Echinococcus multilocularis infection: immunology and immunodiagnosis.

Echinococcus multiloculuris Infection: Immunology and Immunodiagnosis B. GOTTSTEIN

Institute of Parasitology, University of Zurich, CH-80.57 Zurich, Switzerland I. Introduction A. The parasite, its habitat and life cycle B. The prevalence, distribution and speciation of the parasite C. The disease: alveolar echinococcosis (AE) in humans 11. Immunology A. Definitive hosts B. Intermediate hosts 111. Immunodiagnosis A. Immunodiagnosis in definitive hosts B. Antibody detection in human AE C. Immune-complexed and circulating antigens in AE D. Cellular immune response in human AE IV. New developments A. Recombinant E. multilocularis antigens B. Diagnosis by the polymerase chain reaction C. Vaccination against infection with E. multilocularis Acknowledgements References

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I. INTRODUCTION A.

THE PARASITE, ITS HABITAT AND LIFE CYCLE

Echinococcus multilocularis Leuckart, 1863, was definitively established as an independent species by Vogel’s exact morphological and biological investigations, including the completion of the life cycle of the parasite in experimentally infected hosts (Vogel, H., 1957). The natural cycle typically involves foxes (Vulpes and Alopex) as definitive hosts. Other carnivores such as the domestic dog (Canis lupusf.familiaris) or the house cat (Felis silvestris f.familiaris) can occasionally be involved in the cycle as definitive hosts of E. multilocularis. ADVANCES IN PARASITOLOGY VOL. 31 ISBN 0-12-03I 7 3 I I ~

Copyrighr 0 1992 Acodemic Press Limirrd A / / rights of reproducrion in any/orm reserved

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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 components 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 membrane 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

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tion of a germinative membrane with formation of a central cavity and a peripheral laminated layer (Rausch, 1954; Sakamoto and Sugimura, 1970), followed later by endogenous and exogenous proliferation of metacestode tissue (Eckert et al., 1983). Production of protoscoleces may take place within 2-4 months, depending on intermediate host species or strains. However, protoscoleces that arise by a process of asexual budding (Smyth, 1964) are not always produced (Thompson and Lymbery, 1988). For completion of the life cycle, definitive hosts must ingest the mature infective metacestode containing protoscoleces. Digestion of the prey tissue is followed by liberation of protoscoleces with invaginated scoleces, although occasionally some are found to be already evaginated. Pepsin and bile salts stimulate a rapid evagination of the scolex, which is then able to attach firmly to the intestinal mucosa (Smyth, 1979). B.

3

THE PREVALENCE, DISTRIBUTION AND SPECIATION OF THE PARASITE

The natural life cycle of E. rnultilocularis is uniform, although the host assemblages universally differ in accordance with faunal changes southwards from the Arctic (Rausch, 1986). This fact is the principal determinant of the prevalence and distribution of the parasite. Attempts to delineate the approximate geographical range of E. rnultilocularis have been made by Rausch (1967, 1986), Lukashenko (1975) and more locally for Europe by Houin and Liance (1983), Frank (1987), Stossel (1989), Eckert (1990), Auer et al. (1990) and others, but exact distributional data are incomplete. Nevertheless, the geographical distribution of E. rnultilocularis seems to be uniquely restricted to the northern hemisphere. In Europe, the endemic area encompasses central and eastern France, Switzerland, Austria and Germany. Some delineated foci in these countries were previously believed to be in disjunction to each other or to other Eurasian or Asian areas with reported prevalences. Today we assume that prevalence is strongly dependent on many seasonal and biodynamic factors, resulting not only in areas with constant endemicity, but also in certain areas with fluctuating degrees of prevalence, making a gradual conjunction between individually known foci more likely. This assumption is confirmed by the occasional occurrence of adult stage E. rnultilocularis in foxes or other animals from areas such as Nordrhein-Westfalen, Niedersachsen, Hessen, Rheinland-Pfalz and Thiiringen (Frank, 1987; Worbes et al., 1989; Fesseler, 1990). The Asian areas with E. rnultilocularis prevalence include the whole zone of tundra from the White Sea eastwards to the Bering strait and covering much of the Soviet Union. The southern borders are documented by cases reported in the latitudinal zone starting from Turkey eastwards through Afghanistan, Iran, India, China and Mongolia to northern parts of Japan

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(Schantz, 1986; WHO, 1989). In North America, the cestode is present in subarctic regions of Alaska and Canada, including St Lawrence Island (Rausch and Schiller, 1954) and some other islands of North America (Rausch, 1986). The parasite has been discovered in Manitoba and North Dakota (Leiby and Olsen, 1964), and more recently in Illinois, Nebraska (Ballard and Vande Vusse, 1983), Alberta, Saskatchewan, Iowa, South Dakota, Montana, Wyoming, and even in South Carolina (Kazacos and Schantz, 1990), thus indicating an apparent expansion of the focus within the north-central United States. In addition to the classic natural life cycle encountered in the areas noted above (fox-rodent), E. multilocularis may also be maintained in cycles of rarer occurrence involving dog-vole, and to a lesser extent dog/cat-wild rodent interactions (Rausch, 1986; Deblock and Petavy, 1990). The latter interactions may be of relevance for human exposure, as they are no longer separated ecologically from the habitat of humans. In certain areas human exposure is often determined by occupational and avocational pursuits (Schantz, 1986), although there is little exact data. In Switzerland, the incidence of alveolar echinococcosis among persons working in rural areas was found to be four times higher than among urban employees (Gloor, 1988). Foresters, hunters and persons who work with fox fur or fox carcasses in endemic areas may be frequently exposed (Lambert, 1987). Dogs that become infected by capturing and eating infected voles also represent a source of human infection. When infected voles exist in villages as commensal rodents (e.g. in some Eskimo communities of the tundra zone in North America and Siberia) together with dense (sled) dog populations, then such areas may readily become a hyperendemic focus (Wilson and Rausch, 1980; Stehr-Green et al., 1988). An important feature of the biology of E. multilocularis within this complex of transmission cycles can be attributed to the evolutionary behaviour of the parasite itself E. multilocularis probably exists as a complex of intraspecific variants, which differ from one another in a variety of characteristics (reviewed by Thompson and Limbery, 1988). Taxonomically E. multilocularis has been described as three subspecies (Kumaratilake and Thompson, 1982): E. multilocularis multilocularis Vogel, 1957; E. multilocularis sibiricensis (Rausch and Schiller, 1954) and E. multilocularis kazakhensis Shul’ts, 1961, all three having been validated by Rausch (1967). Perhaps more reliable for modern categorization is the division of E. multilocularis into strains (Thompson and Lymbery, 1988): E. multilocularis multilocularis is referred to as the central European strain (Vogel, 1977) and E. multilocularis sibiricensis as the St Lawrence Island strain (Rausch and Bernstein, 1972). Only a few criteria have been used until now for characterizing E.


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multiloculuris in comparison with E. granulosus. Initially, extrinsic criteria such as geographical distribution, range of hosts, host specificity, reproduction biology and pathogenicity have been used to define the categories listed above (reviewed by Thompson and Lymbery, 1988). More recently, intrinsic criteria such as immunological (Gottstein et al., 1986a; Gottstein, 1991), biochemical (McManus and Smyth, 1978, 1979; Kumaratilake et al., 1979) and molecular biology (Rishi and McManus, 1987; Gottstein and Mowatt, 1991; Vogel er al., 1991) techniques have been applied to the characterization of E. multiloculuris isolates. The first immunological and molecular indications of the occurrence of intraspecific variations within E. mulriloculuris isolates were reported by Gottstein (1991) and Vogel et al. (1991), respectively. For the study by Vogel and co-workers, a 0.6 kb DNA fragment was isolated from a genomic library of E. multiloculuris and subsequently subcloned as recombinant plasmid pALl . This recombinant DNA probe hybridized to Southern blots of resolved EcoRI/Pstl digested genomic DNA from E. multiloculuris isolates originating from various geographical areas. The comparison of hybridization banding patterns showed clear differences between these isolates. The value and significance of molecular biological investigations and results needs to be evaluated by comparison of this data with the "classical" characteristics of these isolates, such as morphology and developmental and reproductive biology (and others) of the metacestodes. The practical significance of variation among E. multiloculuris isolates (or strains) for immunology of the host and for immunodiagnosis is discussed later. C.

THE DISEASE: ALVEOLAR ECHINOCOCCOSIS IN HUMANS

Alveolar echinococcosis (AE) of humans is biologically comparable to the disease in the natural intermediate hosts. The primary localization of E. multiloculuris metacestodes (larvae) in humans (as well as in natural intermediate hosts) is almost exclusively in the liver. Local extension of the lesion and metastatic lesion formation in the lungs, brain and other organs can follow (Schantz and Gottstein, 1986). Macroscopically, the hepatic lesion usually appears as a disseminated, firm to solid mass slightly elevated above the surrounding surface of liver tissue. After transection, the lesion area appears as a spongy, pale tissue consisting of scattered small cysts and vesicles. The diffuse borders are not well delineated from the adjacent liver tissue. In advanced chronic cases, a central necrotic cavity may be formed, containing a viscous yellowish to brown fluid, which occasionally may be bacteriologically superinfected. The lesion may contain focal zones of calcification. Microscopically, there is evidence of a vigorous proliferation of fibrous tissue peripherally and of regressive changes centrally, reflecting a

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strong host reaction for suppressing development of the metacestode (Schantz, 1982). The larval mass proliferates continuously by exogenous budding, progressively invading the surrounding tissue. Protrusions of the germinal layer of the metacestode grow within the host tissue, and thus may initiate parasite proliferation and metastasis formation (Eckert et al., 1983; Mehlhorn et al., 1983). In contrast to murine AE, lesions from infected humans rarely exhibit protoscoleces, brood capsules or calcareous corpuscles within vesicles and cysts. Symptoms at diagnosis of human AE are usually non-specific. Mild upper quadrant and epigastric pain with hepatomegaly can occur together with obstructive jaundice. Occasionally the initial manifestations are caused by metastases localized in the lungs or other organs (Ammann, 1983; Schantz and Gottstein, 1986; Schantz, 1986). The clinical signs in AE patients resemble those of hepatic carcinoma and cirrhosis. Non-invasive imaging techniques are usually applied simultaneously with immunodiagnostic procedures for diagnosis of hepatic AE. The AE lesions give rise to typical signs by ultrasound and computed tomography (CT). The CT image of the liver in AE shows indistinct solid masses, often with central necrotic colliquation and central or peripheral plaque-like calcifications (Otto et al., 1982). CT has also proved useful in evaluating the disease and its treatment by quantitative volumetric assessment of the lesion size (Schroder and Robotti, 1986); magnetic resonance (MR) imaging may become of interest in diagnosis and characterization of AE of extrahepatic extension (Mikhael et al., 1985; Claudon et al., 1990). As treatment of AE is beyond the scope of this introduction, immunological, immunopathological and serological aspects will be briefly considered later (see Sections III.B,C). At the global level, scant data exist on the overall prevalence of human AE. Cases have been diagnosed in the populations at risk in western Alaska, including St Lawrence Island, at an average annual rate of 28 per I00000 inhabitants (Wilson and Rausch, 1980). Switzerland reported an annual morbidity rate of 0.18 AE cases per 100000 inhabitants (Eckert and Ammann, 1990). Similar data were reported from France, Germany and Austria (WHO, 1988). In contrast to relatively stable annual morbidity rates in Europe and Alaska, Japan reported spreading of both parasite and disease in its northern areas: from 129 AE cases reported between 1937 and 1982 on Rebun Island, the disease was spread to Hokkaido Island with a total of 264 new AE cases registered up to 1988, and 60 new cases on Honshu Island (Kamiya, M., 1988; WHO, 1988). Insufficient data from many important geographical areas endemic for E. multilocularis prevents a reliable global estimate of the current annual morbidity rate. The importance of the disease, however, is not found in the number of reported cases, but rather in the severity of the disease itself, and in its mostly lethal


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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. A.

IMMUNOLOGY DEFINITIVE HOSTS

1. Intestinal immunity ( a ) Structural andfunctional components of the immune system peculiar to the gastrointestinal tract. The mucosa of the mammalian gastrointestinal tract constitutes a large surface area through which animals are exposed to the external environment. A complex system has evolved to protect animal hosts from invasion and/or damage by potentially pathogenic organisms. The system includes non-immune factors such as physical and physiological barriers formed by a continuous epithelium, a mucus layer and the indigenous flora, in addition to the mucosal immune system. Immune and nonimmune components are intricately intercalated as exemplified by the production of secretory components by epithelial cells (Brandtzaeg, 1985), and T lymphocyte-dependent goblet cell hyperplasia in intestinal helminth infections. The lymphoreticular components of the mature mammalian gastrointestinal tract consist of plasma cells, Peyer’s patches (PP), lamina propria of the villi, and intra-epithelial lymphocytes (IEL). Substantial evidence indicates that intestinal immune responses are initiated in PP (Heyworth, 1988). PP are circumscribed collections of lymphoid nodules in the small intestine. Each nodule consists of an intramucosal dome, covered by dome epithelium, and a submucosal follicle. The peripheral parts of these follicles contain densely packed lymphocytes expressing B and T cell markers. A large proportion of B cells are mimmunoglobulin (Ig) A positive, with variable numbers of membrane mIgM- and mIgG-positive cells (Spencer et al., 1986). mIgA-positive B cells apparently leave the PP and migrate via lymph and blood to the lamina propria and other mucosal localizations, where they mature into IgAproducing plasma cells. IgA is released into the interstitial fluid as a dimer, and the molecule is taken up by specific receptors expressed on the basolateral membrane of the intestinal epithelial cells (Husband, 1987). The antibody complex is then secreted into the mucosal coat overlaying the epithelium, where sIgA can bind to epitopes on pathogenic organisms or

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molecules. T cells in PP are predominantly LY4(CD4)+, endowed with the primary responsibility of stimulating the development of B cell antibody responses to antigens that enter PP from the intestinal lumen. Very few PP cells are LY2(CD8)+ (Ermak and Owen, 1986). IEL reside between the columnar epithelial cells of the villi (Lefranqois and Goodman, 1989) and are mostly LY2(CD8)+, but surprisingly only about half of them are Thyl' (Parrott et al., 1983). Most extra-intestinal peripheral T cells of mature mice express the ap heterodimer form of the T cell receptor (TCR) associated with the CD3 complex of proteins (Staerz et al., 1985). It has been shown that murine IEL are mostly @-bearing T cells (Bonneville et al., 1988), ap' and y6+ lymphocytes may be related to each other by a common precursor (Winoto and Baltimore, 1989). TCR y6 lymphocytes constitute a distinct T cell lineage with cytolytic activity and the capacity to produce lymphokines in mice (Matis et al., 1989) as well as in humans (Carding et al., 1990). y6 T cells may be of crucial importance in modulating the immunological defence against infectious organisms in the small intestine, as their development seems to be independent of the thymus. This latter observation relates not only to previous work with athymic animals and intestinal helminth infections, but also to the elusive physiological functions of these y6+ cells (Ferrick et al., 1989). Antigen-specific priming of PP and IEL lymphocytes has to occur in association with antigen-presenting cells (APC) such as macrophages and/or dendritic cells. Domes of PP contain mixed populations of cells, including lymphocytes, plasma cells, macrophages and dendritic cells (Mayrhofer et al., 1983; Wilders et al., 1983). The epithelium that covers the domes of PP differs from villous epithelium by the presence of absorptive enterocytes, few goblet cells and a high number of M cells, which are specialized epithelial cells that are involved in the preferential uptake of particulate or complexed antigens (Owen and Jones, 1974; Hogenesch and Felsburg, 1990). ( b ) The gastrointestinal tract as a site of immunological interaction with adult stage E. multilocularis. In general, enteric helminths cause internal stress which results in changes in the structure and function of local tissue; these changes result largely from host responses to the parasites (Castro, 1989). The small intestipe is capable of a primary immunological response as it has a rich vascular supply, and its mast cells and other cells of lymphoid origin can interact with the helminth infection. The cells may release substances associated with immediate allergic reactions (especially in the case of intestinal nematode infections) and contraction of smooth muscle. LY4(CD4)+ cell-dependent IgE production in association with mast cells may contribute to these mechanisms. All these mechanisms require a primary sensitization of the local immune system by the APC-B lymphocyte-LY4(CD4)+ (or other T lymphocytes with "helper" functions) path-


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way, and the elucidation of this pathway will help to explain the basic differences between a persistence of infection and the elimination or expulsion 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-dependent immune mechanisms (It0 and Smyth, 1987). H . nana infections in mice result in immunity against adult infection, but do not prevent egg development 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 (Thompson 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 (Thompson, 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

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premature interruption of infection at the end of prepatency (Thompson and Eckert, 1983). Repeated infections (which presumably occur in natural infections) would probably effect a different degree and pathway of immunological sensitization, and would give a different picture of immunological protection. Also, intestinal E. multiloculuris infection may implicate biochemical or physiological alterations of activities in the digestive brush border or of replicative crypt enzymes. Such mechanisms, even if nonspecific, may either favour the parasite by inhibiting the protective properties of host immune reactions, or they may favour the host by resulting in morphological damage and subsequent detachment, or in decrease of viability, productivity and persistence of the parasites. These selectively listed hypothetical modes of action may be dependent on the strains or isolate of parasite, number and time interval of infections, biological status of ingested protoscoleces and various host factors such as age, sex, nutritional and hormonal status and, not least, immunological polymorphism related to major histocompatability complex (MHC) genes. The question of whether resistance to infection and protectivity to reinfection in definitive hosts may occur naturally was approached from the viewpoints of epidemiology and ecology: recent Japanese studies on Hokkaido Island have demonstrated an age-dependent prevalence of intestinal adult stage E. multiloculuris infections in foxes; 63% of foxes younger than 1 year were found to be infected with E. multiloculuris. A gradual decrease in infection frequency was observed in foxes between I and 4 years old, and 5- to 7-year-old foxes were no longer infected. Interestingly, 1O h of foxes between 8 and 11 years old were reinfected with intestinal E. multilocuh i s (M. Takahashi, Sapporo, personal communication). One interpretation of these observations is that younger foxes may achieve a degree of protection to reinfection during repeated infections; this immunity may diminish in older animals in parallel with the ageing and exhaustion of the general immune status. Similar data were recorded in a Swiss area endemic for E. multiloculuris, where the infection intensity and extensity in foxes were both significantly correlated with the age of the animals (M. Siegenthaler, University of Neuchltel, personal communication). Acquired protective immunity to experimental E. grunulosus infections in dogs has been reported by Gemmell et al. (1986). To show this, 16 dogs were repeatedly infected with 87 500 E. grunulosus protoscoleces on eight or nine occasions, with each infection being investigated and terminated by arecoline purge between 5 and 12 weeks after infection. Observations on the worm numbers in the individual dogs suggested that rather than a continuous decline in susceptibility, each animal remained susceptible for a varying number of infections, after which they became less susceptible. Five of the 16 dogs did not show a reduction in infectivity over the length of the trial. However, the 11 (69%) other dogs developed significant protective immu-


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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/excretory 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 investigation 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. Nevertheless, 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-specifically 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). Irradiated 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 strategies 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 intestinal 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. multilocularis 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 treatment) of the animals amplified susceptibility, as shown by increased survival periods and worm numbers. Such work would be greatly advantageous for

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future research on the immunology of adult E. multilocularis infections due to the minimized potential risks for laboratory workers. 2.

Peripheral or systemic immune responses

Information concerning peripheral humoral and cellular immune responses in adult stage E. multilocularis infections of definitive hosts is sparse with regard to the former and practically absent in the case of the latter. Therefore a closer consideration of work done in the field of E. granulosus should give some indication of possible similarities with E. multilocularis. In dogs, adult stage E. granulosus infections stimulate the synthesis of various isotypes of antibodies against various kinds of parasite antigens. Williams and Perez-Esandi (1971) found that dogs infected with E. granulosus produce reaginic IgE specific to hydatid fluid antigen. Movsesijan and Mladenovic (1971) used the indirect immunofluorescent antibody test (IFAT) to demonstrate dog serum antibodies precipitating on the scolex and genital porus of adult E. granulosus worms. The occurrence of antibodies directed to surface structures of adult E. granulosus was also observed by Singh and Dhar (1988) using IFAT. Jenkins and Rickard (1985) and Gasser et al. ( 1989) used excretory/secretory (E/S) antigens collected during maintenance of evaginated scoleces and oncospheral antigens in vitro for the successful detection of dog immunoglobulins against both types of antigens. These experiments indicated that adult E. granulosus tapeworms elicit a marked humoral immune response detectable in the peripheral blood, antibodies being directed against antigens of all stages of the parasite from adults (surface and E/S products) to oncospheres and metacestode. Similar to E. granulosus, adult E. multilocularis is assumed to induce a humoral immune response in definitive hosts such as foxes. In contrast to E. granulosus, which enables the experimental infection of dogs for investigating the specificity of antibody-detection tests, no research in this direction has been undertaken with E. multilocularis, presumably due to the higher risks and health hazards encountered in such work. Antibody detection in naturally infected foxes requires a species-specific antigen, as the prevalence of numerous other cestode species in foxes is very high and, thus, is potentially the source of major cross-reactions. The so-called Em2 (E. multilocularis) antigen (see Section IILB), derived from the metacestode stage of E. multilocularis, has been developed initially for the species-specific immunodiagnosis of AE in humans (Gottstein et al., 1983). This antigen was subsequently used to detect antibodies in serum and body fluids of foxes originating from areas with documented prevalence of E. multilocularis with either presence or absence of intestinal stages of the parasite. Specificity investigations performed with dogs experimentally infected with various cestode species had previously shown that cross-reactions do not occur with


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any Taenia spp. infection. The diagnostic potential of antibody detection in foxes infected with E. multilocularis will be discussed later in this review. One interesting aspect, however, concerns the stage specificity of this Em2antigen. A monoclonal antibody specific to the Em2-antigen has been developed (Deplazes and Gottstein, 199I ) . Subsequent antigen analysis with this antibody showed that Em2 is neither synthesized in adult stages nor in hatched oncospheres, but rather, uniquely at the metacestode stage level. Post-oncospheral development requires a cultivation time in vitro of at least 12 days for inducing primary synthesis of Em2, which then will be potentially available in vivo throughout metacestode development for contacting the immune system. Em2 was localized by direct immunofluorescence and indirect sandwich enzyme-linked immunosorbent assay (ELISA) on the germinal layer, embedded as condensed complexes in the laminated layer as well as in E/S products from metacestode cell suspensions maintained in vitro, but was not found in or on the surface of protoscoleces nor as an E/S product of protoscoleces maintained in vitro. Consequently, the finding of anti-Em2 antibodies in foxes infected with adult stage E. multilocularis could not be directly related to the presence of intestinal parasites inducing an immune response, nor to oncospheral antigens being potentially presented after egg release or as a reaction to oncospheral egg breakdown products. Antibody responses to the metacestode stage-specific Em2-antigen, therefore, were explained by following hypothetical mechanisms. The repeated ingestion of large metacestode masses by foxes in areas with a high prevalence of E. multilocularis in rodent prey may be responsible, due to large quantities of Em2-antigens expressed in metacestode tissues (Gottstein, 1985), for a gastrointestinal antigen challenge resulting in seroconversion. Such mechanisms of peroral antigen challenges have been described for multiple antigen systems (Langevin-Perriat et al., 1988; Kay and Ferguson, 1989; Van der Heijden et al., 1989). However, daily oral administration of 20g of inactivated metacestode tissue for 14 days per fox resulted in no subsequent seroconversion to the Em2-antigen (Aubert and Gottstein, unpublished data). The more (or most) likely explanation, however, may be defined by post-oncospheral development within the definitive host. Previously oncospheres of E. multilocularis were not thought to hatch in the intestine of specific definitive hosts. However, Coman and Rickard (1975) found eggs of T . ovis and T. hvdarigena hatching and subsequently being activated in the small intestine of dogs following peroral ingestion. Similar mechanisms may occur in E. multilocularis infection. Oncospheres may penetrate the mucosa and then invade host tissue for an undefined but prolonged period, and thus be able to synthesize EmZantigen. Considerable support for this suggestion was described in a recent report of three dogs naturally infected with metacestodes of E. multilocularis in Switzerland (J. Eckert and Y. Stingelin, personal communication) and of two dogs in

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southern Germany (Barutzki et al., 1990), proving the capacity for complete metacestode development in definitive hosts. B.

1.

INTERMEDIATE HOSTS

Cellular and humoral immune responses in human A E

No or little information is available of the immune response against migrating and subsequently established oncospheres and their development to the larvae of E. multilocularis in humans. Therefore, only the metacestode at the site of larval proliferation will be considered. At the time of AE diagnosis an already fully developed and often rapidly proliferating metacestode has usually induced, reacted against and influenced an immune response of the host. The cellular and humoral immune response in humans, in contrast to experimentally infected animals, can vary enormously, evidenced by the different patterns of parasite antigens recognized in the course of the immune response (Furuya et al., 1989). These disparities are probably related to human genetic diversity, unlike the uniform genetic background of most experimental animals (Smyth and McManus, 1989). Many human AE patients respond with a marked synthesis of parasitespecific antibodies, including all isotypes of immunoglobulins (measurable at diagnosis); very few patients fail to demonstrate a humoral immune response (Gottstein et a[., 1984; Vuitton et al., 1984, 1988). Consequently, it has not been possible to discern the sequential induction of synthesis of the various antibody isotypes after infection (for more information on this subject see Section 1II.B). From a general point of view, specific antibody synthesis in AE may be associated with a characteristic hyperglobulinaemia, including other perturbations of serum proteins related to inflammatory reactions (Engler and Jayle, 1976; Miguet et al., 1976). Functionally, no evidence indicates that specific antibodies have a direct restricting role on the growth of the metacestode. Parasite-specific antibodies detect a large range of parasite antigens with respect to localization of determinants within the metacestode. Antibodies can be demonstrated by IFAT against surface structures and soluble molecules derived from protoscoleces, germinal layer, laminated layer and vesicular fluid. Detailed characterization of E. multilocularis antigens or their respective epitopes is currently limited exclusively to one antigenic protein called Em2 (Em2 will be discussed later with regard to its immunodiagnostic characteristics and immunochemical properties, see Section 1II.B) (Gottstein, 1985). The EmZantigen was characterized in our group by using an anti-Em2 monoclonal antibody (MAb GI l), which demonstrated its predominance in the laminated layer formed within the metacestode tissue,

ECHINOCOCCUS MULTILOCULARIS INFECTION

335

synthesis starting approximately at day 12 after oncospheral development (Deplazes and Gottstein, 1991). Protoscoleces and oncospheres of E. multilocularis can be lysed by antibody-mediated complement interaction. An association between reaginic IgE antibodies and infection with helminth parasites has long been recognized (Wakelin, 1984). Increased levels of total IgE and parasite-specific serum IgE have been shown in human AE (Ito et al., 1977; Gottstein et al., 1984), as has specific IgE bound to circulating basophils (shown by measuring specific degradation and histamine release in vitro; Vuitton et al., 1988). However, clinical manifestations related to immediate-type hypersensitivity have never been reported, not even during surgical manipulations of liver needlebiopsies (Miguet et al.. 1976). Rather than helping the host in controlling parasite proliferation, antibodies appear to be involved in immunopathological mechanisms responsible for the occasional chronic granulomatous course of the disease. Immune complex-associated membranous nephropathy was reported in human AE by Ozeretskovskaya et al. (1978). The authors found amyloid deposits in the spleen, liver and kidneys of patients with metastatic forms of AE. Similarly, Ali-Khan and Rausch (1987) described histopathological changes related to the incidence of amyloid and immune complex deposits in the liver of several Alaskan AE patients. Immunophysiopathological significance, however, must be attributed primarily to T lymphocyte interactions. A specific cellular immune response had already been shown by proliferation in vitro of peripheral blood mononuclear cells of AE patients (BressonHadni et al., 1989b). More direct information about the potential site of host-parasite reaction was obtained by Vuitton et al. (1989), who showed that the periparasitic granuloma, mainly composed of macrophages, myofibroblasts and T cells, contained a large number of CD4’ lymphocytes in patients with so-called “abortive” or “died-out’’ lesions (lesions are considered to be abortive when no viability can be shown after surgical resection of the parasite lesion and subsequent transplantation of the parasite to susceptible laboratory rodents), whereas in patients with active parasite tissue the number of CD8’ cells was increased. This observation may be related to mechanisms of resistance or susceptibility to E. multilocularis infections; however, the function and significance of these lymphocytes has yet to be assessed. 2. Cellular and humoral immune responses in murine A E

Immune responses should or must be considered in relation to different phases: reactions at the oncosphere penetration site followed by migration is often referred to as an “early phase”, while establishment of oncospheres

336

B. GOTTSTEIN

followed by maturation to metacestode is known as “late phase” immune response. Antibody-mediated, complement-dependent destruction of oncospheres in the gut or at the tissue sites of migration is thought to be the most effective mechanism of host defence. B lymphoblasts, eosinophils, neutrophils and macrophages migrate to and concentrate in the vicinity of the developing oncosphere. In the early stages at least, successful establishment of the parasite depends upon the outcome of a race between the development of the larva versus the establishment of a protective host immune response. In a primary infection, the parasite usually reaches the resistant and survival stage before host mechanisms can act successfully, although the speed of development of a host immune response may depend on different mouse strains, and thus may explain the susceptibility or resistance of various mouse strains discussed later in this review. A pre-existing larval infection can prevent or suppress the development of a secondary infection (Lloyd, 1981). Experimental peroral infections of Microtus arvalis with 10 000 E. multilocularis eggs, and subsequent sequential microscopic analysis of tissues from the small intestine and the liver of these rodents provided relevant insight in the biological and pathological events after infection (Bosch, 1982). Thus, the first oncospheres were detected 30 min after infection in the gut lumen and 8 hours after infection in the liver. Macroscopical lesions were visible 2 days after infection on the surface of the liver, and histological analysis showed solitary vesicles on day 5 after infection, followed later by multilocular proliferation of vesicles, including budding and formation of protrusions invading surrounding tissue. Once established in susceptible laboratory rodents, E. tnultilocularis metacestodes appear well protected from the host immune response; they grow progressively and metastasize despite a marked lymphoproliferative activity in the B and T cell areas of lymphoid tissues. In infected mice, serum antibody levels are related to the initial and resultant parasite biomass (AliKhan, 1974a,b). A similar correlation was shown for haematological parameters including anaemia, reticulocytosis, lymphocytopenia, neutrophilia, monocytosis and eosinopenia, i.e. all changes were directly proportional to the size of the parasite lesion. Specific and non-specific antibodies of various isotypes, as well as C3, have been detected on the surface of metacestode structures as early .as 4 weeks after intraperitoneal inoculation of E. multilocularis metacestode tissue (Ali-Khan and Siboo, I98 I ; Kroeze and Tanner, 1986; Alkarmi et al., 1988). The inability of parasite-specific antibodies alone to control parasite growth and host tissue infiltration may be due in part to “complement neutralizing” factors released by the metacestode causing complement depletion at the host-parasite interface (Hammerberg et al., 1977) or to the inactivation of C3 as it enters the metacestode tissue (Kassis and Tanner, 1976, 1977). Kamiya, H. et al.


337

(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 immunological 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 amyloidogenesis 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. 3. Susceptibility, resistance and immune evasion in murine A E Susceptibility and resistance to infection with E. multilocularis metacestodes may depend upon (genetically based) acquired immunological factors as well as mechanisms of innate resistance, including factors such as species or strain, sex, age and health status of the host and putative intraspecies variation of parasite isolates. Thus, cotton rats (Sigmodon hispidus) and jirds (Meriones unguiculatus) are generally more susceptible to infection by E. multilocularis than mice, but the outbred status and lack of immunological markers within these host species hinder any detailed studies of host-parasite interaction. Kamiya, H. (1972) gave the only report of age-dependent effects on resistance to E. multilocularis metacestode infection, and showed that highly susceptible AKR mice demonstrated decreased susceptibility between 29 and 83 days old (with a peak at age 48 days), with susceptibility returning

338

B. GOTTSTEIN

to normal after 83 days. The same author observed no evident sex difference in resistance or susceptibility of AKR mice. Yamashita et al. (1963) concluded from infection experiments that some mouse strains showed resistance 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 susceptibility 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 fragmentary 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 lymphocytes 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 thymusdependent T lymphocytes, have never been substantiated by the transfer of appropriate cell populations for sequential analysis of this primary phenomenon. 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

+


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 macrophage 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 cytokine 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 establishment of immune responses. 111. IMMUNODIAGNOSIS A.

IMMUNODIAGNOSIS IN DEFINITIVE HOSTS

The examination of definitive carnivore hosts for infection with intestinal stages of Echinococcus spp. classically includes either purging with arecoline hydrobromide followed by examination of purged faecal samples for tapeworms or parasitological examination of small intestines after necropsy. These techniques, besides being time-consuming, are accompanied by a potential infection risk for the investigators, and diagnostic sensitivity may be

340

B. GOTTSTEIN

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 considered 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 oncospheres, which might penetrate the intestinal wall. Serum antibodies to scolex E/S antigens were detected by ELISA 2-3 weeks after experimental infection 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 antibodies 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 oncospheres 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 Em2antigen indicated that its synthesis started at day 12 after oncospheral development after hatching. The Em2-antigen has been evaluated by Em2ELISA 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 intestinal or tissue-dwelling non-Echinococcus cestodes or nematode species. Inves-


34 1

tigations were performed with various fox populations originating from areas with documented prevalence of E. multilocularis as well as from areas presumed or proven to be free of E. multilocularis. Results clearly indicated that detection of anti-Em2 antibodies did not always reflect the presence of intestinal adult E. multilocularis worms (antibodies probably persist following loss of senescent worms), and the presence of intestinal adult E. multilocularis was not always reflected by the formation of anti-Em2 antibodies. The latter point could be explained by induction of anti-Em2 immunoglobulin synthesis during post-oncospheral development of E. multiloculuris, thus reflecting an infection of the definitive host with viable E. multiloculuris eggs, or an immune response to Em2-antigen ingested with metacestodes. The value of the test hinges upon the specificity of the antigen-antibody reaction which directly relates the presence of anti-Em2 antibodies to the presence of E. multiloculuris as a species. The Em2-ELISA test specificity is exemplified in Fig. 1. A close correlation has been reported between the parasitological and serological prevalence in given fox populations for these test features. In southern Germany, a high parasitological prevalence of 55% was reflected by a seroprevalence of 60% in 244 necropsied foxes; 139 foxes from Austria with a relatively low parasitological prevalence of 4% resulted in a seroprevalence of 12%, whereas all foxes without E. multilocularis infection from Norway were serologically negative in Em2-ELISA. Consequently this test permits (i) the reliable identification of fox populations with or without E. multiloculuris infection, (ii) the estimation of the prevalence of infection within the fox populations by extrapolation, and thus is of potential value in assessing sequentially the dynamics of the prevalence in areas under control campaigns. Two alternatives to coprology for direct diagnosis or serology for indirect diagnosis of intestinal cestodes in definitive hosts have been proposed, both consisting of the detection of parasites on a molecular level. The first has been defined as an antibody-sandwich-ELISA (Deplazes et ul., 1990). For this method, polyclonal antibodies were raised against E/S antigens derived from adult Taenia hydatigena maintained in vitro. After affinity purification these act both as “catching” antibodies and as conjugate after alkaline phosphatase labelling. The assay permitted the detection of copro-antigens from T. hydatigenu in dog faecal samples. It proved to be genus specific by the absence of cross-reactions related to infections with Echinococcus spp. or other cestodes or nematodes. The detection of T. hydutigena copro-antigens was possible in dogs during prepatency from day 18 after infection. Elimination of the cestodes in dogs by anthelmintic treatment resulted in seronegativity within 5 days following treatment. Consequently, such a test was suggested as a sensitive and practical tool for the diagnosis of intestinal adult Tuenia spp. infections, as a positive test result reflected prepatency and

342

B. GOTTSTEIN neg. control animals

E. multilocularis. mi. foxes

E. granulosus. mi. dogs

E. granulosus. e.i. dogs

1

Taenia hydaiigena. e.i. dogs

Mesocestoides corii, e.i. dogs Dipylidiurn caninurn. n.i. dogs Dirofilaria immitis. n.i. dogs

4 A N

-

405nm O

-

(

D

e

I

o

D

*

o

N

1

Ancylostoma caninurn. n.i. dogs Toxocara canis, e-i. dogs

SPF-dogs

I

o

FIG. I . Determination of anti-Em2 antibody concentration by enzyme-linked immunosorbent assay (ELISA) in serum from carnivores infected with various helminth species. The specific anti-E. multilocularis reaction is represented by foxes naturally infected (n.i.) with E. multilocularis, all resulting in a positive and higher ELISAvalue than the lower resolving limit, which is determined as the mean of 60 negative control animals + 4 S.D. Dogs naturally o r experimentally infected (e.i.) with various other helminth species show no cross-reactivity with the exception of one dog infected with E. granulosus. (After Gottstein et al., 1991a.)

patency including temporary periods of patency where eggs or proglottids were not excreted. Preliminary results (unpublished data) using a similar test adapted by our research group for Echinococcus spp. copro-antigen indicated that genus specificity could also be attained, thereby allowing discrimination between potentially cross-reacting Tueniu spp. One of the major advantages of copro-antigen detection, besides the ease of performance and sample preparation, is the stability of the immunogenic parasite components revealed in the faecal samples. The fact that the copro-antigens of T. hydutigenu remain stable in native faeces for at least 5 days at room temperature renders this assay both practical and feasible under field conditions. Such versatility permits large-scale epidemiological investigations simply by collecting faecal samples (which can be frozen to -80°C


343

before processing to kill cestode eggs), an invaluable method compared to classic parasitological examination after necropsy of animals. In addition, an immunological diagnostic method has been developed to identify the generic origin of taeniid eggs using monoclonal anti-oncosphere antibodies (Craig et al., 1986). However, this method requires the handling of viable eggs, including hatching procedures in vitro, and thus, due to the risks involved in these manipulations for the investigator, may not prove very suitable for parasites with a high degree of hazard and mortality such as E. multilocularis. Alternatively, infection by adult cestodes could theoretically also be demonstrated by the detection of parasite-specific DNA fragments originating either from parasite eggs or from cells of adult tapeworms. This approach is rapidly attracting attention, especially since the advent of highly sensitive techniques such as the polymerase chain reaction which allows the demonstration of single copy genes even from individual cells. Molecular biology techniques will be discussed later (see Section 1V.B). B.

ANTIBODY DETECTION IN HUMAN AE

Historical and more recent developments of immunodiagnosis of AE have been included comprehensively in review articles by Schantz and Kagan (l980), Rickard and Lightowlers (1986) and Schantz and Gottstein (1986) among others. 1.

Clinical immunodiagnosis

Alveolar echinococcosis is usually drawn to the attention of the clinician by a complex of non-specific liver manifestations often mimicking those of carcinoma and cirrhosis. Imaging techniques reveal hepatomegaly with indistinct solid tumours, occasionally associated with central necrotic areas and peripheral perinecrotic plaque-like calcifications. In clinically symptomatic cases, the delineation and extent of the lesion is mostly characteristic and obvious enough for considering E. multilocularis as the potential causative agent, therefore immunodiagnosis becomes a secondary diagnostic tool useful in confirming the nature of the aetiological agent. Unfortunately, the lesion in many individuals presenting with clinical symptoms is not radically resectable due to its extension into the liver and invasion of or metastasis formation in surrounding organs (Stehr-Green et al., 1988). In this respect, 63% of the first 33 patients with AE diagnosed in Alaska died as a result of the disease (Wilson and Rausch, 1980). In southern Germany, Schicker (1976) reported that 92% of AE patients without radical surgery and chemotherapy died within 10 years of primary diagnosis between 1960

344

B. GOTTSTEIN

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 strategies, 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’ immunoglobulins (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


345

called “antigen 5”, which was initially demonstrated by Capron et al. (1967) in immunoelectrophoresis with the respective precipitation of arc-5. Antibodies 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-reactions, 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. multilocularis 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

(Gottstein, 1983, 1985) the antigenic component of the Em2-antigen fraction was purified and characterized immunochemically (see Fig. 2). A major molecule of M , 54 000 and PI 4.8 was deemed responsible for the following immunodiagnostic characteristics. Diagnostic sensitivity was determined to be 94% when investigating sera from 78 patients with AE. The sera from patients with potentially cross-reacting nematode or trematode infections showed absolutely no reactivity to the Em2-antigen, resulting in a class specificity of 100%. Genus specificity was calculated to be 92% as 2 of 26 patients with cystic echinococcosis showed quantitatively minor crossreactions with the Em2-antigen (Gottstein er al., 1983). Additional immunological characteristics of the EmZantigen related to sero-epidemiological and post-treatment follow-up studies will be discussed in later sections.

A

B

C

M

B1

M

82

83

M

FIG. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the affinity-purified Echinococcus rnultilocularis antigen Em2. Silverstained SDS-PAGE patterns (A, B, C, M) from (A) E. rnultilocularis metacestode crude pratein extract, (B) affinity-purified Em2-antigen and (C) Em2-antigen purified by immunosorption. Lanes M show molecular weight standards. Lane B1, the fluorographic pattern of '4[C]labelled, affinity purified Em2-antigen in vitro; lanes B2 and B3, as BI but after irnmunoprecipitation with human anti-E. rnultilocularis serum (B2) and negative control human anti-E. granufosus serum (B3). Arrows indicate migration site and relative molecular mass of the Em2-antigen. (After Gottstein, 1985.)


347

Attempts to serologically differentiate both forms of echinococcosis were undertaken by Knobloch et al. (1984) using crude antigens derived from E. granulosus versus crude antigens from E. multilocularis, the resulting discrimination rate being 96%. However, such crude antigens demonstrated marked cross-reactivities with serum antibodies from patients with various other helminth infections. Such a test would not be very suitable for routine serodiagnosis as it requires a prediagnosis of echinococcosis. Auer et al. (1 988a) isolated E/S antigens produced by E. multilocularis protoscoleces maintained serum-free in vitro. Immunodiagnostic properties of the antigens in question were as follows: ELISA combined with the indirect haemagglutination assay (IHA) (both test systems with E. multilocularis E/S-antigens) enabled serodiagnosis of 89% of cases with cystic echinococcosis or AE (Auer et al., 1988b). For discriminating cystic echinococcosis from AE the same authors used a Western-blot test for defining a presumably speciesspecific antibody reactivity to a E. multilocularis polypeptide of M , 62 000 (Auer et al., 1988a). Unfortunately, the diagnostic sensitivity of this 62 kDa band has not yet been described (Auer et al., 1988a), thus limiting its application in practical diagnosis. Western-blotting was also used by Furuya et al. (1989) to analyse banding patterns of serum antibodies from Japanese AE patients. The authors reported specific humoral immune responses to E. multilocularis polypeptides of M , 55 000 and 66 000 with diagnostic sensitivity of 86% and a specificity of 88% (1 of 8 patients with cystic echinococcosis reacted positively with both bands in question). More recently, highly specific E. multilocularis antigens have been synthesized by using recombinant DNA technology (see section 1V.A). As well as separating parasite antigens for epitope specificity and sensitivity, several approaches have been undertaken to individually analyse antibody classes for parasite antigens. The IgE humoral immune response has attracted particular attention due to its well-known relevance to the closely related cystic echinococcosis (Dessaint et al., 1975; reviewed also by Schantz and Gottstein, 1986). Ito et al. (1977) demonstrated parasite-specific IgE using the radioallergosorbent test (RAST) in 30 sera of 34 patients (88%) with AE. Vuitton et al. (1988) used similar techniques to demonstrate parasite-specific serum IgE in 9 out of 18 patients, with a significant correlation observed in 1 1 out of the 18 patients with elevated total serum IgE ( > 150 KIU/l). Similar diagnostic findings were reported by Gottstein er al. (1984): 1 1 out of 16 patients with AE tested positive for parasite-specific IgE with ELISA. One conclusion from these studies is that the diagnostic sensitivity of specific serum IgE detection is relatively low (61% and 69%, respectively in the two studies above), and apparently does not confirm previous preliminary results by Loscher (1983). In general, specificity analyses of IgE detection have never been extensively performed and reported.

348

B. GOTTSTEIN

Presumably, however, problems similar to those found with E. granulosus infection (Afferni et al., 1984) have been observed. Further comparative analysis of specific serum IgE versus basophil-bound IgE (assessed by histamine release and degranulation tests in vitro) demonstrated frequent positivity for the cell assay, despite the absence of clinical symptoms of immediate-type hypersensitivity in these patients (Vuitton et al., 1988). 2. Sero-epidemiology Early serological diagnosis and subsequent treatment of humans with (asymptomatic) AE may reduce mortality of the disease (Kasai et al., 1980). A consequence of this knowledge was the offer of serological screenings to populations and communities in endemic areas such as Alaska (Wilson and Rausch, 1980; Schantz et al., 1983) and Japan (Sato et al., 1983). Initially the use of E. granulosus antigens permitted positive identification in a proportion of diagnosed cases. More recent findings, however, suggested significant improvement not only of diagnostic sensitivity, but also of specificity by the application of crude and subsequently purified E. multilocularis antigens. A first direct comparison between homologous purified E. multilocularis EmZantigen (Gottstein, 1985) and E. granulosus hydatid fluid antigen (using an ELISA technique adequate for both antigens) was performed by Gottstein et al. (1987). The study showed that E. granulosus antigen exhibited false-positive reactions (= no AE detectable) in 0.31 % of healthy blood donors tested, whereas the corresponding rate was 0.03% for the Em2-antigen. Similar findings were reported by Lanier et al. (1987) from Alaskan studies. Sera from 21 patients with histologically confirmed AE were all (100%) positive by Em2-ELISA, whereas 18 (86%) were positive by (E. granulosus antigen) IHA and only 5 (24%) precipitated arc-5 in a doublediffusion test. The same study also confirmed specificity findings as Em2ELISA showed no cross-reactions with sera from patients with non-echinococcal parasitic infections, while 3 1% were positive by IHA. In general, the epidemiological situation of low prevalences in AE requires a serological test system with not only high diagnostic sensitivity but also very high species specificity, as positive and negative predictive values have to result in justifiable clinical investigation of seropositive individuals. Falsepositive reactions lead to unnecessary psychological impairment and stress of the individuals in addition to the expense of unnecessary imaging and other clinical investigations. Results of the sero-epidemiological study in Switzerland mentioned earlier (selected results are briefly summarized in Table 1) resulted in the detection of two asymptomatic, but clinically confirmed, cases of AE by Em2-ELISA (Gottstein et al., 1987). Alaskan studies have shown that Em2-ELISA detected not only asymptomatic AE


349

TABLE1 Sero-epidemiological prevalence of serum antibodies to species-spec@ Em2-antigen assessed by ELISA in Swiss adult blood donors, and the resulting clinical findings in seropositive personsa Blood donors Percentage

Serological results (anti-Em2-IgG detection)

17 160 4

99.97 0.02

Negative Positive

2

0.01

Positive

No.

Total 17166

Clinical findingsb No investigation performed No liver lesion detected by US/CT Liver AE confirmed clinically

100.00

After Gottstein el a/. (1987). US, Ultrasound examination; CT, computer-assisted tomography examination; AE, alveolar echinococcosis. a

cases not discovered by other serological techniques (Gottstein et al., 1985), but also cases in which the metacestode lesion was very small and died out at an apparently early stage of infection (Rausch et al., 1987). These abortive lesions were assessed through immunohistochemical tests (Condon et al., 1988) and by inoculation of parasite material isolated from hepatic lesions into susceptible laboratory rodents. The spontaneous death of E. rnultilocularis metacestodes, which is known to be immunologically mediated in laboratory mouse strains with high resistance against this parasite, had been postulated for humans for many years, but had never been demonstrated. Such spontaneous rejection of the infection would have valuable consequences for future research in the immunology of E. multilocularis infection, as well as having important clinical relevance for the appropriate treatment of the respective patients. During the last decade, following those surveys listed above, multiple sero-surveys (using various kinds of homologous or heterologous Echinococcus antigens) were initiated and carried out in different endemic areas (Zeyhle and Frank, 1982; Jacquier et al., 1986; Miihling, 1986; Nakao et al., 1986; Zhang, 1987; Auer et al., 1988c; Bresson-Hadni et al., 1990a). Many of these studies addressed specificity problems due to the use of crude antigens. Two studies deserve further consideration by virtue of their attempt to solve these problems. Auer et al. (1988b) were able to blanket major non-specific or cross-reactive antigen components by generating E. multilocularis E/S antigens produced in vitro. Subsequent resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and immunoblot analysis revealed the presence of a polypeptide with an M , of 62000 that showed species specificity for E. multilocularis

350

B. GOTTSTEIN

infection of humans. Similar techniques were applied by Furuya et al. (1989) to determine the presence of polypeptides with M , of 55 000 and 66 000 with species-specific properties using Western blotting. Such assays may prove useful in confirming the specificity of antibody reactions after primary screening with relatively non-specific crude antigens in sero-surveys. Initial studies using Western blotting for sero-epidemiology were reported by Furuya et al. (1990), and showed its suitability in confirming clinical cases of AE in human patients. According to World Health Organization (WHO) recommendations a strategy is sorely needed to render different sero-surveys comparable (WHO, 1988). Comparability was deemed achievable by referring data from individual validated test procedures to data obtained by a standardized reference test. Standardization of the Em2-ELISA was suggested (WHO, 1988) and comparatively included in sero-epidemiological studies. Three reference standard sera with different defined anti-E. multilocularis antibody concentrations have been created and are presently available upon request at the WHO Collaborating Center for Parasitic Zoonoses (Institute of Parasitology, University of Zurich). 3. Post-treatmept control Experience with E. multilocularis post-surgical cases without chemotherapy is limited, as most cases are currently treated with a combination of surgery and chemotherapy whenever possible. Nevertheless, only complete surgical removal of the entire parasite lesion offers a prospect for curative treatment. Radical resectability is presently estimated to vary between 26% and 58% for central Europe, the USA and Japan, and is heavily dependent upon the efficacy of screening programmes for early diagnosis. The “radicality” of the surgical resection is very difficult to estimate macroscopically by surgeons, as microlesions and root-like parasite protrusions (Eckert et al., 1983; Mehlhorn et al., 1983) and cell complexes may remain unseen in apparently healthy tissue of liver or other organs. Some resection patients will develop recurrences subsequent to surgery, justifying the consequent chemotherapy of all patients with AE until proof of cure. Serological tests have been used, among others, for post-operative follow-up studies. Generally, a decrease of parasite-specific serum antibody concentration can be observed after surgery. Schantz et al. (1983) revealed that recurrence of disease following surgery was consistently associated with persistence or increase in antibody concentrations using IHA and E. granulosus crude antigens. Using more purified preparations such as Em2-antigen, a preliminary study performed on three patients with AE indicated that anti-Em2 antibody concentration declined dramatically within months after successful radical operation


35 1

(Lanier et al., 1987). A subsequent larger study confirmed these findings (Gottstein et al., 1989). Of nine patients with successful radical surgical resection (no recurrence observed for the following decade(s)), six converted to negative within 1 year and the remaining three patients within 4 years after surgery. On the other hand, six of seven patients who showed recurrences in an average of 6 years after surgery, despite assumed complete surgical resection, were positive by Em2-ELISA at the time of recurrence. Such significant data were not obtained when employing crude E. multilocularis or E. granulosus antigens. Consequently the anti-Em2 antibody concentration reliably reflected complete or incomplete surgical resection, depending upon the viability of the resected metacestode tissue (Rausch et al., 1987). More recent investigations with a new recombinant E. multilocularis antigen (II/3-lO-antigen; Miiller et al., 1989a) have indicated that the presence of anti-II/3-10 antibodies may uniquely reflect the presence of viable metacestodes; thus, this test may provide additional information of clinical relevance (unpublished data). An exceptional immunological situation is encountered when serologically monitoring AE patients receiving orthotopic liver transplantation (Gillet et al., 1988; Miguet and BressonHadni, 1989). Abundant blood transfusions coupled to immunosuppressive therapy usually resulted in an immediate conversion to negative of Echinococcus serology. Recurrence rates were high in cases with remaining residual foci of extrahepatic parasite tissue, due to immunosuppression and interruption of chemotherapy with antiparasitic benzimidazoles (Bresson-Hadni et al., 1990b). Such recurrences were also accompanied by reappearance of anti-Echinococcus serum antibodies. Post-chemotherapy monitoring by classic serological methods (with crude E. multilocularis or E. granulosus antigens) has been difficult to evaluate (Schantz et al., 1983; Knobloch et al., 1985; Gottstein et al., 1986b; Rausch et al., 1986; Lanier et al., 1987; Auer et al., 1988b; Ammann et al., 1988). Generally a tendency toward decrease in specific antibody concentrations was observed in chemotherapeutically treated patients with partially resected lesions. In contrast, specific antibody concentrations in serum of patients with non-resectable lesions and/or palliative surgery remained elevated or fluctuating. To date, the need of clinicians for a clearly predictive interpretation of serology of individual patients with regard to recurrence has not been granted by classic serology. There are some indications that speciation of parasite-specific immunoglobulin classes may reflect more reliably the assessment of drug efficacy, and thus may better correlate with clinical findings than results of classic serological tests. Gottstein et al. (1985) measured parasite-specific immunoglobulin class reactivity to echinococcal antigens by ELISA in sera of 16 patients with AE treated with mebendazole (Table 2). A restricted decrease

352

B. GOTTSTEIN

of specific IgG antibody concentration was observed in most cases with favourable clinical courses, whereas specific IgA and IgE disappeared within 2 years in the same patients after initiation of chemotherapy. In contrast, patients with progressive AE under chemotherapy showed reversed tendencies with significantly increasing specific IgG, IgA and IgE antibody concentration during the same period. Vuitton et al. (1984) reported transient changes in parasite-specific serum IgA and IgM antibodies in patients with AE following chemotherapy with flubendazole. The same authors suggested that specifically basophil-bound IgE could also reflect positive or negative response to therapy (Vuitton et al., 1988). C.

IMMUNE-COMPLEXED AND CIRCULATING ANTIGENS IN AE

In cystic echinococcosis (caused by the metacestode of E. grandoms) there is evidence that glomerular deposituf-parasite antigen may be associated with membramms-qhrq%athy (Ibarrola et al., 1981). The possibility of an association of antigens released by the E. multilocularis metacestode with glomerulonephritis and other pathological conditions must be considered. Immune-complexed antigen and amyloid deposits were then reported by AliKhan and Rausch ( I 987) in liver and kidney samples from Alaskan patients with AE. The determination of circulating parasite antigens with their potential for immune-complex formation in the serum of patients might be useful for monitoring the disease, as it may reflect the viability and biological activity of parasites in the host more reliably than antibody titres (Eckert and Gottstein, 1983). For E. granulosus, circulating specific immune complexes (Craig and Nelson, 1984) and circulating soluble antigens (Craig and Nelson, 1984; Gottstein, 1984) have been diagnostically detected in 33-85% of sera from patients with cystic echinococcosis. Surprisingly, the diagnostic potential of circulating E. multiiocuiaris antigens has been neglected. Leikina et ai. (1982) reported that ruptured lesions (AE) with central necrotic areas resulted in the release (“leaking”) of parasite antigens that could be subsequently demonstrated in the serum by double gel diffusion. It can be assumed that the use of highly sensitive techniques such as sandwich-ELISA should enable the detection of antigenis molecules released by active and proliferating metacestode tissue. Such a hypothetical antigen detection may be useful for monitoring therapy as a substantial decrease in circulating antigen, reflecting active metabolism of the parasite, is to be expected, whereas antigens released upon degradation of parasite tissue (damaged by drugs) is anticipated to increase in concentration. Such hypotheses should be investigated in the future by identification and characterization of the relevant E. multilocularis antigens with the subsequent elaboration of diagnostic tests.

353


TABLE 2 Parasite-specijic immunoglobulin' isotypes in patients with echinococcosis before and after 2 years of continuous chemotherapy with mebendazole.b Testprocedure was ELISA using Echinococcus granulosus hydatidjluid antigen' Group/ patient No.

Mebendazole concentration' (Pmolll)

IgG IgE IgA IgM -Parasite

rb rib r rr

E. multilocularis, partial removal 63 18 1 0.25 2 0.35 10 14 3 0.90 33 3

0 0 4

E. multilocularis, radically operated upon 53 5 3 4 0.20 50 8 6 5 0.11 34 10 0 6 0.12 70 35 15 7 0.59 82 70 13 8 0.89

r rr

0 15 0 0 0 0

E. multilocularis, bile drainage surgery 16 0.28 57 40 18

0

E. granulosus, inoperable 17 0.26 18 0.31 19' 0.10'

0 48 0 37 0' 7'

a

localization

0 0 0 0 0 0

Liver Liver Liver

0 8 0 0 0 30 0 0 0 0 0 0 0 30 0 0 0 28 18 18

E. multilocularis, not operated upon 9 0.17 23 10 5 0 8 10 0.49 78 73 17 12 31 11 0.45 100 63 5 0 84 0.10' 70'54' 20' 0' 34' 12' 13 0.59 56 45 4 0 22 14' 0.13' 52'83' 0' 38' 18' 15' 0.07' 65'91' 0' 22' 13'

91 40 6 92 42 81 64'67' 15'

0 0 0

r rr

15

0 0 0 0 0

0 54 0 26 0 0 22 10 0 14' 12' 0' 14 0 0 28' 0' 0' 22' 0' 0' 4

0 0

0 0 0 0 18 0 7' 0' 0'

Liver Liver/rpd Liver Liver Liver Liver Liver Liver Liver/kidney Liver/rp Liver Liver Liver Liver/pancreas Liver/lungs Liverlkidney Intraperitoneal

Immunoglobulin (Ig) concentration in antibody units (AU) referring to a standard of

100 AU, negative is 0 AU.

I, Initial examination; 11, examination 2 years after initiation of chemotherapy.

'Mebendazole; determination of plasma level 4 h after morning dose.

rp, Retroperitoneal tissue. Failure in chemotherapy, i.e. progression of parasite lesion. Unlettered numbers represent patients with successful chemotherapy, i.e. constancy or regression of parasite lesion. After Gottstein er al. (1984).

354

B. GOTTSTEIN D.

1.

CELLULAR IMMUNE RESPONSE IN HUMAN AE

Clinical diagnosis

The induction (and importance) of cellular immune responses and reactions are suggested by the granulomatous infiltration surrounding E. multilocularis lesions in infected human livers (Vuitton et al., 1985). Thus, the determination of parasite-specific lymphocyte reactivity has been proposed as a diagnostic alternative to antibody-detection in human AE. In a French study (Bresson-Hadni et al., 1989a), a lymphocyte proliferation assay in vitro with crude E. multilocularis antigen stimulation was used to study 48 AE patients. A significantly positive stimulation index was found in 47 (98%) of the patients. This high diagnostic sensitivity was restricted to some extent in its value by the observation that 5 of 35 (14%) healthy control subjects included in the study also reacted “positive” in the same range of stimulation indices. The authors explained these reactions by prior contact of the persons with E. multilocularis, followed by an unapparent spontaneous elimination of the parasite. From a statistical and epidemiological point of view, this explanation appears rather unlikely, assuming that the healthy control subjects had been randomly and casually selected. Given that T cell epitopes are generally well conserved, a more probable explanation would be cross-reacting epitopes of other infecting organisms. In a Swiss study (Gottstein, 1990), where diagnostic sensitivity similar to that described above was reported, the investigation of 10 healthy control subjects resulted in the identification of two persons with non-specifically positive stimulation indices. Interestingly, both persons were laboratory employees who handled dogs infected with different adult Taenia spp. (unpublished data), thus indicating a possible cross-reaction due to non-symptomatic infection with Tuenia eggs. With respect to these findings, the aspect of cross-reactive lymphocyte stimulation should be investigated, as none of the present studies tested patients with cystic echinococcosis (E. granulosus), Tuenia solium cysticercosis or taeniasis for cross-reactive lymphoproliferative responses to E. multilocularis antigens. Another aspect of interest not yet studied is the analysis of specific E. multilocularis protein antigens with T cell epitopes for identification of their individual immunodiagnostic characteristics. Such questions can be approached by employing purified E. multilocularis polypeptides such as the Em2-antigen (Vuitton et al., 1990) for cell stimulation in vitro, or by analysing the antigen profile of E. multilocularis with T cell blotting as described for Giardia lumblia (Gottstein et ul., 1990b). An additional main subject to be investigated concerns the determination of parasite-reactive T lymphocyte subsets and their pattern of released lymphokines, both points being expected to provide insight into immunological mechanisms controlling or failing to control progression of disease.


355

2. Post-treatment control

Concerning chemotherapy of human AE, Vuitton et al. (1984) assessed parameters of non-specific cellular immunity in 12 patients with hepatic AE during a follow-up period of 2 years with and without flubendazole treatment. A significant decrease of total numbers of peripheral B lymphocytes and total circulating lymphocytes was observed, linked to an impairment of the functional activity of T cells assayed by a leucocyte migration test with purified protein derivative (PPD) and Candidin antigen stimulation. The nature of these impairments was not elucidated and was suggested to be related to flubendazole as causative substance. In a subsequent study (Bresson-Hadni et al., 1989a), 20 patients with AE were investigated for their E. multilocularis-specific cellular immune response during a 24-year period of mebendazole treatment. A progressive decrease of the specific lymphocyte proliferation stimulation index was observed in most of the patients, 15 of 20 patients being “negative” after 4 years. The long-term persistence of specific lymphocyte reactivity was also emphasized by the results obtained from patients who underwent a radical surgical resection of the parasite lesion (no recurrence for the following 5 years). In such patients a significant reduction of stimulation indices was demonstrated 4 years after surgery; reactivity, however, still persisted at the end of the study. However, an increase of the stimulation index was usually shown to be associated with a progression of the liver lesion (Bresson-Hadni et al., 1989a). Assessment of the viability of the parasite at diagnosis or following treatment by cellular immunological parameters was attempted by Gottstein et al. (1991). The study showed that the in vitro lymphoproliferative response to E. multilocularis antigen stimulation was very high in cured patients who had radical surgery or patients with “died-out’’ lesions. Lymphoproliferation was significantly lower in patients with still active parasite lesions following partial surgical or no resection, indicating potentially immunosuppressing activity by the active metacestode. IV. NEWDEVELOPMENTS A.

RECOMBINANT E. MULTILOCULARIS ANTIGENS

It is now evident that techniques in molecular biology have great pc en .~ ial as tools for synthesizing defined protein antigens. The difficulty in obtaining sufficient amounts of parasite antigens by classic immunochemical methods can be circumvented by cloning and expressing parasite genes in suitable vectors. The synthesis of recombinant parasite antigens generally begins with the construction of a complementary deoxyribonucleic acid (cDNA)

356

B. GOTTSTEIN

expression library using messenger ribonucleic acid (mRNA) obtained from the appropriate parasite stages. Such libraries are subsequently screened with immune sera (affinity-purified), polyclonal or monoclonal antibodies to identify bacterial clones producing recombinant antigens bearing the relevant B cell epitopes. Similar screening can be performed today with T cell lines or clones for identification of the respective T cell epitopes. The most interesting recent developments concern the various gene expression systems for the efficient synthesis of the antigen in an appropriate biological form. This strategy requires a suitably constructed vector into which the cDNA fragment can be inserted. For cloning experiments, a wide range of vectors is available from simple plasmid cloning vectors to mammalian expression systems. Helminth antigens are generally complex protein molecules; thus, the expression of certain genes in bacteria may exhibit distinct limitations for biochemistry (e.g. glycosylation), stability, yield or applicability. Hence the selection of suitable and optimal bacterial or other expression systems presently remains the major point to be investigated. The first published E. multilocularis cDNA library was constructed by Vogel et al. (1988) using the Escherichia coli expression vector hgt 1 1. As the study was aimed at the generation of E. multilocularis species-specific recombinant clones, the library was screened with serum from patients with AE for the selection of clones synthesizing E. multilocularis recombinant antigens. Species specificity was controlled by the exclusion of clones reacting with serum antibodies from patients with cystic echinococcosis. The authors were able to identify 11 antibody-binding clones, and one of these clones (clone II/3) demonstrated optimal immunodiagnostic characteristics assayed by Western-blotting. The resulting recombinant antigen (antigen II/3) was able to bind antibodies from 40 out of 41 patients infected with E. multilocularis (a diagnostic sensitivity corresponding to 98%). The overall specificity was 96%, with minor cross-reactions occurring with serum antibodies from patients with cystic echinococcosis (3 1 patients tested, 1 positive) and with Taenia solium neurocysticercosis (15 patients tested, 2 positives). No cross-reactions occurred for nematode and trematode infections. Unfortunately, poor bacterial expresion and the lack of an appropriate purification protocol excluded the application of antigen II/3 in routine diagnosis. This problem was solved in a subsequent study by using two different molecular biologic methods leading to increased bacterial production with subsequent purification of the antigen in question (Miiller er al., 1989a). In a preliminary study the initial 1.0 kilobase (kb) cDNA sequence encoding for the antigen II/3 was shortened by sonication to a 0.6kb fragment, which provided a much higher expression level after recloning into hgtl 1. In a second step, the shortened fragment was sub-cloned into the plasmid vector pAR3038, resulting in a further increase in antigen synthesis; pAR3038 was chosen on the basis of its expression mode. In this vector the

357


shortened recombinant antigen II/3- 10 was synthesized as a polypeptide fused to a short (1 1 amino acid) N-terminal peptide of phage T7 origin. This short phage peptide was shown to be immunologically irrelevant, in contrast to bacterial proteins such as P-galactosidase, to which recombinant proteins are mainly fused when employing E. coli expression systems. An efficient biochemical purification by a two-step ion-exchange chromatography was essential to provide a final product (see Fig. 3) for direct application in immunodiagnostic test systems such as ELISA. A preliminary large-scale evaluation of the recombinant antigen 11/3-10 with ELISA (see Table 3) gave satisfactory results\as operating characteristics were similar to the previously discussed Em2-antigen. Another E. multilocularis cDNA library was subsequently constructed and published by Hemmings and McManus (1 989) resulting in the identification of two bacterial clones with immunodiagnostic potential. M r i

2

3

1

2

3

1

2

3

926645-

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 DEAESephacel 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

One major problem encountered when recombinant parasite antigens are produced in E. coli is the extensive purification step needed in order to avoid immunological cross-reactions of the antigen preparations with anti-E. coli antibodies that occur frequently at high concentrations in human sera (Stahel et a/., 1984). TABLE 3 Determination of diagnostic sensitivity and specificity regarding the Echinococcus multilocularis recombinant antigen 11/3-10 versus the E. multilocularis afinitypurified antigen Em2 by ELISA" Antigen 11/3-10 Origin of patients Diagnostic sensitivityb Switzerland Alaska France

Total

Number Positives of patients (%)

Antigen Em2

Negatives

Positives

(%)

(%)

Negatives

f %)

67

10 11

60 (90) 10 (100) 9 (82)

7 0 2

62 8

(93) (80) 11 (100)

5 2 0

79

79 (90)

9

81

7

Specificityb Infecting parasites E. granulosus T. solium Trematodes Nematodes

108 15 26 71

1 2 0 0

107 13 26 71

Total

220

3

217 (99)

(99) (87) (100) (100)

(92)

5 0 0 0

103 15 26 71

(95) (100) (100) (100)

5

215 (98)

After Muller el al. (1989). Sera were from human patients with alveolar echinococcosis for sensitivity testing and with cystic echinococcosis, T. solium neurocysticercosis or infection with various other nematode or trematode species for specificity testing. a

In expression systems such as those listed above, the recombinant protein accumulates within the bacterial cell, either as a soluble protein or in the form of insoluble precipitates. Purification of recombinant E. multilocularis antigens was greatly simplified by their excretion into the periplasmic space, from which they could be extracted without simultaneously solubilizing the large major bacterial proteins (Muller et al., 1989b). This was achieved by employing the plasmid pVB2 (Scholle et ul., 1987) which carries a part of the E. coli mgl operon. pVB2 contains the mgl promoter region and the first gene of the operon, mglB, which encodes the periplasmic galactose-binding protein (GBP). The mglB gene contained a single EcoRI restriction site close to its 3'-terminus allowing the direct in-frame insertion of E. multilocularis cDNA fragments from recombinant hgtl 1 phages. Recombinant GBP was


359

synthesized in high yield and was quantitatively exported into the periplasmic 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 advantages 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 hybridomas secreted parasite antigens as demonstrated by indirect immunofluorescence analysis. The hybrid E. multilocularis antigens were investigated by ELISA for the potential immunodiagnostic value. Results indicated a genusspecific 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. B.

DIAGNOSIS BY THE POLYMERASE CHAIN REACTION

Rapid developments in molecular biology have resulted in a variety of

360

B. GOTTSTEIN

technical innovations with potential applications in diagnostic investigations. Thus, the use of specific parasite DNA probes for identification and demonstration of the respective disease by hybridization to DNA from diagnostic samples has rapidly become commonplace in many laboratories. Apart from the restricted availability of specific probes, one major problem may be the limited sensitivity of the hybridization and labelling techniques used. This lack of sensitivity can usually be attributed to the low level of target sequences in diagnostic samples, to the poor quality of this DNA, or to the very small amounts of DNA obtained (de Bruijn, 1988). These limitations can now be essentially eliminated by an extraordinary new tool: the polymerase chain reaction (PCR). PCR was originally developed by Saiki et al. (1985) at Cetus Corporation, providing a method for rapid amplification of specific DNA target fragments in vitro. PCR depends upon the availability of appropriate sequences that flank regions of interest (Bell, 1989). Two synthetic oligonucleotide primers are designed based on these flanking sequences, one complementary to each of the original strands. In the reaction, the diagnostic template DNA is denaturated at high temperature (95"-100"C) and then reannealed in the presence of excess primers. The oligonucleotide primers, oriented with their 3' ends pointing to each other, hybridize to the corresponding target template strands. Enzymatic primer extension occurs subsequently in the presence of deoxyribonucleotide triphosphates. The synthesized end product is then denaturated for another cycle (Innis et al., 1990). Consequently, by the selective experimental amplification of specific DNA fragments, these targets can be readily demonstrated, manipulated and visualized. The crucial point in this technique was the identification of Taq polymerase (obtained from the thermophilic aquatic bacterial species Thermus aquaticus), which is stable up to DNA denaturating temperatures of 95°C. More recently, similar enzymes have been identified with heat-stability at IOO'C. DNA techniques for the identification and characterization of E. multiloculuris have already provided, among others, a DNA probe (pAL1) which showed species-specific polymorphic hybridization patterns to genomic DNA of E. multilocularis and E. granulosus (Vogel et al., 1991). In a subsequent study the complete nucleotide sequence of the E. multilocularis DNA insert of pALl was determined with a view toward deriving oligonucleotide primers suitable for use in PCR amplification of specific target sequences from diagnostic Echinococcus genomic DNA (Gottstein and Mowatt, 1991). The initial PAL1 cloning process, partial mapping of the resulting sequenced DNA probe in question and the location site of oligonucleotide primers are depicted schematically in Fig. 4. In this analysis two E. multilocularis oligonucleotides, BGl and BG2, defined a 2.6 kilobase pair (kbp) fragment in the genome of E. multilocularis. A PCR study


36 1

FIG.4. Derivation of the plasmid subclone PAL1 from a hEMBL4 genomic E. rnultiIocuIaris clone as deduced by a combination of PCR, restriction and Southern

hybridization analyses. The following restriction endonucleases were employed: E, EcoRI; H, HaeIII; N, Nsil; P, PstI; S, Sau3A. Note that all Sau3A sites were not mapped. BGl, BG2 and BG3 represent the location sites of the respective oligonucleotide primers for sequencing and PCR. (After Gottstein and Mowatt, 1991.)

including 14 independent E. multilocularis isolates (originating from Switzerland, Alaska, Canada, France, Germany and Japan) in addition to E. granulosus, E. vogeli, different Taenia spp. and other cestodes, revealed that the 2.6 kbp PCR product was amplified only from genomic DNA of all E. multilocularis isolates, but from genomic DNA of none of the other cestode species (see Fig. 5). Another E. multilocularis primer set (BGl and BG3) was used which resulted in the genus-specific amplification of a 0.3 kb PCR product, i.e. from E. multilocularis, E. granulosus and E. vogeli genomic DNA only (see Fig. 6). The diagnostic sensitivity of the E. multilocularis PCR using both primer sets was experimentally determined to range between 2.5 and 50.0 pg template DNA. It was assumed that for about 50% of the template DNA consisted of heterologous host DNA due to the nature of the metacestode tissue used for DNA extraction. Thus, PCR was estimated to reach a diagnostic level of sensitivity corresponding to one single Echinococcus egg, which contains about 8 pg of nuclear DNA (Rishi and McManus, 1987). The diagnostic application of the E. multilocularis PCR in question was suggested to include (i) the identification of fine-needle biopsy material obtained from patients with liver lesions of unknown aetiology, (ii) the rapid and easy identification of E. multilocularis liver lesions from rodents in epidemiological studies, and (iii), perhaps the most promising and important approach, the demonstration and identification of

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adult stage parasites or eggs in samples derived from the faeces or the small intestines of definitive hosts.

Echinococcus multilocularis isolates

5.0

4.0k

3.0 2.0L 1.6 1.0

-

0.51-

5.0 4.0k 3.0 2.01.6 1.0 0.51-

5.0 d4.0 - 3.0 -2.0 - 1.6 - 1.0

- 0.51

5.0

d4.0

- 3.0 -2.0 - 1.6 - 1.0 - 0.51

FIG.5. Analysis of Echinococcus spp. and other cestode isolates by diagnostic polymerase chain reaction (PCR) using the BGl and BG2 primer set. Ethidium bromide-stained agarose gel fractionation of PCR products derived from amplification of 500 ng genomic template DNA of various and different cestode species and isolates. The 2.6 kb BG 1/BG2 target amplification product is species-specific for E. rnultiloculuris. M, DNA size markers. (After Gottstein and Mowatt, 1991 .)

363

ECHINOCOCCUS MUL TILOCULARIS INFECTION €chinococcus

multilocularis isolates

- 3.0 - 2.0 - 16

302016-

-

10-

0 51-

0.51

- 0.39 - 0.30 - 0.22

0.39030-

0.22 -

1.6 -

- 3.0 - 2.0 - 1.6

3.0 2.0 1.0

10

- 1.0

-

0 51-

- 051 - 0.39

0 390 300 22-

- 0.30 - 022

w

l

i

-

G

FIG. 6. Analysis of Echinococcus spp. and other cestode isolates by diagnostic polymerase chain reaction (PCR) using the BGI and BG3 primer set. Ethidium bromide-stained agarose gel fractionation of PCR products derived from amplification of 500 ng genomic template DNA of various and different cestode species and isolates. The 0.3 kb BGI/BG3 target amplification product is genus-specific for Echinococcus spp. M, DNA size markers. (After Gottstein and Mowatt, 1991.) C.

VACCINATION AGAINST INFECTION WITH E. MULTILOCULARIS

Vaccines for prevention of adult stage intestinal E. multilocularis o r larval stage E. multilocularis metacestodes would hypothetically be valuable for

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disease control, including the decrease of prevalence or eradication of the disease. However, like almost all of the few other parasite vaccines available to date, Echinococcus spp. have not yet yielded any candidate for inducing protective vaccination. However, it is well established that intermediate host animals, such as sheep and cattle, develop a strong immunity within about 2 weeks after primary infection against reinfection with eggs of Taenia and Echinococcus spp. (reviewed by Heath, 1986). These observations represented the basis for vaccine development against metacestode infection of intermediate hosts. Only very recently has a recombinant gene product demonstrated protection against a cestode (Johnson et al., 1989) of veterinary importance; a vaccine against Taeniu ovis cysticercosis in sheep stands as the first recombinant model helminth vaccine to be commercialized. For E. multilocularis, research on inducing protective immunity has been restricted almost entirely to prevention of infection with the larval stage of the parasite. Hence, non-specific stimulation of peritoneal cells with bacillus Calmette-Guerin (BCG) or phytohemagglutinin (PHA), in vitro and in vivo, respectively, protected cotton rats against infection by E . multilocularis (Reuben and Tanner, 1983). Before this study it had been shown that BCG suppressed growth and metastasis formation of E . multilocularis infection (Rau and Tanner, 1975). Attempts to specifically induce protective immunity were described by Adel’shin et al. (1983) by investigating purified highM , E. multilocularis antigens, but the approach failed in that immunized animals showed faster parasite proliferation than control animals. Despite these experiments, the recent epidemiological findings of spontaneously “aborted” or “died-out’’ E. multilocularis lesions in human patients (Rausch et al., 1987) may have indicated a potential for immunologically mediated protective immunity in humans. Evidence for the existence of protective immunity against intestinal adult tapeworms in foxes, dogs or other definitive hosts is less clear. At least partial resistance to infection with E. granulosus was reported by Gemmell et al. (1986) and by Movsesijan et ul. (1968). Vaccination trials using various E. granulosus antigens to immunize dogs against challenge to infection with E. granulosus have resulted in varying degrees of protective immunity (reviewed by Lightowlers, 1990). For E. rnultilocularis, no such experiments have been performed yet. The only indication for, at least partial, protective immunity are epidemiological investigations with a decrease of prevalence observed dependent on the age of infected foxes (see Section 1I.A). As far as a hypothetical effective immune response to E. multilocularis is concerned, this must be divided into B and T cell-dependent mechanisms (Cryz et al., 1989). The host gives rise to the synthesis of antibodies which can co-operate with complement and various cell types leading to antibody-dependent cellular cytotoxiciry. Stimulated T cells may be directly generated as cytotoxic lymphocytes or may produce lymphokines that stimulate macrophages and


365

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 hostprotective 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 (macrophages) 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 immunized 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 incompatibility 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, including alternative expression and presentation systems such as hybrid, live attenuated Salmonella or vaccinia virus, should rapidly stimulate at least

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partial successes. The trend to use recombinant subunit vaccines or synthetic antigens has also resulted in new highly potent adjuvant formulations which elicit strong humoral and cell-mediated immunity, such as detergents or threonyl analogues of muramyl dipeptide (Byars and Allison, 1987) or immunostimulating complexes (ISCOMs) (Morein et al., 1987). Consequently, the major and crucial aspect remains concentrated on the identification of E. multilocularis antigens to protect hosts.

n,

control

11/3-10 control

11/3-10

control

11/3-10

control

11/3-10

I14K-

66K 45K 92K

31K21K14K

protein stain (Indla ink)

E. multiloculerlr serum pool

E,granulosus serum pool

serum pool from healthy blood donors

FIG.7. Western-blot analysis of three batches of recombinant Salmonellu typhimurium vaccine strain (LT2 M 1C) expressing the Echinococcus multilocularis antigen II/3-10. The Western-blot analysis depicts resolved S. typhimurium transformed with pVM 11/3-10(which encodes for the E. multilocularis antigen 11/3-10)and expressing/

synthesizing the respective antigen 11/3-10 (arrowhead indicates the location of the recombinant ~-gal-II/3-10antigen). Control S. typhimurium were transformed with the initial plasmid pUR 278. Immunoreactions were done individually with human serum pools from patients with AE or CE (infection with E. multilocularis or E. granulosus, respectively),and a pool of control sera from healthy blood donors. (After Gottstein et al., 1990.) 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

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demischen Nachwuchses, Kanton Zurich and the “Schweizerische Stiftung fur Medizinisch-Biologische Stipendien”. I thank Professor J. Eckert and Dr M. Mowatt for critical comments on the manuscript. I owe a debt to Professor Eckert whose enthusiastic support of my research of alveolar echinococcosis provided a major stimulus for the present review. REFERENCES Adel’shin, F. K., Ballad, N. E. and Kovalenko, F. P. (1983). Evaluation of the protective activity of purified fractions of alveococcosis antigens in experimental alveococcosis of mice. Meditsinskaya Parazitologiya i Parazitarnye Bolezni 61, 21-26. Afferni, C., Pini, C., Misiti-Dorello, P., Bernardini, L., Conchedda, M. and Vicari, G. (1984). Detection of specific IgE antibodies in sera from patients with hydatidosis. Clinical and Experimental Immunology 55, 587-592. Ah-Khan, Z. (1974a). Host-parasite relationship in echinococcosis. I. Parasite biomass and antibody response in three strains of inbred mice against graded doses of Echinococcus multilocularis cysts. Journal of Parasitology 60,23 1-235. Ali-Khan, Z. (1974b). Host-parasite relationship in echinococcosis. 11. Cyst weight, hematologic alterations, and gross changes in the spleen and lymph nodes of C57L mice against graded doses of Echinococcus multilocularis cysts. Journal of Parasitology 60, 236242. Ah-Khan, Z. and Rausch, R. L. (1987). Demonstration of amyloid and immune complex deposits in renal and hepatic parenchyma of Alaskan alveolar hydatid disease patients. Annals of Tropical Medicine and Parasitology 81, 38 1-392. Ah-Khan, Z. and Siboo, R. (1980). Pathogenesis and host response in subcutaneous alveolar hydatidosis. I. Histogenesis of alveolar cyst and a qualitative analysis of the inflammatory infiltrates. Zeitschrift f i r Parasitenkunde 62, 241-254. Ali-Khan, Z. and Siboo, R. (1981). Echinococcus multilocularis: distribution and persistence of specific host immunoglobulins on cyst membranes. Experimental Parasitology 51, 159-168. Alkarmi, T. 0.and Ah-Khan, Z. (1984). Chronic alveolar hydatidosis and secondary amyloidosis: pathological aspects of the disease in four strains of mice. British Journal of Experimental Pathology 65,40541 7. Alkarmi, T. 0. and Ah-Khan, Z. (1989). Phlogistic and chemotactic activities of alveolar hydatid cyst antigen. Journal of Parasitology 14, 71 1-719. Alkarmi, T. 0. and Behbehani, K. (1989). Echinococcus multilocularis: Inhibition of murine neutrophil and macrophage chemotaxis. Experimental Parasitology 69, 1622. Alkarmi, T. O., Alshakarchi, Z. and Behbehani, K. (1988). Echinococcus multilocularis: the non-specific binding of different species of immunoglobulins to alveolar hydatid cysts grown in vivo and in vitro. Parasite Immunology 10, 443-457. Ammann, R. ( 1983). Diagnose und Therapie der Echinokokkose. Schweizerische Rundschau , f i r Medizin (Praxis) 12, 1568-1 572. Ammann, R., Tschudi, K., von Ziegler, M., Meister, F., Cotting, J., Eckert, J., Witassek, F. and Freiburghaus, A. (1988). Langzeitverlauf bei 60 Patienten mit alveolarer Echinokokkose unter Dauertherapie mit Mebendazol (19761985). Klinische Wochenschrift 66, 106Ck1073.

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Auer, H., Hermentin, K. and Aspock, H. (1988a). Demonstration of a specific Echinococcus multilocularis antigen in the supernatant of in vitro maintained protoscoleces. Zentralblatt fur Bakteriologie und Hygiene A268, 416-423. Auer, H., Picher, 0. and Aspock, H. (1988b). Combined application of enzymelinked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA) as a useful tool for the diagnosis and post-operative surveillance of human alveolar and cystic echinococcosis. Zentralblatt fur Bakteriologie und Hygiene A270, 313-325. Auer, H., Hermentin, K., Picher, O., Lexer, G., Weitensfelder, W., Wilhelmer, S. and Aspock, H. (1 988c). Parasitologisch-serologische Screening-Untersuchung der Bevolkerung in einem Herd von Echinococcus multilocularis in Oesterreich. Mitteilung der osterreichischen Gesellschaft fur Tropenmedizin und Parasitologie 10, 151-158. Auer, H., Bohm, G., Dam, K., Frank, W., Ferenci, P., Karner, J. and Aspock, H. (1990). First report on the occurrence of human cases of alveolar echinococcosis in the northeast of Austria. Tropical Medicine and Parasitology 41, 149-156. Ballard, N. B. and Vande Vusse, J. (1983). Echinococcus multilocularis in Illinois and Nebraska. Journal of Parasitology 69, 79&79 I . Baron, R. W. and Tanner, C. E. (1976). The effect of immunosuppression on secondary Echinococcus multilocularis infections in mice. International Journal of Parasitology 6, 3 7 4 2 . Baron, R. W. and Tanner, C. E. (1977). Echinococcus multilocularis in the mouse: the in vifro protoscolicidal activity of peritoneal macrophages. International Journal of Parasitology 7, 489495. Barutzki, D., Loscher, T., Geisel, O., Minkus, G. and Hermanns, W. (1990). Metazestoden von Echinococcus multilocularis (Leuckart, 1983) Vogel, 1955, bei Hunden. Abstract No. P63. Proceedings of the 14th Meeting of the German Society of Parasitology, 4-6 April 1990, Marburg. Bell, J. (1989). The polymerase chain reaction. Immunology Today 10, 351-355. Bonneville, M., Janeway, C. A., Ito, K., Haser, W., Ishida, I., Nakanishi, N. and Tonegawa, S. (1988). Intestinal intraepithelial lymphocytes are a distinct set of y6 T cells. Nature 336, 4 7 9 4 8 1. Bosch, D. (1 982). Tierexperimentelle Untersuchungen zur Entwicklung von Echinococcus multilocularis. In “Probleme der Echinokokkose unter Berucksichtigung Parasitologischer und Klinischer Aspekte” (R. Bahr, ed), pp. 3 W . Hans Huber Verlag, Berne. Brandtzaeg, P. (1985). Role of J chain and secretory component in receptor-mediated glandular and hepatic transport of immunoglobulins in man. Scandinavian Journal of Immunology 22, 11 1-146. Bresson-Hadni, S., Vuitton, D. A., Lenys, D., Liance, M., Racadot, E. and Miguet, J. P. (1989a). Cellular immune response in Echinococcus multilocularis infection in humans. I. Lymphocyte reactivity to Echinococcus antigens in patients with alveolar echinococcosis. Clinical and Experimental Immunology 78, 6 1 4 6 . Bresson-Hadni, S., Vuitton, D. A., Lenys, D., Liance, M., Racadot, E. and Miguet, J. P. (1989b). Lymphocyte reactivity to Echinococcus antigens in patients with alveolar echinococcosis. Clinical and Experimental Immunology 78, 6 1 4 6 . Bresson-Hadni, S., Lenys, D., Vuitton, D., Laplante, J. J., Robert, S., Liance, M., Gottstein, B., Jacquier, P., Meyer, J. P. and Miguet, J. P. (1990a). Serological screening for Echinococcus multilocularis (E.m.) infection using ELISA in eastern France. Bulletin de la Societe Franqaise de Parasitologie 8, 948.


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Bresson-Hadni, S., Franza, A., Lenys, D., Miguet, J. P., Paintaud, G., Monnet, E. and Vuitton, D. (1990b). Treatment of human alveolar echinococcosis (AE) by liver transplantation: indications, clinical and serological follow-up. Bulletin de la Societe FranGaise de Parasitologie 8, 418. Bresson-Hadni, S., Liance, M., Meyer, J. P., Houin, R., Bresson, J. L. and Vuitton, D. A. ( 1990c) Cellular immunity in experimental Echinococcus multilocularis infection. 11. Sequential and comparative phenotypic study of the periparasitic mononuclear cells in resistant and sensitive mice. Clinical and Experimental Immunology 82, 378-383. Byars, N. E. and Allison, A. C. (1987). Adjuvant formulation for use in vaccines to elicit both cell-mediated and humoral immunity. Vaccine 5, 223-228. Capron, A., Vernes, A. and Biguet, J. (1967). Le diagnostic immunoelectrophoretique de I’hydatidose. In: Le kyste hydatique du foie, pp. 2740. Journtes Lyonnaises d’Hydatidologie, SIMEP Editions, Lyons. Carding, S. R., McNamara, J. G., Pan, M. and Bottomly, K. (1990). Characterization of y8 T cell clones isolated from human fetal liver and thymus. European Journal of Immunology 20, 1327-1335. Castro, G. A. (1989). Immunophysiology of enteric parasitism. Parasitology Today 5, 11-19. Claudon, M., Bessieres, M., Regent, D., Rodde, A., Bazin, C., Gerard, A. and Bresler, L. (1990). Alveolar echinococcosis of the liver: MR findings. Journal of Computer Assisted Tomography 14, 608-6 14. Coman, B. J. and Rickard, M. D. (1975). The location of Taenia pisiformis, T . ovis and T. hydatigena in the gut of the dog and its effect on net environmental contamination with ova. Zeitschrift fur Parasitenkunde 47, 237-248. Condon, J., Rausch, R. L. and Wilson, J. F. (1988). Application of the avidin-biotin immunohistochemical method for the diagnosis of alveolar hydatid disease from tissue sections. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 731-735. Craig, P. S. and Nelson, G. S. (1984). The detection of circulating antigen in human hydatid disease. Annals of Tropical Medicine and Parasitology 78, 219-227. Craig, P. S., Macpherson, C. N. L. and Nelson, G. S. (1986). The identification of eggs of Echinococcus by immunofluorescence using a specific anti-oncospheral monoclonal antibody. American Journal of Tropical Medicine and Hygiene 35, 152-1 58. Cryz, S. J., Granstrom, M., Gottstein, B., Perrin, L., Cross, A. and Larrick, J. (1989). “Immunotherapy and Vaccines,” Voi. A14. Ullmann’s Encyclopedia of Industrial Chemistry, Weinheim. Deblock, S. and Petavy, A. F. (1990). Donnes recentes sur I’epidemiologie de I’echinococcose alveolaire en France. Bulletin de la Socittt de Pathologie Exotique 83,242-248. de Bruijn, M. H. L. (1988). Diagnostic DNA amplification. No respite for the elusive parasite. Parasitology Today 4, 293-295. Deplazes, P. and Gottstein, B. (1991). A monoclonal antibody against Echinococcus multilocularis Em2 antigen. Parasitology 103, 41-49. Deplazes, P., Gottstein, B.. Stingelin, Y.and Eckert, J. (1990). Detection of Taenia hydatigena copro-antigens by ELISA in dogs. Veterinary Parasirology 36,91-103. Dessaint, J. P., Bout, D., Wattre, P. and Capron, A. (1975). Quantitative determination of specific IgE antibodies to Echinococcusgranulosus and IgE levels on sera from patients with hydatid disease. Immunology 29, 813-823.

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