E.
multilocularis infection is optimistic. Recombinant DNA techniques, includ-
ing alternative expression and presentation systems such as hybrid, live
attenuated Salmonella or vaccinia virus, should rapidly stimulate at least
366 B. GOTTSTEIN
I14K-
92K -
66K -
45K -
31K-
21K-
14K -
ACKNOWLEDGEMENTS
Our research was supported by grants from the Swiss National Science
Foundation, the Thomas Stanley Johnson Foundation, the Hoffmann-La
Roche Research Foundation, the “Kommission zur Forderung des aka-
ECHINOCOCCUS MUL TILOCULAR IS INFECTION 367
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Nematodes as Biological Control Agents: Part I1
IRENE POPIEL
Paravax Inc., 2301 Research Boulevard, Suite 110. Fort Collins, Colorado
80526. USA
AND
WILLIAM M. HOMINICK
I. Introduction ............................................
11. Mermithidae . . . . . . . . . . . . .............................. 382
111. Allantonematidae ........................... .......... 385
IV. Steinernematidae and Heterorhabditidae ...............................
A. Morphology and taxonomy ............................. 387
B. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 390
C. Production and stora
D. Efficacy ........................... ................. 411
E. Potential and future ..................................
Acknowledgements . . . . . ................................. 421
References .........................................
I. INTRODUCTION
11. MERMITHIDAE
refute their conclusions. There is still uncertainty if vector control alone can
interrupt disease transmission (Webb, 1985) or lead to its eradication (Duke,
1990). Indeed, in integrated control programs, it is possible that insecticides
cause such a reduction in insect populations that the threshold required to
maintain mermithids is breached and mermithids are eliminated from the
system (Hominick and Tingley, 1984; Walsh and Ocran, 1985). Thus, while
survival of postparasites and adults of Romanomermis culicivorax, and
infectivity of their F1 progeny, appear not to be affected by some commer-
cial pesticides and fertilizers (Walker and Meek, 1987), the possibility of
indirect effects via reductions in the host population must also be con-
sidered.
There is some support for the notion that mermithids can reduce the
transmission of onchocerciasis, at least at certain seasons (Davies et al.,
1984; Walsh and Ocran, 1985), and of malaria (Rojas et al., 1987), if climatic
conditions are suitable (frequent rains and maximum temperatures below
35°C). Although Rojas et al. (1987) alleged a reduction in prevalence of
malaria in children because the mermithids killed mosquitoes, their results
showed persistence of the nematodes in one pond, but not in two other test
ponds. It is hard to envisage how such erratic and short-term mortality
could significantly affect transmission. Furthermore, results are not always
predictable, so that Zaim et al. (1988), working in Iran, showed high rates of
mortality of mosquitoes immediately after application, but 38 days post-
treatment and thereafter, the prevalence of mermithids was usually between
0 and 10%. A year later, the nematodes had established in 2 out of 13 sites,
showing a 6 8 % prevalence. They concluded “effective, long term control is
unlikely to occur from a few artificially created epizootics. Further, the
technical procedures of production, storage and transportation of the
nematode make it costly to use for periodic inundative releases for immed-
iate control.” They felt that R. culicivorax offered limited use in antimalarial
campaigns in southern Iran, based on present knowledge. Hominick and
Tingley (1984) came to the same conclusion-that the mermithids offered
potential in programs where periodic inundative releases were deemed
suitable, provided that problems of mass production, storage and shipment
could be overcome.
Use of mermithids for control of agricultural pests may be more practical
than vector control because the level of insect population suppression
required is less. It is ironic that in China, mermithids cause high losses of
oak-silkworms, so the parasites must be controlled with chemicals (cited in
Wang and Li, 1987). In any case, workers in China appear to be investi-
gating the usefulness of mermithids in agriculture (e.g. Chen et al., 1989), and
their conclusions are that there is potential for control. Similarly, Agamermis
unka parasitizes brown planthoppers (Nilapavata lutens) in rice paddies in
NEMATODES AS BIOLOGICAL CONTROL AGENTS 385
111. ALLANTONEMATIDAE
is one reason why mermithids remain largely unused for mass biocontrol
programs, and why there is so much current interest in entomopathogenic
rhabditids.
feltiae. As Poinar (1990) points out, unless authors used precise designations
for the nematodes in their studies, the identity of their specimens may never
be known. He lists the nine currently recognized species with their synonyms
and considers the first species described, S. kraussei (Steiner, 1923) as species
inquirendae. Keys for identifying infective juveniles and adult males are also
provided.
Heterorhabditids have not escaped taxonomic revision. Heterorhabditis
heliothidis has been synonymized with H . bacteriophora, while other isolates
previously designated H . heliothidis have been described as H . zealandica.
Currently, there are three Heterorhabditis species, namely H. bacteriophora
Poinar, 1976, H. zealandica Poinar, 1990, and H. megidis Poinar, Jackson
and Klein, 1987. As for the steinernematids, reasons for these changes are
documented by Poinar (1990) to whom readers should refer if they require
details, descriptions or keys. As Hominick and Reid (1990) emphasized,
Poinar’s (1990) contribution is a bench mark for entomopathogenic nema-
todes, and it is essential that his proposals are followed or refuted in the
literature so that a universal system evolves.
Identification at the species level is based on the morphology of adult
males and infective stages. D. Sturhan (personal communication) has
observed characters of the infective stages of Steinernema that supplement
those provided by Poinar ( 1 990) to permit their identity to be determined. This
work is being prepared for publication and its usefulness remains to be
determined. Furthermore, such identities must be confirmed by investigating
the morphology of adults so that the confusion of separate juvenile and
adult descriptions, which so pervades and confuses mermithid taxonomy, is
avoided.
Molecular techniques are making contributions to entomopathogenic
rhabditid taxonomy with DNA sequence analysis offering the most promise
to characterize isolates at the species level and lower (Curran, 1990a). This is
because the DNA of a species remains constant in all stages and is
unaffected by the environment, while methods depending on protein analysis
require careful controls to dismiss effects due to developmental changes,
nutrition, environmental effects and, in the case of these nematodes, the
bacterial symbiont proteins. Nevertheless, A. Burnell and colleagues at St
Patrick’s College, Maynooth, Ireland (personal communication) are finding
that isoelectric focusing (IEF) of proteins of Heterorhabditis isolates is
useful for determining the degree of relatedness between isolates. Dendro-
grams, based on IEF of general proteins and specific enzymes, can be
constructed with the aid of computer software. Other workers have shown
the usefulness of electrophoresis of proteins in the taxonomy of entomo-
pathogenic rhabditids (see references in Curran, 1990a). The difficulty of
testing mating compatibilities for Heterorhabditis isolates, together with the
NEMATODES AS BIOLOGICAL CONTROL AGENTS 389
B. BIOLOGY
The third stage juvenile, sometimes called the dauerlarva, is the survival and
infective stage. These juveniles are non-feeding and can survive in the soil for
extended periods, the duration of which depends on the species and physical
conditions. Infective juveniles harbor specific symbiotic bacteria (Xenorhab-
dus spp.), and can locate hosts with varying degrees of efficiency. Stimuli that
induce aggregation in steinernematids include temperature, carbon dioxide,
host excretory products, plant roots and the symbiotic bacteria of the
nematode (Gaugler et af., 1991). Once they contact a potential host, they
enter through natural openings (mouth, anus, spiracles) and, in the case of
Heterorhabditis spp. whose infectives possess a tooth, across the body wall
(Bedding and Molyneux, 1982). Some authors disagree about the import-
ance of this method of entry (Mracek et al., 1988). In any case, the infectives
enter the host hemocele and initiate development by releasing cells of
Xenorhabdus which rapidly multiply. The host dies soon thereafter, and the
nematodes develop by feeding on the bacteria. Infectives of Steinernema
develop into males or females which mate, while those of Heterorhabditis
develop into hermaphroditic females. A second generation of smaller adults
consists of amphimictic males and females. The nematodes go through
several generations in the cadaver and then produce infective juveniles. It
has been shown that high population densities and/or lack of nutrients
induce infective juvenile formation (Popiel et af., 1989b) and a pheromone
inducing infective juvenile formation was implicated. Infectives leave the
host to enter the soil where they remain until they contact another host or
perish.
Once the nematodes enter the soil, our knowledge of their population
biology becomes limited. The reader is referred to several recent reviews for
details which are beyond the scope of this review (Curran, 1990b; Hominick,
1990; Hominick and Reid, 1990; Kaya 1990; Klein, 1990). Major contem-
porary issues include how best to quantify the nematodes in soil (flotation,
Whitehead trays, or counting the numbers that penetrate into insect baits:
see Fan and Hominick, 1991a; Curran and Heng, 1992), the role of soil
antagonists in controlling nematode populations, the role of the second
stage cuticle ensheathing the infective nematodes, host-searching and mo-
NEMATODES AS BIOLOGICAL CONTROL AGENTS 39 1
2. Geographical distribution
only the characters sandy, loamy or clay and find that clay soils are poorest
in supporting entomopathogenic rhabditids; Hominick and Briscoe ( I 990),
Kaya (1990) and Hara et al. (1991), supply details. However, soil is a
complex medium and other important characteristics must be considered.
For example, in the UK, Steinernema feltiae ( = bibionis) and a Steinernema
sp. were associated with calcareous soils (Hominick and Briscoe, 1990). The
Hawaiian survey of Hara et al. (1991) showed that Heterorhabditis sp. was
also associated with calcareous soils. Finally, Lindegren et al. (1990) found
that S. carpocapsae persisted significantly longer in coral sand than soil, and
coral is a source of calcium. As Hara et al. (1991) point out, the association
of these nematodes with, and their persistence in, calcareous and sand soils
needs further investigation, which could provide a means for increasing the
effectiveness of these nematodes as biological control agents.
Another intriguing association for entomopathogenic nematodes is that of
some heterorhabditids and the sea. In the Hawaiian islands, 24 of 351 sites
(6.8%) were positive for entomopathogenic nematodes, with 22 of the
sites producing Heterorhabditis sp. and only two producing Steinernema sp.
(Hara et al., 1991). Presence of heterorhabditids was highly correlated with
samples taken from ocean beaches within 100 m of the seashore. In the UK,
only 1 of 403 sites chosen randomly (0.25%) proved positive for a hetero-
rhabditid (Hominick and Briscoe, 1990), and the site was within 1000 m of
the sea. A subsequent targeted survey of 61 sites having sandy soil and
within close proximity to the coast produced two more sites with hetero-
rhabditids, and both were within 100 m of the sea (W. M. Hominick and B.
R. Briscoe, unpublished observations). This prevalence of 3.3% is much
higher than the prevalence obtained from random samples. Colleagues in
Eire also tend to find heterorhabditids close to the sea, both there and in
Scotland (C. T. Griffin and M. J. Downes, personal communication). This
coastal association is unexplained and deserves further investigation as it
could elucidate factors restricting efficacy of at least some heterorhabditids.
3. Environmental limitations
factor for nematode survival in nature. Although high and low temperatures
are lethal, it is not known how capable these nematodes are of moving away
from lethal extremes. Behavioral studies indicate that steinernematids pos-
sess programmed responses to temperature. Burman and Pye (1980a)
showed that infective juveniles of S. carpocapsae tended to migrate on the
surface of agar from cooler and warmer temperatures towards the tempera-
ture at which they were cultured when tested immediately after harvesting
from culture. The authors suggested that having a conditioned temperature
preference might help the nematodes locate new hosts in the same soil
stratum as the insect they left. This behavior may also ensure the avoidance
of temperature extremes. Seasonal vertical migration in response to moisture
and temperature changes are likely to occur.
To what extent infective juveniles can reduce their metabolism to prevent
exhaustion of food reserves is also not known. Ishibashi and Kondo (1986a)
found that the recovery of nematodes maintained at 25°C for 1 month in
sterilized soil appeared constant by using the sucrose flotation recovery
method, but seemed to decline if the Baermann funnel method was used. As
Baerman funnel recovery requires nematode migration from the soil, the
authors assumed that over time the nematodes became quiescent. However,
deterioration and loss of motility as a result of maintenance at 25°C could
equally account for these results (Fan and Hominick, 1991b). Under
experimental conditions infective juveniles are often observed in character-
istic non-motile postures. The most common is a straight configuration with
the tail at an angle to the body. Some nematodes, most notably S. gfaseri,
have a tendency to become immobile in a coiled position. Molyneux (1985)
attributed the longer survival of S. gfaseri,in comparison to other species, to
its ability to become quiescent in the coiled position. However, it remains to
be shown whether the metabolism of hydrated nematodes in these postures
is merely reduced by lack of mobility, or whether the nematodes actually go
into a physiological quiescent state.
In soils with water contents which allow nematodes to remain hydrated,
survival is best at low soil moistures, e.g. S. carpocapsue and S. gfaseri
survive best at soil moistures of 2% and 4%, respectively (Kung et af.,
1991). In waterlogged soils movement is impaired due to the absence of a
discrete water film around soil particles, and oxygen becomes a limiting
factor for nematode survival. Molyneux and Bedding (1984) showed that the
optimum soil water potential for infectivity of the host varied depending on
the soil type. In fine sand, infection of sheep blowfly larvae by Heterorhabdi-
tis sp. D1 and S. gfaseri took place at water potentials of 0.0034.4 bars,
whereas in loamy sand, the range for infection was greater, that is 0.01-100
bars. From a physiological point of view, the most important parameter
with regard to the water content of soil is the water potential (pF value). In
NEMATODES AS BIOLOGICAL CONTROL AGENTS 395
different soils with the same per cent water content, the p F value may be
different making per cent water content a useless comparative measurement.
Thus, p F values should be used by all researchers in this field who measure
water in soil.
The earliest observations on the effect of desiccation on S. carpocapsae
were made in order to determine the nematodes’ physical limitations when
applied against insects in environments in which water loss would occur.
Schmiege (1963) and Kamionek et af.,(1974) showed that infective juveniles
of S. carpocapsae are rapidly killed by direct exposure to dry air, e.g. for
infective juveniles individually exposed to 20% relative humidity, the LT,,
was 1.5 h. Simons and Poinar (1973) attempted to simulate nematode
dehydration in soil by exposing individual infective juveniles of S. carpo-
capsae to 96% relative humidity for 12 h, then to 93% for 12 h, followed
by transfer to final relative humidities ranging from 79.5% to 0%. Survival
was inversely proportional to the final relative humidity. At 79.5% relative
humidity, survival was 90% after 12 days and 50% after 17 days; at 48.4%
relative humidity, survival was 80% after 4 days and 0% after 12 days. In
comparing their results to those of Schmiege (1963) and Kamionek et a f .
(1974), Simons and Poinar (1973) attributed the extended survival in their
experiment to the effect of gradual desiccation. They concluded that the
application of steinernematids to soil was more practical than on above-
ground plant surfaces, a conclusion borne out by numerous field trials. It
should also be noted that survival and infectivity are two different processes
and need not be related (Fan and Hominick, 1991b).
Although it is recognized that successful insect control cannot be achieved
when nematodes are applied to environments in which they will dehydrate,
unpublished observations of many workers show recovery of viable S.
carpocapsae and S. felriae from dry soil (e.g. G . 0. Poinar; J. H. White;
B. R. Briscoe; J. Curran). Thus, it cannot be disputed that, in natural
conditions at least, some proportion of nematode populations can survive in
conditions of extreme desiccation.
Soil type has an effect on both nematode survival and infectivity. In an
evaluation of the survival of S. carpocapsae and S. gfaseri in different soil
types, Kung et al. (1990a) found the lowest survival to be in clay. They
suggested this was because of the utilization of more energy reserves during
movement in the small pore spaces of clay, combined with low oxygen
tension. Nematode infectivity is also adversely affected by soils with a high
clay content (Molyneux and Bedding, 1984; Geden er al., 1985). Infectives of
S. gfaseri, the largest steinernematid, are the least effective in soils with a
small pore diameter (Molyneux and Bedding, 1984).
Infective juveniles of S. carpocapsae are acutely sensitive to sunlight
(Gaugler and Boush; 1978). Exposure of infective juveniles to sunlight for
396 I. POPIEL AND W. M. HOMINICK
(c) Biotic factors. Predation, competition for space and parasitism are
the major biotic factors which could affect natural and applied nematode
populations. Most studies on nematode antagonists have been performed
under laboratory conditions and little is known about their impact in the
field.
The survival of S. glaseri and S. carpocapsae was significantly greater in
sterilized soil than non-sterilized soil (Ishibashi and Kondo, 1986a), but the
biotic factors responsible were not investigated. Potential nematode antagon-
ists identified under laboratory conditions include nematophagous fungi
(Poinar and Jansson, 1986; Timper and Kaya, 1989), nematode trapping
fungi (Poinar and Jansson, 1986), microsporidian parasites (Poinar, 1988),
predatory nematodes (Ishibashi et al., 1987b) and arthropods (Ishibashi et
al., 1987b; Epsky er al., 1988).
NEMATODES AS BIOLOGICAL CONTROL AGENTS 397
cation criteria include dye uptake on nutrient tryptone broth agar (phase
one colonies of most species will be blue) (Akhurst and Boemare, 1988),
pigment production (Khan and Brooks, 1977; Grimont et al., 1984; Richard-
son et al., 1988) antibiotic production (Akhurst, 1982; Richardson et al.,
1988), colony morphology and the presence of intracellular protein crystals
(Boemare et af., 1983b; Couche et af., 1987; Couche and Gregson, 1987). X .
luminescens is luminescent (Khan and Brooks, 1977).
Xenorhabdus growth has been reported in a variety of complex media
(Gotz et al., 1981; Dunphy et al., 1985; Bleakley and Nealson, 1988; Schmidt
et al., 1989; Poinar et af., 1990), insect hemolymph (Gotz e f af., 1981; Poinar
et al., 1990) and a proline minimal medium (Bleakley and Nealson, 1988).
Temperature optima for growth range from 24 to 30°C (Dunphy and
Webster, 1989).
Phase one Xenorhabdus spp. produce several unusual secondary metab-
olites including pigments, bioluminescence, antibiotics, intracellular protein
crystals and extracellular enzymes. The occurrence and nature of these
products within the genus display a singular heterogeneity which com-
pounds attempts to assign specific functions to them. Their properties and
putative roles, # discussed in detail by Nealson et af. (1990), are summarized
below.
Most species of Xenorhabdus produce pigments ranging in colour from
buff to brown to red (Grimont et al., 1984; Akhurst and Boemare, 1988).
These pigments cause a colour change in infected insects, the most dramatic
being the red coloration of insects infected with X. luminescens released by
Heterorhabditis spp. A red pigment has been isolated from X. luminescens
and identified as an anthraquinone derivative (Richardson et al., 1988). The
pigment genes have been isolated and cloned and expressed in E. coli
(Frackman and Nealson, 1990). Pigment function is unknown. The color-
ation of infected insects could function to attract potential hosts (Nealson et
al., 1990), or as camouflage, or as a deterrent to insect predators. However,
such visual signals could only operate when infected insects occur in the
light.
Bioluminescence, which only occurs in X. luminescens, was first discovered
by Khan and Brooks (1977). The luminescence is catalyzed by a luciferase
which is structurally and functionally similar to the luciferases of luminous
marine bacteria (Schmidt et af., 1989). However, gene regulation appears to
be different (Levisohn and Nealson, 1988). Phase two X. luminescens
produces significantly less luminescence than phase one; in some strains this
is due to lower levels of luciferase, whereas in others it is due to differences in
cellular expression of the enzyme (Nealson et al., 1990). Cloned lux genes
have been expressed in E. cofi (Nealson et al., 1990). A non-luminous strain
of X. luminescens has been described (Akhurst and Boemare, 1986). Thus,
the tools are in place for further study of this intriguing property, the func-
NEMATODES AS BIOLOGICAL CONTROL AGENTS 399
5 . Safety
1. Production
tion occur at lower nematode densities (I. Popiel and W. Lanier, unpublished
observations). A second round of reproduction maximizes conversion of
insect tissue to nematode tissue. The ability of the nematodes to regulate
reproductive rate in response to nematode density results in reliably high
yields of infective juveniles irrespective of inoculum size. The formation of
infective juveniles, as opposed to non-infective third stage juveniles, is
influenced by increased nematode densities and decreased levels of nutrients
(Popiel et af., 1989b). About 14 days after infection, infective juveniles will
conveniently migrate from the insect cadavers and associated debris towards
a water source, producing a relatively contaminant-free preparation. The
high cost of in vivo production makes it generally inappropriate for commer-
cial use (an Italian company is using Galleria for production), but it is a
useful simple method for research-scale production.
Entomopathogenic nematodes were first produced in vitro by Glaser
( 1 93 I , 1940a) and Glaser et al. ( I 940). They produced S. glaseri on a variety
of agar-based media pre-inoculated with yeast and the natural flora of the
nematode which was, unbeknownst to them, Xenorhabdus. Unreliable yields
due to contamination and loss of Xenorhabdus as a result of unwitting use of
antibiotics, provided the motivation for Glaser to develop an axenic culture
method. In this method rigorously disinfected infective juveniles were
inoculated onto 1 YOagar overlaid with slices of raw kidney (Glaser, 1940a;
Glaser et al., 1942). This medium remains the most commonly used for the
production of axenic nematodes.
Glaser (l940b) and Stoll(l953) showed that nematodes could also grow in
liquid medium containing raw kidney or liver extract, respectively. Stoll
(1953) obtained optimum yields in test tube cultures by shaking them at
100 r min- '. The beneficial effect of aeration of axenic liquid cultures was
also demonstrated by Hansen and Cryan (1966) and Hansen et al., (1968)
who grew S. feltiae and S. carpocapsae in thin films of a medium containing
3% soy peptone, 3% yeast extract and 10% liver extract or 20% fresh yeast
extract adsorbed on the surface of glass wool. The defined basal medium
CbMM (Caenorhabditis briggsae maintenance medium) supplemented with
heated liver extract or chick embryo extract and serum was also used,
achieving a five-fold increase in yield. Aeration of the same media in bottles
gave rise to similar yields (Buecher and Hansen, 1971). S. glaseri has been
grown in a completely defined medium (Jackson, 1962, 1973), but with only
low levels of multiplication. The severely reduced pathogenicity of axenic
compared to monoxenic infective juveniles, and the high cost of axenic
culture make this an unacceptable approach for mass culture. Initial
methods of monoxenic culture involved systems in which the nematodes and
the symbiont were grown on the surface of a variety of media in tubes, Petri
plates or trays. Glaser (1940a) grew S . glaseri on its symbiont, on fermented
406 1. POPIEL AND W. M. HOMINICK
potato mash or veal infusion agar. House et al., (1965) used a dog food
medium for the culture of Xenorhabdus and S. carpocapsae strain DD136.
In response to the need for a larger scale and more economical culture
method, Bedding (198 1, 1984b) devised a system with increased surface area
for medium adsorption by using crumbed plastic foam coated with pig
kidney, beef fat, or chicken offal media. A medium containing soy flour,
nutrient broth, yeast extract and corn oil was developed by Wouts (1981) for
heterorhabditid production. The Bedding method can be used with many
types of vessel ranging in size from 500 ml Erlenmeyer flasks each yielding,
for example, 56 x lo6 Heterorhabdifis sp. (Bedding, 1981), to I x 0.5 m
polypropylene bags each yielding 2 x lo9 Heterorhabditis sp. (Bedding,
1984b). For larger scale production, self-aerating trays are used (Bedding,
1988). For nematode harvesting, 5-cm deep foam crumbs are placed on
sieves, and the nematodes are allowed to migrate out and into water below.
Although the entire method is very labor intensive, it has been used
successfully for commercial production.
Using an economic model of the Bedding process, Friedman (1990)
showed that high labor costs limit the economies of scale to yields of no
greater than 10 x 10l2 infectives per month. Although this demonstrates that
the process is’ ultimately uneconomical for industrialized countries, it is
appropriate in countries where the cost of labor versus capital equipment is
relatively low. In China, for example, S . carpocapsae is produced cost
effectively by this method (Bedding, 1990) (Fig. 2).
A semi-fluid dog-food agar medium has been described for the culture of
S. kushidai (Ogura and Mamiya, 1989), a new species recently isolated in
Japan. The ammonia content of spent medium from S . kushidai cultures was
six times greater than in S. felfiae spent medium, suggesting a greater protein
requirement. This was supported by the fact that addition of peptone to the
medium resulted in greater yields. Peptone also stimulated the initial
development of infective juveniles.
A detailed study of the physical and chemical requirements of S . carpo-
capsae and H . heliothidis in monoxenic culture showed differences in the
reproductive responses of the nematodes to many nutrients (Dunphy and
Webster, 1989). Although this allowed for improvements to be made to the
basic medium, the presence of metabolizing bacteria did not allow for
unambiguous conclusions to be made about nematode nutrition. Indeed,
because of the advantage of growing nematodes with their symbionts, their
direct nutritional requirements are never likely to be defined.
Monoxenic liquid culture of entomopathogenic nematodes has been
developed recently, but because aspects of the technology are proprietary,
little of the process is described in the scientific literature. Buecher and
Popiel (1989) described the complete development of S. carpocapsae in a
semi-defined medium containing tryptic soy, yeast extract, cholesterol and
NEMATODES AS BIOLOGICAL CONTROL AGENTS 407
2 . Storage
derives from the commercial need to extend the shelf life of nematodes at
ambient temperature beyond their normal life span in the hydrated state.
Desiccation-tolerant organisms survive extreme water loss in a state of
arrested metabolism called anhydrobiosis. Several groups of organisms,
including some plant parasitic and free-living nematodes, are capable of
anhydrobiosis (Crowe and Clegg, 1973). For the soil-dwelling mycophagous
nematode, Aphelenchus avenae, ahydrobiotic survival is dependent on slow
drying, which allows the nematodes time to prepare biochemically for
extreme water loss (Madin and Crowe, 1975). This involves accumulation of
glycerol to a level of 5% and'the disaccharide trehalose to a level of 12%.
There was a direct relationship between the level of trehalose and the ability
to survive exposure to 0% relative humidity.
Attempts to induce similar biochemical changes by slow drying have
formed the basis of an extensive effort to achieve anhydrobiosis in steiner-
nematids. Ishibashi et a / . (1987~)showed that slow drying infective juveniles
of S . carpocapsae strain DDI 36 was only beneficial for short-term desic-
cation survival. Survival for 6 days at 34% relative humidity was 21 YOwhen
nematodes were pre-exposed to 98% relative humidity for 3 days whereas it
was < l o % without pre-exposure. During exposure of the nematodes to
98% relative humidity for 6 days, trehalose content increased to a level of
0.1 pm glucose equivalents (per mg dry weight), a level significantly lower
than that achieved in A . avenae.
Popiel et a / . (l989a) showed that trehalose and glycerol levels equivalent
to those in A . avenae could be induced in S . carpocapsae by exposure of
pellets of infective juveniles to 96% relative humidity for 3 days. But once
again, slow drying resulted only in improved short-term desiccation survival,
as measured at 53Yo relative humidity. Prolonged maintenance of desiccated
infective juveniles at a range of relative humidities led to a decline in survival
which was inversely proportional to water content. Similar survival charac-
teristics were described by Womersley (1990) for S . carpocapsae. It would
appear, therefore, that either the trehalose levels that can be induced in S .
carpocapsae are insufficient to protect the nematodes from desiccation, or
that trehalose is spatially prevented from acting as a water replacement
molecule, or that trehalose levels are unrelated to desiccation survival in
these nematodes. It is also possible that natural anhydrobiotes possess
additional, as yet unknown, adaptations to protect themselves from water
loss or degradation in the dry state.
It was shown for A . avenae that the longer the nematodes were stored in
the desiccated state, the more dependent their recovery became on slow
rehydration at 100% relative humidity for 24 h (Crowe and Madin, 1975). If
such slow rehydration, thought to allow for the repair of damaged mem-
branes, were a requirement of desiccated entomopathogenic nematodes,
NEMATODES AS BIOLOGICAL CONTROL AGENTS 41 1
D. EFFICACY
1. Field trials
In the USA, extensive efforts have been made to control Popillia japonica,
the Japanese beetle, a major pest of turf grass. Beetle larvae emerge to
feed on grass roots in the spring and autumn. Nematodes are applied in the
autumn because temperatures in the spring are usually too low for the
nematode to be effective. S . carpocapsae and Heterorhabditis sp. have been
field tested the most, simply because of availability. Heterorhabditids have
been generally more effective, although their performance has not been
consistent (Shetlar et al., 1988; Georgis and Poinar, 1989). Although
approximately 100 field trials against P . japonica have been performed, some
notable gaps in the knowledge of the interactions between nematodes,
’
insects and the environment remain. Published data from laboratory screen-
NEMATODES AS BIOLOGICAL CONTROL AGENTS 413
ing of different nematode species and strains (Klein, 1990) is scanty, little is
known about the ability of different nematode species and strains to pass
through the thatch layer (a dense layer of dead roots and organic matter that
accumulates above the living root zone) to the root zone where the insects
occur, few experiments have been performed to identify the physical factors
which limit nematode effectiveness in turf, and the effect of biotic factors is
unknown. Consequently, low efficacy in field trials often goes unexplained.
Improvements in efficacy may come from subsurface injection of nematodes
(Berg et a[., 1987), which delivers them directly to the zone of insect activity,
and spring applications of strains that are infective at low temperatures, e.g.
S. feltiae (Wright and Jackson, 1988). However, what is most required is a
redirection of effort from repetitive field trials to the acquisition of more
knowledge on the interactions between different nematode species and
strains with the target insect and the turf environment.
The results of attempts to control the corn rootworm, Diabrotica sp., a
major pest in the USA, have also been variable. Results of field tests with
various strains of S. carpocapsae have ranged from no control (Munson and
Helms, 1970) to control superior to that achieved with chemicals (Poinar et
al., 1983). Once again, the factors contributing to success and failure were
not always identified, and the use of nematodes in this application remains
unpredictable.
Cryptic habitats within plants, although not the natural habitat for
nematodes, provide ideal conditions for their survival and infectivity. In-
deed, some of the most reliable results have been achieved against plant
boring insect pests. The blackcurrant borer, Synanthedon tipuliformis, was
successfully controlled by applying S. feltiae to blackcurrant cuttings (Bed-
ding and Miller, 1981). In China, the tree boring cossid moth, Holcocercus
insularis, has been successfully controlled by manual application of nema-
todes to the uppermost entry and exit holes on the tree (Fig. 5) (Bedding,
1990). This species of borer produces interconnecting galleries which facili-
tate nematode recycling; insect mortalities in excess of 90% are common. In
developed countries, lack of a cost-effective method of delivery to gallery
openings, which are often difficult to find, is a major limitation for the use of
nematodes against boring insects.
Application of S. carpocapsae to artichoke plume moth larvae infesting
artichoke leaf stalks has been successful (Bari and Kaya, 1984). This part of
the plant provides conditions suited to nematode survival as does the cool
foggy climate of the artichoke growing area.
In contrast to their use in cryptic habitats, attempts to use nematodes for
insect control in foliar, manure and aquatic habitats have met with little
success, largely because the environmental conditions are not suitable for
nematode survival and/or infectivity (for further details see Begley, 1990).
414 I. POPIEL AND W. M. HOMINICK
FIG. 5. Manual application of steinernematids for the control of the tree boring
cossid moth, Hokocercus insuluris, in the galleries of shade trees in China.
2 . Ecological considerations
lia cuprina) larvae between Heterorhabditis sp. (LD,, = 18) and S. feltiae
(LD,, = 53 490). Differences in LT,, as great as 50-fold were also observed
between strains of the same species in more extensive bioassays by the same
authors (Bedding et al., 1983). It is now generally accepted that a number of
nematode species and strains should be tested against a particular insect
prior to field testing. Bedding (1990) recommended a preliminary determi-
nation of LT,, against individual insects in sand. Two or three nematodes
which are the most effective should then be evaluated in pot tests using
appropriate soil and plants, followed by small-scale field trials. Although
this is all very well in theory, in practice few nematode species and strains are
available in large enough numbers for field trials, making it impossible to
field test some nematode strains which show most promise in laboratory
tests. For example, S . glaseri and H. megidis were the most effective species
against P.japonica larvae in laboratory tests (Klein, 1990), but they have yet
to be produced in sufficient numbers for field testing. Thus, for many insect
pests, acceptable control with nematodes will not be achieved until appro-
priate production methodology has been developed.
While strain variability of entomopathogenic nematodes is a recognized
phenomenon, the possibility of strain variability of the hosts has been
neglected. This is no doubt a complicating factor which will play a part in
affecting the efficacy of nematodes. Although details of the effects are not
published and have been communicated to us personally, they are suffi-
ciently important to be mentioned here. R. A. Bedding, R. A. Sikora, N.
Treverrow and P. F. Parniski conducted a co-operative project to control
adult Cosmopolites sordidus, the banana weevil borer, in plantations in
Australia and the Kingdom of Tonga. They tested populations of this insect
for differences in susceptibility to nematodes and found that there were
differences in susceptibility when tested with identical nematode species and
strains. N. Renn tested steinernematids against strains of adult houseflies
which had been selected for resistance to certain insecticides or unselected,
and found that these housefly strains varied in their susceptibility to the
nematodes. Finally, D. A. Bohan and J. Curran tested larvae from 10 strains
of the sheep blowfly for their susceptibility to steinernematids, and found
differences in susceptibility. While we must await publication of details, it is
clear that successful results using a known strain of nematode against a
particular pest in a particular locality need not guarantee success against the
same pest in another part of its range.
It is necessary to time nematode applications to coincide with or slightly
precede peak occurrence of the most susceptible stage of the insect’s life cycle.
This is especially critical where the life span or accessibility of the target
stage is short, e.g. root maggots. More than one application may be required
when insects feed on plants for longer than 2 months, e.g. root weevils and
416 1. POPIEL AND W. M. HOMlNlCK
3. Quality control
compared to other limiting factors. This brings us back full circle to the
problem of interpreting field trials. Until we know precisely which factors
limit efficacy, nematode quality will remain a matter of opinion, and trial
and error will remain the guiding principle in the use of nematodes as agents
of insect control.
I. Commercial development.
ambient temperature shelf life made on product labels until they are
substantiated with relevant data.
Because entomopathogenic nematodes are multicellular organisms, they
are exempt from government registration in most countries, and this adds to
their commercial attractiveness. However, naturally occurring organisms
cannot be patented and this leaves the production companies vulnerable to
competition. Alternatives to product patents are proprietary production
processes and formulations. Some companies have patented their produc-
tion process, but because infringement of process patents is difficult to
detect, others have chosen to keep theirs a trade secret. With the nematode
storage problem unsolved, the greatest potential for proprietary technology
lies in the development of novel formulations that increase shelf life.
Commercial producers of nematodes are currently introducing their
products to niche markets in which environmental conditions are conducive
to efficacy and in which there is little competition from conventional
pesticides. However, profitability from large-scale nematode production by
fermentation will depend on penetration of market segments in large-scale
agriculture. For this, progress is required in understanding and responding
to the factors which limit efficacy, mass production of the most efficacious
strains and improved storage methods.
ACKNOWLEDGEMENTS
Thanks are extended to Drs H. K. Kaya and J. Curran for their valuable
comments o n the draft of this review. Figure 1 was kindly provided by R.
Hurlbert, Washington State University, USA, Figures 2 a n d 5 by J. Curran,
CSIRO, Australia, a n d Figures 3 a n d 4 by BIOSYS Inc., USA.
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NEMATODES AS BIOLOGICAL CONTROL AGENTS 43 1