Fasciola hepatica and Fasciola gigantica - NRC Research Press

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REVIEW / SYNTHÈSE

Immunology of the host–parasite relationship in fasciolosis (Fasciola hepatica and Fasciola gigantica)1 D. Piedrafita, H.W. Raadsma, R. Prowse, and T.W. Spithill

Abstract: The protective resolution of liver fluke (Fasciola hepatica and Fasciola gigantica) infection is a dynamic interplay between the host’s effector responses and the parasite’s defence and immunomodulatory systems. The evidence suggests that the juvenile or immature parasite is the target of protective host immune responses but the effector mechanisms employed vary between hosts. Moreover, F. hepatica and F. gigantica differ in their susceptibility to these killing mechanisms. In the rat, in vitro killing of juvenile F. hepatica involves an antibody-dependent cell cytotoxicity mediated by nitric oxide produced by activated monocytes and (or) macrophages. However, monocytes and (or) macrophages from Indonesian sheep do not produce nitric oxide yet can effectively kill juvenile F. gigantica in vitro and in vivo by a mechanism that is ineffective against F. hepatica. These data show that disease progression or resolution in fasciolosis is determined both by biochemical differences between Fasciola species and by host-dependent factors. Understanding the genetic basis for these differences is a key question for the future. Fasciola hepatica and F. gigantica actively modulate the host immune response, downregulating type 1 responses during infection. It is important to determine whether such modulation of the immune response by Fasciola spp. directly leads to enhanced parasite survival in the various hosts. Résumé : La résolution par protection d’une infection causée par les douves du foie (Fasciola hepatica et Fasciola gigantica) est une interaction dynamique entre les réactions effectives de l’hôte et les systèmes de défense et d’immunomodulation des parasites. Il y a des indices que le parasite jeune ou immature est la cible des réactions immunitaires de protection de l’hôte, bien que les mécanismes effecteurs employés varient selon les hôtes. De plus, F. hepatica et F. gigantica n’ont pas la même susceptibilité à ces mécanismes de destruction. Chez le rat, la destruction in vitro de jeunes F. hepatica implique une cytotoxicité cellulaire dépendant des anticorps par l’intermédiaire d’oxyde nitrique produit par des monocytes et (ou) macrophages activés. Cependant, les monocytes et (ou) macrophages de moutons indonésiens ne produisent pas d’oxyde nitrique, mais ils sont capables de détruire de jeunes F. gigantica, tant in vitro qu’in vivo par un mécanisme qui reste inefficace contre F. hepatica. Ces données démontrent que la progression de la maladie ou la résolution de la fasciolose sont déterminées à la fois par des différences biochimiques entre les espèces de Fasciola et des facteurs dépendants de l’hôte. La compréhension des facteurs génétiques responsables de ces différences est un problème central pour l’avenir. Fasciola hepatica et F. gigantica modulent de façon active la réaction immunitaire de leur hôte, réduisant par régulation les réponses de type 1 durant l’infection. Il est important de déterminer si une telle modulation de la réaction immunitaire par Fasciola spp. entraîne directement une survie accrue du parasite chez les divers hôtes. [Traduit par la Rédaction]

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Received 28 May 2003. Accepted 27 October 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 16 April 2004. D. Piedrafita. Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Victoria 3010, Australia. H.W. Raadsma. Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, Camden, New South Wales 2570, Australia. R. Prowse. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia, and the Cooperative Research Centre for Vaccine Technology, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. T.W. Spithill.2 Institute of Parasitology and the Centre for Host–Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, QC H9X 3V9, Canada. 1

This review is one of a series dealing with aspects of the biology of the phylum Platyhelminthes. This series is one of several virtual symposia on the biology of neglected groups that will be published in the Journal from time to time. 2 Corresponding author (e-mail: [email protected]). Can. J. Zool. 82: 233–250 (2004)

doi: 10.1139/Z03-216

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1. Introduction 234 2. Fasciola spp. life cycle following infection 234 3. Immune responses during Fasciola spp. infection 234 3.1. Immune responses in rats 234 3.2. Immune responses in ruminants 235 3.2.1. Immune responses in sheep 235 3.2.2. Immune responses in cattle 236 4. Potential effector mechanisms of killing of juvenile Fasciola spp. by sheep and rats 237 4.1. Putative effector mechanisms of rats identified ex vivo and (or) in vitro 238 4.1.1. Early studies 238 4.1.2. Free radical-mediated killing of REJ F. hepatica in vitro 238 4.2. Putative effector mechanisms of ruminants identified ex vivo and (or) in vitro 239 5. Differences in the host–parasite relationship determining the differential susceptibility of rats and sheep to Fasciola spp. infection 240 5.1. Host-dependent factors 240 5.2. Parasite-dependent factors 241 5.2.1. Production of antioxidant defence enzymes 241 5.2.2. Glycocalyx turnover 242 5.2.3. Release of excretory/secretory products 242 5.2.3.1. Effect of Fasciola spp. infection on immune responses 242 5.2.3.2. Effect of Fasciola spp. ESPs 243 5.2.3.3. Cathepsin proteases and immune modulation 244 6. Conclusions 244 7. Acknowledgements 245 8. References 245

1. Introduction Fasciola hepatica and Fasciola gigantica (temperate and tropical liver flukes, respectively) are recognised as two of the most economically important helminth parasites of production animals (reviewed in Fabiyi 1987; Spithill et al. 1999a). Recently, worldwide losses in animal productivity due to fasciolosis were conservatively estimated at over US$3.2 billion per annum (Spithill et al. 1999a). The global demand for food of animal origin in developing countries is predicted to grow by 2.8% per annum from 1993–2020 (Delgado et al. 1999) and the control of major helminth diseases such as fasciolosis could contribute significantly to improved animal production. Recent studies have suggested that a complex interaction between the host and Fasciola spp. determines whether infection will lead to a detrimental (chronic infection) or beneficial (parasite expulsion) host immune response. It appears that differences between various host factors and putative differences between the temperate and tropical liver fluke have a major influence on the host–parasite relationship and successful resolution of disease. Here, we review the immunology of the host–parasite relationship in fasciolosis and discuss the implications of recent results in the context of understanding immune effector mechanisms active against Fasciola spp. in different animal hosts and the immunomodulatory properties of the parasite.

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2. Fasciola spp. life cycle following infection The infective phase of the Fasciola spp. life cycle involves ingestion of metacercariae found either encysted on infected plants or floating in drinking water, which excyst in the small intestine, releasing the juvenile flukes into the gut. The juvenile parasites migrate through the intestinal wall, the peritoneal cavity, and the liver parenchyma, where they feed and develop, before burrowing into the biliary ducts in the liver, where they mature into adult flukes and commence egg laying (Dawes and Hughes 1964). Fasciola spp. have been estimated to produce 20 000 to 50 000 eggs per fluke per day (Boray 1969). The behaviour of F. hepatica and F. gigantica in the same host differs in that the time taken to reach the bile ducts and subsequently mature in sheep is 7–10 weeks post infection for F. hepatica versus 12–16 weeks for F. gigantica (Grigoryan 1958; Dawes and Hughes 1964; Guralp et al. 1964; Sewell 1966; Boray 1969). These parasites also differ in size, with adult F. hepatica and F. gigantica typically measuring up to 30 mm and 75 mm in length, respectively. Fasciola spp. infections can lead to chronic or acute disease within human and ruminant hosts (cattle, buffalo, and sheep). Chronic infections — the result of a small intake of parasites over a long period of time — are the most common form of liver fluke infection in sheep (Boray 1969).

3. Immune responses during Fasciola spp. infection 3.1. Immune responses in rats Large-scale studies involving sheep and cattle are often impractical for immunological research because of the expense and size of the animals; therefore, infection models using small laboratory animals, such as mice, rats, and rabbits, have been developed. Rats develop immunological resistance to reinfection with F. hepatica (reviewed in Hughes 1987) and have, therefore, been used more than any other experimental model to study both host immune mechanisms and evasion strategies used by this parasite. A suitable laboratory model for the study of F. gigantica, however, has not been identified, and rats almost completely reject F. gigantica infections. In rats, recovery of F. gigantica (0%–5%) is much lower than recovery of F. hepatica (20%–30%) and Japanese Fasciola spp. (36%–47%) (Mango et al. 1972; Gupta and Chandra 1987; Itagaki et al. 1994). Whether the mechanism of this resistance of rats to F. gigantica infection is mediated by an immune response is unknown. Consequently, most studies of this parasite have used large natural hosts such as sheep (see section 3.2) and resulted in fewer studies and little knowledge of the immunobiology of F. gigantica infections. Resistance to primary and challenge infection with F. hepatica is routinely seen in rats (Hughes 1987). The level of resistance varies considerably and is dependent on several factors including the strain, age, and sex of the rats (Hayes et al. 1974a; Hughes et al. 1976; Rajasekariah and Howell 1977a, 1981). The level of resistance in rats is the same whether they are challenged with homologous or heterologous clones of the parasite; a clone is defined as the metacercariae produced from a single snail infected with a © 2004 NRC Canada

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single miracidium (Chapman et al. 1981). Primary infections are commonly expelled within 6–12 months and may result from crowding effects and the inhospitable nature of the distended bile duct space (Hughes et al. 1976; Hughes 1987). Rats infected orally with metacercariae rapidly become resistant to a secondary challenge within 2–3 weeks (Hughes 1987). However, in the absence of a further challenge infection, rats harbouring a primary infection for about 6 months become susceptible to reinfection with F. hepatica; interestingly, this correlates with a decline in antibody levels, suggesting a potential role of anti-Fasciola spp. antibodies in determining resistance (see below). If chronically infected rats are subsequently reinfected, the ability to kill a further challenge infection is restored within 2 weeks (Hughes et al. 1977). There is good evidence to suggest that resistance to challenge infection in the rat can occur with little or no preexisting damage to the liver and, hence, a physical barrier due to liver fibrosis can be excluded as the mechanism for resistance in challenged animals. For example, rats are protected from challenge infections when living adult flukes, metacercariae, or eggs are implanted subcutaneously (Rajasekariah and Howell 1978; Haroun et al. 1980). In these experiments, sensitization of the rat was achieved with no damage to the liver. Furthermore, 2–3 weeks after oral infection with a single metacercaria, which results in only slight liver damage, rats were also protected from a challenge infection (Hayes et al. 1973). Resistance to F. hepatica infection in rats appears to involve both antibodies and lymphoid cells. Immunity can be transferred from infected animals by both sera and lymphoid cells (Armour and Dargie 1974; Hayes et al. 1974b, 1974c; Rajasekariah and Howell 1979; Mitchell et al. 1981). Passive transfer of either homologous or heterologous sera can protect naive rats but only against a challenge infection occurring on the day of transfer (Hayes et al. 1974b, 1974c; Chapman and Mitchell 1982). One possible explanation may be that, when the challenge occurs on the same day, the concentration of specific antibodies is high enough to allow effective antibody-dependent cell cytotoxicity (ADCC) mechanisms that may mediate killing of excysting juvenile flukes in the rat (see section 4.1). Protective responses seen in active transfer experiments involving lymphoid cells appear to arise only when donor rats are infected for more than 4 weeks (Corba et al. 1971). Protective responses induced in rats following vaccination with parasite antigens or exposure to a primary parasite infection suggest that the juvenile stage may be most susceptible to host killing and is the target of effective immunity. For example, Van Milligen et al. (2000) vaccinated rats with lysates of adult or newly excysted juvenile parasites. They found that significantly fewer flukes were recovered from rats vaccinated with newly excysted juvenile antigens than from rats vaccinated with adult antigens. A similar result was seen in one of the few studies carried out in rodents infected with F. gigantica, where vaccination with metacercariae extracts conferred significantly greater protection to rats than immunization with extracts from adult worms (Yoshihara et al. 1985). In F. hepatica infections of challenged rats, juvenile parasites are killed within 24–48 h of challenge and this involves two basic mechanisms (Hayes and Mitrovic 1977;

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Rajasekariah and Howell 1977b; Doy et al. 1978). The first involves the gut wall, through which about 25%–75% of juvenile flukes are unable to penetrate after being coated with host antibodies (Burden et al. 1983; Van Milligen et al. 1998a). Several studies suggest that the mechanism of resistance at the gut wall is thymus-independent and that nonspecific and hypersensitivity reactions may play a role (Rajasekariah and Howell 1977a, 1977b; Doy et al. 1981a, 1981b; Doy and Hughes 1982a; O’Malley et al. 1993). However, Van Milligen et al.’s (1998a) demonstration that resistance at the gut wall was induced by juvenile parasite migration through the peritoneum or early after entering the liver, without the necessity of crossing the gut wall in a sensitizing infection, also suggests that the acquired memorybased immune response that effectively kills juvenile flukes in the peritoneum may also be involved in the killing of juvenile parasites within the gut wall. The second mechanism determining resistance in rats, effective once the juvenile fluke has penetrated the gut wall, is T cell dependent and acts in the peritoneal cavity (Rajasekariah and Howell 1977b; Kelly et al. 1980; Doy and Hughes 1982a). Juvenile flukes that penetrate the gut wall of sensitized rats are coated with antibodies and appear to be irreversibly damaged by host cells, including macrophages, eosinophils, neutrophils, and mast cells, and death follows within a matter of hours after entry into the peritoneal cavity (Hughes 1987). Migration through the gut wall is not necessary for killing of juvenile flukes in sensitized rats; juvenile flukes injected intraperitoneally into resistant rats are also killed, suggesting that effector mechanisms present in the peritoneal cavity alone are able to kill recently excysted flukes (Rajasekariah and Howell 1977b; Kelly et al. 1980; Doy and Hughes 1982a). Antibodies are likely to be important for resistance to F. hepatica infection in rats in this T cell dependent cytotoxicity mechanism. In fact, Hanna (1980a) suggested that the loss of resistance of rats to infection with F. hepatica coincides with the decline in the level of specific antibodies in circulation. These findings suggest that there is a parasite-killing mechanism in the peritoneal cavity of rats that is dependent on the presence of parasitespecific antibodies (Armour and Dargie 1974). Recently, protection against F. hepatica in rats was associated with accumulation of eosinophils and IgE-positive cells in the gut wall and with juvenile antigen-specific IgG1 induced within 2 weeks of infection (Van Milligen et al. 1999; Tliba et al. 2000). 3.2. Immune responses in ruminants 3.2.1. Immune responses in sheep Infection of European sheep with F. hepatica causes a large infiltration of white blood cells into the liver and production of antibodies to the parasite (Sinclair 1962, 1971a; Boray 1967, 1969; Ross et al. 1967; Movsesijan et al. 1975; Rushton and Murray 1977; Knight 1980; Sandeman and Howell 1980a, 1980b; Wedrychowicz et al. 1984; Boyce et al. 1987). However, there is little evidence of immunological resistance of sheep to a challenge infection with F. hepatica when judged by worm burdens (Boray 1969; Sinclair 1971a, 1971b, 1973, 1975; Meek and Morris 1979; Sandeman and Howell 1981; Haroun and Hillyer 1986). Initial studies, in © 2004 NRC Canada

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which resistance of sheep to F. hepatica reinfection was reported, have not been reproducible (Hughes 1987). The severity of the disease due to Fasciola spp. infection may, however, be reduced in previously exposed sheep. A challenge infection with F. hepatica after a sensitizing primary infection can result in decreased worm size, reduced egg production by adult worms, delayed onset of anaemia, an earlier rise and a greater number of eosinophils, and earlier lymphocyte infiltration into the liver (Boray 1969; Sinclair 1962, 1971a,1973, 1975; Meeusen et al. 1995). As first suggested by Boray (1967, 1969), it is likely that the reduction in the severity of the pathological changes in the liver of challenged sheep is due to the fibrosis caused by the primary infection, and this fibrotic response appears to be a result of the immunological response of sheep to F. hepatica infection (Sinclair 1968, 1970). Interestingly, despite the lack of evidence for resistance (as measured by decreased worm burdens) to challenge infection by F. hepatica in sheep, there are significant differences in the ability of Fasciola species to establish in different sheep breeds, and resistance to F. gigantica infections is well described in sheep (Boyce et al. 1987; Wiedosari and Copeman 1990; reviewed in Spithill et al. 1999a). The factors responsible for this apparent natural resistance to Fasciola spp. infection in certain sheep breeds are currently unknown. A significant reduction in parasite numbers was observed in Sudanese desert sheep vaccinated with irradiated metacercariae of F. gigantica (A’Gadir et al. 1987), and high resistance to F. gigantica infection has been observed in Indonesian thin tail (ITT) sheep (Wiedosari and Copeman 1990; Roberts et al. 1997a, 1997b, 1997c). ITT sheep express resistance to F. gigantica within the prepatent period of a primary infection and acquire a higher level of resistance after exposure (Roberts et al. 1997a, 1997c). The acquired resistance of ITT sheep, expressed within 3– 4 weeks of infection, can be suppressed by dexamethasone, implying that this resistance is immunological and that the killing of many migrating parasites occurs within 2–4 weeks of infection (Roberts et al. 1997c; Spithill et al. 1999a). The resistance of ITT sheep to F. gigantica appears to be genetically determined (Roberts et al. 1997b; Raadsma et al., unpublished data) but the immune mechanisms involved in resistance remain unclear. Immunological analyses of responses in ITT and Merino sheep following a primary infection showed that, at 1– 3 weeks post infection, ITT sheep exhibited significantly greater eosinophilia than Merino sheep, whereas neutrophilia was significantly greater in ITT sheep than Merino sheep at 5–9 weeks after infection (Hansen et al. 1999). Both sheep breeds demonstrated IgM, IgG1, and IgE responses to F. gigantica, whereas ITT sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds, with the first response detected by week 2 and a second response developing 4–9 weeks after infection. Western blot analysis of sera showed a similar pattern of adult fluke antigens recognised by IgG1 and IgE antibodies from both sheep breeds, with the IgE response associated with a major 92 kDa antigen. These results suggested that IgG2 could act as a blocking antibody in sheep and that ITT sheep have an enhanced capacity to kill F. gigantica in vivo

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owing to down-regulation of the IgG2 response (Hansen et al. 1999). The overall data are consistent with the view that resistance of ITT sheep to F. gigantica is associated with a type 2 immune response. 3.2.2. Immune responses in cattle In general, cattle are resistant to infections with F. hepatica and are able to expel both primary and challenge infections (Boray 1969; Dawes and Hughes 1970; Doyle 1971, 1972, 1973a; Anderson et al. 1978; Kendall et al. 1978; Doy and Hughes 1984). The expulsion of the majority of parasites from a primary F. hepatica infection in cattle usually occurs within 30 weeks of infection (Doyle 1971, 1972). The development of resistance to reinfection with F. hepatica is dependent on the duration of the primary infection. Cattle infected with a primary F. hepatica infection for 12 weeks were resistant to challenge with the parasite, whereas those with a primary infection for 7 weeks were not resistant to reinfection (Doyle 1973a). Doy and Hughes (1984) found a 56% reduction in fluke burdens when cattle with an 18-week-old primary infection received a subsequent challenge infection. Resistance to reinfection increased to 94% in cattle given a primary infection with F. hepatica 26 weeks previously. The presence of live adult worms in the bile ducts at the time of challenge is not necessary for the development of resistance (Kendall and Parfitt 1975). Collectively, the above studies suggest that in cattle given a primary F. hepatica infection, a long infective period with the parasite is required before most of the parasites are expelled from the initial infection or from a subsequent challenge infection. Intense fibrosis of the liver tissue and calcification of the bile ducts occurs as a result of a primary F. hepatica infection, and parasite rejection begins at the same time as calcification of the bile ducts (Boray 1969; Doy and Hughes 1984; Hughes 1987). It has, therefore, been suggested that physical barriers including fibrosis of the liver and calcification of the bile ducts, due to migration and feeding activities of flukes from a primary infection, are responsible for the resistance to challenge infections with F. hepatica in cattle (Boray 1969; Hughes 1987). Only a small number of studies clearly implicate immunological responses in resistance to infection with F. hepatica (Corba et al. 1971; Dargie et al. 1973, 1974). Elimination of a primary infection in cattle coincided with the highest levels of homocytotrophic antibodies at 20–28 weeks post infection (Doyle 1973b). However, whether these antibodies play any functional role in the expulsion of the parasite is unknown. Corba et al. (1971) were able to confer 80% resistance to a recipient monozygotic twin calf by transfer of lymphoid cells and, therefore, suggested the involvement of cellular immune mechanisms in resistance; transfer of serum was not successful. Unfortunately, validation of this important observation has not been carried out owing to the unavailability of suitable animals. Unlike cattle, rats acquire resistance to F. hepatica before most flukes reach the liver and, therefore, the presence of a physical barrier such as a fibrotic liver can be discounted in the rat (see section 3.1). Furthermore, the two sites of parasite killing demonstrated in the rat, the gut wall and the peritoneal cavity (Hughes 1987; Van Milligen et al. 1998a, 1998b, 1999), are not in© 2004 NRC Canada

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Table 1. Summary of host susceptibility to infection with either Fasciola hepatica or Fasciola gigantica. Parasite Host

F. hepatica

F. gigantica

Indonesian thin tail sheep Merino sheep Rat

SUS, NPIR; primary or secondary infection SUS, NPIR; primary or secondary infection RES, PIR; secondary infection

RES, PIR; primary or secondary infection RES, UPIR; secondary infection RES, UPIR; primary or secondary infection

Note: SUS, host susceptible to infection; NPIR, no protective immune response; RES, host resistant to infection; PIR, protective immune response (i.e., resistance is induced in this host and studies show that an immune response is involved in this resistance); UPIR, unknown protective response (i.e., host develops resistance but whether an immune response is involved in this resistance has not been determined). Resistance is expressed against primary or secondary infections.

volved in resistance in cattle (Burden and Bland 1983; Doy and Hughes 1984). A recent study has provided indirect evidence that a degree of resistance to F. hepatica can be induced in cattle by an abbreviated sensitizing infection that does not produce significant liver fibrosis (Hoyle et al. 2003). Sensitization of cattle with one or three drug-abbreviated infections (of duration 5 days or 1 day, respectively) significantly reduced liver damage (as estimated by glutamate dehydrogenase (EC 1.4.1.2) and γ-glutamyltransferase (EC 2.3.2.2) activity in serum) following a secondary challenge. This suggests that preexposure to juvenile antigens (i.e., those expressed during the 5-day exposure) reduced migration of flukes into the liver and bile ducts. Although fluke burdens in these vaccinated cattle were not reported, this experiment suggests that previously exposed cattle can mount a protective (presumably immunological) response that reduces parasite invasion into the liver. Strong evidence in support of an immunological basis of acquired resistance in ruminants comes from multiple vaccine studies in naive animals showing that cattle and sheep can be protected against F. hepatica infection by vaccination with defined antigens (reviewed in Spithill and Dalton 1998; Piacenza et al. 1999; Spithill et al. 1999b; Mulcahy and Dalton 2001). Fluke burdens were reduced by 38%–72% in cattle (with some vaccinates showing >90% reduction in fluke burdens) and up to 89% in sheep vaccinated with the leucine aminopeptidase vaccine (Piacenza et al. 1999). These results strongly suggest that vaccinated cattle and sheep can mount protective immune responses that can kill invading parasites before significant liver fibrosis occurs, since protected animals show reduced liver damage (Sexton et al. 1990; Dalton et al. 1996; Piacenza et al. 1999). Results from J.P. Dalton’s laboratory have suggested a correlation between the IgG2 response to cathepsin L vaccines and protection in cattle, whereas the IgG1 response has been correlated with susceptibility (Mulcahy et al. 1998). A logical conclusion from these studies would be that protective responses induced in sheep and cattle following vaccination are not induced during a natural infection of these hosts by F. hepatica. Type 1 responses are down-regulated in cattle infected by Fasciola spp. Brown et al. (1994) showed that antigen-specific T cell clones isolated from chronically infected cattle express only a type 0 or type 2 helper phenotype. Type 1 helper T cells were isolated from genetically identical cattle infected with Babesia bovis. An antigenspecific T cell clone isolated from immune cattle also expressed a type 0 helper response (Shoda et al. 1999). Clery et al. (1996), using cattle chronically infected with F. hepat-

ica, also showed that lymphocytes proliferating in response to F. hepatica antigens failed to produce IFN-γ. These results suggest an inverse correlation between chronic F. hepatica infection and induction of parasite-specific type 1 T cells. The IgG2 response in cattle is positively regulated by IFN-γ, whereas the IgG1 response is positively regulated by interleukin (IL) 4 (Estes and Brown 2002). Type 0 helper T cell clones specific for F. hepatica predominantly stimulated IgG1 production in vitro (Brown et al. 1999). Thus, the observation of elevated parasite-specific IgG1 levels but low IgG2 levels in infected cattle is consistent with these observations (Clery et al. 1996; Spithill et al. 1997; Brown et al. 1999; Estes and Brown 2002). Basic studies of immune responses following F. gigantica infections are less frequent. Earlier studies using radiationattenuated metacercariae as vaccines support the view that cattle express acquired protective immune mechanisms against F. gigantica; i.e., fibrosis is not the basis of resistance in cattle to this parasite (reviewed in Spithill et al. 1999a). Cattle acquire resistance to F. gigantica after a primary exposure to irradiated metacercariae (reviewed in Haroun and Hillyer 1986; Spithill et al. 1999a). Cattle develop specific IgG responses, and sensitization of cattle by a primary infection with irradiated metacercariae resulted in 45%–98% protection from a subsequent F. gigantica challenge infection (Spithill et al. 1999a). Vaccine studies in cattle with several F. gigantica antigens showed that the fatty acid binding protein complex could induce a significant 31% reduction in fluke burdens in cattle; although this level of protection appears modest, it was observed in cattle that received a very high challenge dose (386 flukes), confirming that cattle can mount protective immune responses against F. gigantica (Estuningsih et al. 1997; Spithill et al. 1999a). Recently, protection against F. gigantica has been observed using a Schistosoma bovis glutathione S-transferase (GST) (EC 2.5.1.18) vaccine (unpublished data cited in De Bont et al. 2003). Such studies suggest that ruminants are capable of mounting effective immune responses that can block F. gigantica infection.

4. Potential effector mechanisms of killing of juvenile Fasciola spp. by sheep and rats From the above literature and our studies to date, there is a strong suggestion that the two Fasciola species differ dramatically in their ability to infect rats and sheep (Table 1); broadly, F. hepatica will establish a primary infection in rats and sheep, whereas rats and sheep show resistance to F. gigantica (Boyce et al. 1987; Spithill et al. 1999a). If rats © 2004 NRC Canada

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are first primed with a sensitizing infection of F. gigantica, the challenge infection is almost completely rejected (Mango et al. 1972; Gupta and Chandra 1987; Itagaki et al. 1994); this has also been observed in ITT sheep (Spithill et al. 1999a). These findings suggest that F. gigantica is more susceptible to killing by host responses than F. hepatica. Our laboratories are interested in determining why two apparently closely related parasites differ in their ability to establish infection, and we have begun to elucidate the resistance mechanisms expressed by hosts against F. hepatica and F. gigantica. To understand the immune mechanisms that may eliminate Fasciola spp. in vivo, we performed comparative in vitro killing studies using immune cells from rats and sheep with juveniles of F. gigantica and F. hepatica. 4.1. Putative effector mechanisms of rats identified ex vivo and (or) in vitro 4.1.1. Early studies As stated above, putative effector mechanisms expressed by rats (or other laboratory animals) against F. gigantica infection have not been studied. However, a number of studies have been carried out with F. hepatica. The protective role, if any, of complement in acquired immunity to F. hepatica infection is unknown. Rat serum containing complement was unable to kill recently excysted flukes in vitro (Duffus and Franks 1980; Davies and Goose 1981). Davies and Goose (1981) found that rat C3 bound neither to the surface of recently excysted flukes incubated in immune rat serum in vitro nor to recently excysted flukes recovered from the peritoneal cavity of sensitized rats. Both Duffus and Franks (1980) and Hanna (1980a) suggested that the rapid turnover of the glycocalyx may prevent complement attachment and activation on the surface of the parasite. It has been suggested that the eosinophil is one of the main effector cells involved in the killing of recently excysted flukes by challenged rats. This is based on the finding of large numbers of eosinophils in the intestinal wall and peritoneal cavity after challenge of resistant rats; the large numbers of eosinophils attached to damaged juvenile flukes recovered from challenged rats (Doy et al. 1978; Davies and Goose 1981; Doy and Hughes 1982b; Burden et al. 1983; Charbon et al. 1991); and the correlation between eosinophil levels in the gut wall and protection in rats (Van Milligen et al. 1998b, 1999). However, these studies provide only circumstantial evidence to implicate the eosinophil as an important effector cell. In vitro studies have been unable to demonstrate irreversible damage to recently excysted flukes by sera and eosinophils obtained from rats (Doy et al. 1980; Duffus and Franks 1980; Doy and Hughes 1982b). Doy et al. (1980) found selective adherence of eosinophils, in the presence of immune serum, from mixed populations of cells derived from either normal or fluke-infected rats. An IL5-like molecule, which could directly stimulate eosinophil maturation in rat bone marrow cells, was found among F. hepatica excretory/secretory antigens (Milbourne and Howell 1990, 1993). Stimulated eosinophils were functionally normal and matured in vitro (Milbourne and Howell 1993). The significance of these observations to the host–parasite relationship is unknown. However, it has been suggested that stimulation of a particular effector cell by parasite-derived material

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might allow the parasite to avert potent host cellular effector mechanisms (Milbourne and Howell 1990). Several criteria must be met before a particular immune effector pathway can be postulated to play a pivotal role in determining the resistance of rats to F. hepatica infections. One of the obvious criteria is demonstration that the parasite is killed by the end product of the putative immune effector pathway. However, one of the problems in defining the components of effective immunity in the F. hepatica–rat model has been that, until our recent studies, no effector mechanism capable of killing the parasite had been identified. This was in direct contrast to studies involving the equivalent developmental stage of a related parasite, schistosomula of Schistosoma mansoni. Many different effector mechanisms that kill schistosomula have been identified, including complement, antibodies, reactive species of oxygen and nitrogen, and various cytotoxic proteins (Pearce and MacDonald 2002). In the case of S. mansoni, the problem has been determining which mechanism(s) are biologically relevant in the host rather than identifying a cytotoxic mechanism capable of killing Schistosoma spp. larvae. The preceding literature suggested that the peritoneal cavity of rats was important in killing recently excysted juvenile (REJ) F. hepatica within 24–48 h of infection. REJ liver flukes are killed when injected intraperitoneally into resistant rats, suggesting that all the constituents necessary to kill the migrating parasite are present in the peritoneal cavity (Davies and Goose 1981). It was noted that prior to their destruction within the peritoneal cavity, REJ F. hepatica that penetrated the gut wall of resistant rats were coated with antibodies and host cells including eosinophils, neutrophils, macrophages, and mast cells (Davies and Goose 1981; Burden et al. 1983; Hughes 1987; Van Milligen et al. 1999). This suggested that parasite-specific antibodies and immune cells were important in mediating this killing mechanism. Studies in vitro, however, were unable to demonstrate any killing of REJ liver flukes by sera and eosinophils or neutrophils (Doy et al. 1980; Duffus and Franks 1980; Doy and Hughes 1982a; Glauert et al. 1985), suggesting that this antibody-dependent cellular mechanism might not be relevant in vivo. However, it was also noted that intraperitoneal passive transfer of sera from F. hepatica-infected sheep, cattle, or rats could protect naive rats from F. hepatica infections. Resident peritoneal cells of rodents consist mainly of monocytes and (or) macrophages (MMs) and the antibodydependent cytotoxicity of these cells had not been specifically studied in resistant rats, suggesting that a common effector pathway dependent on MMs and parasite-specific antibodies could be operating in both naive and resistant rats. Other studies have demonstrated that MMs are one of the major effector cell types involved in the killing of parasites by the production of free radicals (reviewed in Piedrafita and Liew 1998), so we decided to look at free radicals produced by MMs as immune mediators correlated with resistance in rats. 4.1.2. Free radical-mediated killing of REJ F. hepatica in vitro Two major classes of free radicals produced by immune cells are reactive oxygen species (ROS) and reactive nitrogen species (RNS). We initially demonstrated that REJ © 2004 NRC Canada

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F. hepatica and F. gigantica stimulated production of both classes of free radicals by MMs and then tested whether these parasites were susceptible to killing by the free radicals. Cell-free, chemically generated ROS and RNS were shown to mediate killing of REJ liver flukes (Piedrafita et al. 2000, 2001). However, the levels of ROS required to kill REJ F. hepatica were very high, and direct comparisons with a closely related juvenile trematode, schistosomula of S. mansoni, confirmed that REJ F. hepatica are relatively resistant to killing by ROS (Piedrafita et al. 2000). This suggested that ROS may not actually be effective at killing REJ F. hepatica in vivo. This was confirmed when MMs from rats were shown to be unable to mediate killing of REJ F. hepatica in vitro by production of ROS (Piedrafita et al. 2001). We therefore investigated a role for RNS in killing and demonstrated a mechanism of host cell killing of REJ F. hepatica that was dependent on RNS production by rat peritoneal cells but only in the presence of sera from F. hepatica-infected animals, i.e., ADCC (Piedrafita et al. 2001). Evidence that cytotoxicity was dependent on the production of RNS and not ROS was threefold. Firstly, the level of killing of REJ liver flukes was directly proportional to the level of one of the stable end products of nitric oxide catabolism, nitrite, in culture supernatants. Secondly, the killing of REJ liver flukes was inhibited by the addition of the nitricoxide synthase (EC 1.14.13.39) inhibitor NG-monomethyl-Larginine, as seen with killing of schistosomula of S. mansoni (James and Glaven 1989). Finally, the killing of REJ F. hepatica was not inhibited by the addition of superoxide dismutase (EC 1.15.1.1), which prevents the formation of a reactive oxygen species, superoxide anion radicals (O•2− ) (Piedrafita et al. 2001). These studies established ADCC mediated by RNS and peritoneal MMs as a key effector mechanism acting against juvenile F. hepatica in vitro. These studies identified an effector mechanism that was fully consistent with earlier observations of the resistance mechanisms expressed by rats against F. hepatica. The protection of naive rats from F. hepatica infection by intraperitoneal injection of sera from F. hepatica-infected sheep, cattle, or rats suggested a killing mechanism at that site dependent on the presence of parasite-specific antibodies (Armour and Dargie 1974; Hayes et al. 1974a, 1974b; Rajasekariah and Howell 1979; Mitchell et al. 1981; Boyce et al. 1987). The protective response in these studies was evident only when challenge infection occurred on the day of transfer, with killing of REJ liver flukes occurring within 48 h, prior to entry into the liver (Armour and Dargie 1974; Hayes et al. 1974a, 1974b; Rajasekariah and Howell 1979); therefore, it is likely that the response involved resident peritoneal cells. We observed killing of REJ liver flukes that was not dependent on homologous sera from F. hepaticainfected animals and was mediated by RNS produced by resident peritoneal cells of naive rats. We believe that the production of RNS by resident peritoneal cells of naive rats is likely to be the mechanism of killing of REJ liver flukes in these earlier studies. An extension of these studies would then be to ask whether this antibody- and RNS-dependent mechanism operates in naturally resistant rats following a primary infection with F. hepatica. The various studies cited above, in which

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REJ F. hepatica were incubated with peritoneal cells isolated from resistant (i.e., previously exposed) rats and with antibodies, have not demonstrated killing of REJ liver flukes in vitro (Doy et al. 1980; Doy and Hughes 1982b). This at first appears to be inconsistent with our observations that REJ F. hepatica stimulate high levels of RNS production by peritoneal cells isolated from naive rats and that RNSmediated killing of REJ liver flukes occurs in vitro in the presence of immune sera (Piedrafita et al. 2001). This apparent discrepancy could be an experimental artefact due to the fact that the previous studies incubated peritoneal cells from Fasciola-resistant rats with REJ F. hepatica for only short periods (4 h). The REJ liver flukes were washed, cells were removed, and REJ liver flukes were incubated with freshly harvested peritoneal cells and sera for a further 4 h, and so on. The authors proposed that this experimental approach would preclude depletion of any putative effector mechanism of the harvested immune cells, comparable to the in vivo situation in the host where effector cells are continuously recruited. However, a 4-h incubation would have been insufficient time for cells to synthesize enough RNS to elicit killing of REJ F. hepatica in these earlier experiments, since maximal RNS production by rat MMs occurs 24–48 h after parasite stimulation and almost no RNS is induced within a 4-h incubation (Piedrafita et al. 2001). We are currently assessing whether this is the principal reason for the lack of direct cytotoxicity to REJ F. hepatica in these earlier studies. In contrast, preliminary experiments in our laboratory have shown that REJ F. gigantica are susceptible to RNS produced by rat peritoneal cells, independent of the presence of parasite-specific sera. REJ F. gigantica are more susceptible to killing by ROS and (or) RNS than REJ F. hepatica using peritoneal cells harvested from uninfected rats (D. Piedrafita et al., unpublished data). This susceptibility in the absence of antibodies specific to Fasciola spp. suggests that the low F. gigantica infection rates observed in rats result from a greater susceptibility of REJ F. gigantica to innate cell-mediated mechanisms in this host. The biological significance of these observations for the host–F. gigantica relationship in the rat model is unknown owing to the few immunological studies carried out with this parasite. 4.2. Putative effector mechanisms of ruminants identified ex vivo and (or) in vitro To date, no published results have identified putative effector mechanisms against F. gigantica in ruminants, and very few studies with F. hepatica have been carried out. Bovine complement does not damage juvenile flukes in vitro, and no damage to recently excysted F. hepatica was observed following incubation in vitro with excess antibodies and bovine neutrophils or eosinophils (Duffus and Franks 1980). However, incubation with 1 µM purified eosinophil major basic protein caused both damage to and death of recently excysted flukes in vitro (Duffus et al. 1980). Glauert et al. (1985), on the other hand, demonstrated that bovine eosinophils in the presence of immune sera bound to REJ F. hepatica and then degranulated. Despite degranulating, these eosinophils were unable to kill the parasite in vitro, and the authors suggested that the presence of a protective layer of antigen–antibody complexes on the parasite surface © 2004 NRC Canada

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Table 2. Effector mechanisms of rats and Indonesian thin tail (ITT) sheep mediating toxicity to juvenile F. hepatica and F. gigantica in vitro. Rat Effector mechanism Macrophages Eosinophils Nitric oxide Superoxide anion radical Hydrogen peroxide Antibodies

F. hepatica Mediate killing Ineffective Produced and effective Produced but ineffective Produced but ineffective Required for killing

Surface contact Complement

Required for killing Ineffective

ITT sheep F. gigantica Mediate killing Not tested Produced and effective Produced and effective Not tested Not required for RNS killing; required for ROS killing Not tested Not tested

F. hepatica Ineffective Ineffective Not produced Produced but ineffective Not tested Required for cell attachment but ineffective

F. gigantica Mediate killing Mediate killing Not produced Produced and effective Not tested Required for killing

Ineffective Not tested

Required for killing Not tested

Note: RNS, reactive nitrogen species; ROS, reactive oxygen species.

was preventing close attachment of the eosinophils to the juvenile parasite. Thus, the potential role of ruminant eosinophils in mediating F. hepatica killing is equivocal. Our studies to date in Indonesia have demonstrated that the target stage of effective immunity in ITT sheep is the early migrating immature F. gigantica and that the peritoneum is likely to be an important site of parasite attrition (Roberts et al. 1997a, 1997c); in contrast, we have found no effective immunity in the ITT host to repeated F. hepatica infections (Roberts et al. 1997a; Spithill et al. 1999a; section 3.2.1). This observation leads to the prediction that the effector mechanisms expressed in ITT sheep, which are capable of killing immature F. gigantica, should be less effective in mediating killing of immature F. hepatica. We have recently demonstrated that peritoneal cells from F. gigantica-infected ITT sheep could indeed effectively kill immature F. gigantica in the presence of sera from F. gigantica-infected ITT sheep (D. Piedrafita et al., unpublished data). Next, we investigated the potential effector molecules that might be mediating this cytotoxic mechanism. By using a series of inhibitors, we were able to determine that the primary cytotoxic molecules produced by these cells were O•2− radicals (Table 2; D. Piedrafita et al., unpublished data). The important observation that ITT sheep are not resistant to F. hepatica infection (Roberts et al. 1997a) suggested that this effector mechanism expressed by ITT sheep should be ineffective in mediating killing of immature F. hepatica. This was indeed the case, and we recently demonstrated that REJ F. hepatica were not susceptible to killing by the superoxide-dependent cytotoxicity of ITT peritoneal cells (Table 2; D. Piedrafita et al., unpublished data). This is consistent with previous observations that rat peritoneal cells produced ROS that was unable to mediate significant killing of F. hepatica in vitro (Piedrafita et al. 2000, 2001).

5. Differences in the host–parasite relationship determining the differential susceptibility of rats and sheep to Fasciola spp. infection The relative degree of killing of Fasciola spp. in vitro by cells from sheep or rats (F. gigantica >> F. hepatica) corre-

lates with the higher resistance of sheep and rats to F. gigantica infection in vivo. In light of our findings, there are a number of possible explanations as to why the two parasites differ in their capacity to infect various hosts. These could include (i) host-dependent factors such as differences in the magnitude, timing, or expression of effector mechanisms between hosts (see section 5.1) and (ii) parasite-dependent factors such as an immune response that is ineffective against F. hepatica because of some defence mechanism operating in this species (see sections 5.2.1 and 5.2.2) or an immune response that is suppressed by F. hepatica as a result of some factor(s) released by the parasite during infection (see section 5.2.3). 5.1. Host-dependent factors As discussed above, Fasciola parasites have varying degrees of success at establishing within different hosts and this success varies between parasite species. This observation suggests that differences between host immune responses may play a role in parasite survival. Many different host-dependent factors play a critical role in either the resolution or progression of infection by parasites, and host free radical production is a critical component of this outcome in many of these parasitic diseases (reviewed in Piedrafita and Liew 1998). Our laboratories have become interested in the role of nonspecific defence mechanisms, including free radicals, as current evidence suggests that they may be important in Fasciola spp. infections of various hosts (Doy et al. 1981b; Oldham and Hughes 1982; Oldham 1983; Ford et al. 1987; Hughes 1987; Smith et al. 1992; Baeza et al. 1994a, 1994b). However, few studies of Fasciola spp. infections have looked at the critical importance of the protective or pathological role that hostgenerated free radicals play in relation to susceptibility to infection expressed by different hosts. Smith et al. (1992) demonstrated that free radical production in response to crude adult antigens of F. hepatica in challenged rats was greater than that in naive rats and rats with primary infections and was 30 times higher than that of a susceptible mouse host. It has also been suggested that several mammalian hosts potentially recognize protective F. hepatica antigens but that only rats possess the necessary cellular effector mechanisms for killing of juvenile F. hepatica (Sandeman © 2004 NRC Canada

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and Howell 1982). Accordingly, we have shown that lung lavage cells from susceptible Merino sheep were unable to kill REJ F. hepatica and that this was due to the lack of RNS generation by these cells; conversely, lavage cells from a resistant rat were able to generate RNS and kill REJ F. hepatica in vitro (Piedrafita et al. 2001). These results are supported by studies with REJ F. gigantica in which both resistant ITT sheep and resistant rats killed the parasite by means of ROS produced by lavage cells. To date, these limited studies suggest that the ability of Fasciola spp. to infect a host or be killed may be, in part, host-dependent and may involve differences in the ability to express certain effector mechanisms, the ability of different hosts to rapidly initiate the effector response, and (or) the magnitude of this effector mechanism in hosts. Further studies are required to confirm these interesting observations. 5.2. Parasite-dependent factors 5.2.1. Production of antioxidant defence enzymes Several studies have provided direct and indirect evidence of a role for antioxidant defence (AOD) enzymes in protecting Fasciola spp. against attack by free radicals, both ROS and RNS (Baeza et al. 1993; Jefferies et al. 1997; Cervi et al. 1998, 1999; El Ghaysh et al. 1999; Piedrafita et al. 2000, 2001). Piedrafita et al. (2000) compared the cytotoxicity of chemically generated ROS and RNS to S. mansoni schistosomulae and REJ F. hepatica in vitro. Schistosoma mansoni schistosomulae were shown to be more susceptible than F. hepatica juveniles to killing by free radicals. Furthermore, REJ F. hepatica expressed AOD enzyme levels up to 10 times higher than those of the schistosomula, suggesting that these enzyme levels were linked to the relative resistance of F. hepatica to free radical attack. Jefferies et al. (1997) showed that increasing concentrations of excretory/secretory products (ESPs) released by F. hepatica in vitro correlated with increasing suppression of O•2− and H2O2 production by sheep neutrophils. In other work from this group (El Ghaysh et al. 1999), F. gigantica ESPs were also shown to inhibit ROS production by sheep neutrophils. The authors proposed that both observations were linked to superoxide dismutase (SOD); however, both reports measured little or no SOD among the respective Fasciola spp. ESPs. The majority of F. hepatica AOD enzyme work has focussed on F. hepatica GSTs (FhGSTs). FhGSTs exist as a mix of homo- and hetero-dimers with subunit sizes ranging from 24–29 kDa (Howell et al. 1988; Hillyer et al. 1992; Wijffels et al. 1992). FhGSTs are expressed in a range of tissues including the parenchyma, gut, and tegument (Howell et al. 1988; Wijffels et al. 1992; Creaney et al. 1995). FhGSTs exist as at least six isoenzymes, as detected by agarose–starch gel electrophoresis (Howell et al. 1988), chromatofocussing (Brophy et al. 1990), two-dimensional gel electrophoresis (Wijffels et al. 1992; Jefferies et al. 2001), and cDNA cloning (Panaccio et al. 1992; Muro et al. 1993). The level of GST expression is lower in REJ liver flukes than in immature and adult F. hepatica (Piedrafita et al. 2000). Interestingly, Miller et al. (1993) showed variations in isoenzyme expression and activity of FhGSTs in adult flukes recovered from different hosts. Lower FhGST activities were observed in flukes removed from resistant

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hosts (cattle and rats) as opposed to susceptible hosts (sheep and mice). FhGSTs are proposed to play three roles. Firstly, FhGSTs are involved in detoxifying cytotoxic aldehydes produced during lipid peroxidation (Brophy et al. 1990); secondly, FhGSTs are involved in the absorptive function of the adult parasite’s gut (Creaney et al. 1995); and thirdly, FhGSTs interact with haematin and this is proposed to prevent blockage of the parasite’s gut by haematin crystal formation (Brophy et al. 1990). Noteworthy is the lack of cellular responses against FhGSTs during F. hepatica infections in sheep (Moreau et al. 1998a), as opposed to infections in rats (Howell et al. 1988). There are conflicting reports of antibody responses to FhGSTs in sheep (Sexton et al. 1990; Hillyer et al. 1992), and not all hosts generate anti-FhGST antibodies during fluke infections (Hillyer et al. 1992). This is unexpected, as GST is a major constituent of F. hepatica ESPs (Cervi et al. 1999; Jefferies et al. 2001). However, when sheep are injected with GST as a vaccine, high antibody titres are observed along with a reduction in worm burdens (Sexton et al. 1990). SOD is another AOD enzyme expressed by F. hepatica (Sanchez-Moreno et al. 1987; Piedrafita et al. 2000). Three isoenzymes have been observed (Sanchez-Moreno et al. 1987), and Kim et al. (2000) identified two identical cytosolic Cu/Zn SOD subunits of 17.5 kDa. SOD activity has been detected in ESPs, detergent-soluble fractions, and somatic fractions of F. hepatica. The highest activities were observed in F. hepatica ESPs, where two or three bands of Cu/Zn SOD with sizes of 16 and 60 kDa were measured (Piacenza et al. 1998; Jefferies et al. 2001). In contrast to other parasites such as Brugia malayi (Ou et al. 1995) and S. mansoni (Nare et al. 1990), F. hepatica SOD activity in ESPs decreases as the parasite develops to adulthood (Piacenza et al. 1998) but increases in somatic tissues (Piedrafita et al. 2000). The authors proposed that this may relate to the parasite moving from an aerobic to a relatively anaerobic environment and becoming less exposed to toxic oxygen intermediates. Cu/Zn SOD invokes a cellular immune response within bovine and human hosts (Kim et al. 2000). The main function of SOD is to catalyse the spontaneous dismutation of O•2− radicals to H2O2 and molecular oxygen (Piacenza et al. 1998). However, H2O2 is still toxic to parasites; hence, other AOD enzymes are needed to break this down into less harmful products. Typically, catalase (EC 1.11.1.6) and glutathione peroxidase (GSH-Px) (EC 1.11.1.9) are involved in this second stage. Catalase activity has not been detected in F. hepatica (Sanchez-Moreno et al. 1987; Piedrafita et al. 2000) and only low levels of a selenium-dependent GSH-Px have been detected in the cytosol of F. hepatica (Brophy et al. 1990). McGonigle et al. (1997) identified a cDNA from F. hepatica that encoded a protein from the most recently identified antioxidant family Peroxiredoxin (Chae et al. 1994). Peroxiredoxins remove toxic H2O2 and could be the “missing link” for F. hepatica. This was the first report of a peroxiredoxin in a trematode. The cDNA was isolated using antisera raised against adult F. hepatica ESPs to screen a cDNA expression library, suggesting that the protein is likely to be released and may be involved in the parasite’s defence mechanism. © 2004 NRC Canada

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In F. gigantica, GST (Estuningsih et al. 1997; Paykari et al. 2002) and SOD (El Ghaysh et al. 1999) activity have been described in adult flukes. Adult F. gigantica released two products (less than 10 kDa and greater than 50 kDa) that were able to suppress the release of toxic oxygen intermediates by neutrophils (El Ghaysh et al. 1999). This result implies the presence of factors, possible AOD enzymes, in F. gigantica effective against reactive oxygen intermediates. However, in our laboratory, REJ F. gigantica have been found to be more susceptible to killing by ROS and RNS than REJ F. hepatica (sections 4.1 and 4.2; D. Piedrafita et al., unpublished data). This could imply differences in the levels of AOD enzymes between the two species, which could result in different susceptibilities to free radicals. Preliminary results in our laboratory suggest that the activities of several AOD enzymes are lower in F. gigantica than in F. hepatica (R. Prowse et al., unpublished data), but these results await confirmation. Overall, the literature indicates that there is variation in AOD enzymatic activities between F. hepatica isolates and among flukes removed from different hosts (e.g., Miller et al. 1993). To date, there have been no published comparisons of AOD enzyme activities of different Fasciola spp. isolated from the same host. 5.2.2. Glycocalyx turnover The surface of F. hepatica is covered by a syncytial epithelium called the tegument. The tegument contains three types of bodies (T0, type 0; T1, type 1; T2, type 2) that secrete the glycocalyx (Threadgold 1963, 1967; Bennett and Threadgold 1973, 1975). In contrast, preliminary studies of adult F. gigantica suggest that only one type of body secretes the glycocalyx (Sobhon et al. 1994). The glycocalyx consists of two layers: a continuous layer lying next to the apical plasma membrane, and another layer of filaments arising from the continuous layer (Threadgold 1976). The glycocalyx is rich in glycoproteins with ganglioside and oligosaccharide side chains (Threadgold 1976). The glycocalyx, as the interface between the parasite and the host, is likely to be the site of important biochemical and physiological interactions (Threadgold 1976). Possibly because of this, it has been proposed that the glycocalyx contributes to immune evasion, and three mechanisms have been suggested. Firstly, the composition of the glycocalyx changes as the parasite matures (Threadgold 1963, 1967; Bennett and Threadgold 1973, 1975). Juvenile flukes contain only T0 bodies within the tegument secreting T-0 granules (Bennett and Threadgold 1973, 1975). As F. hepatica matures, T0 bodies are replaced by T1 and T2 bodies secreting T-1 and T-2 granules, respectively (Threadgold 1963, 1967). Such compositional changes are likely to delay relevant production of antibodies specific for the constantly changing glycocalyx. Secondly, the glycocalyx is continuously sloughed off and replaced by molecules released by secretory vesicles (Hanna 1978). Sloughing of the glycocalyx results in shedding of components of the immune system that attach themselves to the parasite (e.g., antibody-mediated attached eosinophils, neutrophils, or macrophages) (Hanna 1980a; Burden et al. 1982; Piedrafita et al. 2001), resulting in prevention of successful cellular attack (Duffus and Franks 1980; Hanna 1980b).

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Although the previously discussed mechanisms may contribute to the survival of F. hepatica in susceptible hosts such as sheep, they do not protect the parasite in resistant hosts such as the rat. Some of these parasite survival mechanisms operating in sheep may not be as effective in “unnatural” definitive hosts such as rats (Hanna 1980a; Hughes 1987). For example, Rickard and Howell (1982) have suggested that the kinetics of glycocalyx turnover may vary between rats and sheep and thus render glycocalyx shedding an ineffective mechanism for parasite survival in rodents. Whether the apparent lack of different tegument-secreting granules identified to date in F. gigantica juveniles, when compared with F. hepatica juveniles, plays a role in the greater susceptibility of F. gigantica to killing by different hosts is unknown. However, data from rat studies showing protective responses induced following vaccination with parasite antigens or exposure to a primary parasite infection suggest that the juvenile stage is most susceptible to host killing and is the target of effective immunity (Yoshihara et al. 1985; Van Milligen et al. 2000). Thus, if the ability of F. hepatica juveniles to survive in different mammalian hosts is dependent on the ability of the migrating parasite to alter the composition of the glycocalyx during development and thus thwart an effective immune response by the host, then the apparent lack of this antigenic variation in F. gigantica juveniles could be crucial. 5.2.3. Release of excretory/secretory products 5.2.3.1. Effect of Fasciola spp. infection on immune responses Fasciola hepatica infections, like other parasitic infections discussed previously, result in modulation of lymphocyte proliferative responses. A short-lived, early increase in mitogen-stimulated lymphocyte proliferation is commonly observed during F. hepatica infections in cattle and rats (Oldham 1985; Oldham and Williams 1985; Poitou et al. 1992; Clery and Mulcahy 1998). Poitou et al. (1992) found significant increases in proliferation of rat splenocytes between 2 and 4 weeks post infection (WPI) in the presence of concanavalin A (ConA), pokeweed mitogen (PWM), or antigens of F. hepatica adults or metacercariae. The stimulation by these mitogens suggests that a range of lymphocyte populations is induced, and the results with parasite antigens suggest that the response is antigen specific. This work is partly supported by reports by Cervi et al. (1998), who observed a sustained increase in lipopolysaccharide-stimulated proliferation of splenocytes removed from F. hepatica-infected rats, compared with uninfected control rat cells. In contrast, this paper and two other papers from the same group (Cervi et al. 1996, 1998; Cervi and Masih 1997) reported a decrease in proliferation of splenocytes from F. hepatica-infected rats when these cells were stimulated with ConA or F. hepatica antigens. This suppression was linked to RNS and (or) H2O2 production, since the addition of either aminoguanidine (a nitric oxide synthase inhibitor) or catalase (which decomposes H2O2 and inhibits RNS production) restored proliferation. Potential reasons for the variation in the response of lymphocytes to mitogen stimulation were not alluded to. What is apparent is that F. hepatica infections modulate lymphocyte proliferation within the rat host. Overall, modulation of mitogen-stimulated proliferation of lymphocytes © 2004 NRC Canada

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from cattle and rats was observed early after F. hepatica infection, and these responses do not correlate with host resistance. Fasciola hepatica infections in sheep cause significant suppression of mitogen-stimulated lymphocyte proliferation in vitro (Zimmerman et al. 1983; Chauvin et al. 1995). Zimmerman et al. (1983) observed significant suppression of proliferation during F. hepatica infections with either ConA or phytohemagglutinin at 4 and 8–10 WPI. A similar trend in the suppression profile was observed with PWM, suggesting that the suppressive phenomenon is likely to be occurring with all lymphocyte populations (e.g., T and B cells). This suggestion was supported by Chauvin et al. (1995), who revealed a similar significant suppression of sheep lymphocyte proliferation at 4 and 11 WPI using ConA as the mitogen. Furthermore, the proliferative capacity of lymphocytes after secondary infection was examined in this study. There was a significant increase in proliferation 1–2 weeks after secondary infection, followed by a return of proliferation levels to those observed in sheep with a primary infection. In conclusion, the significance of an effect on lymphocyte proliferation at 4 and 8–10 WPI is that these times correspond to growth phases and migration of F. hepatica within the liver parenchyma (Zimmerman et al. 1983). This suppressive response may aid the parasite’s establishment within the host. Sheep, unlike cattle and rats, have not been shown to acquire resistance to F. hepatica. The significance of increased cellular proliferation after a secondary infection with F. hepatica has yet to be determined. The effects of an F. gigantica infection on sheep lymphocyte proliferation in the presence of mitogens have to date not been studied. In F. hepatica-infected sheep, IFN-γ was produced by ESP-stimulated peripheral blood mononuclear cells and hepatic lymph node mononuclear cells at 7–14 days post infection. Interleukin 10 activity was detected within the first 6 weeks of infection (Moreau et al. 1998b). Studies examining cytokine profiles in rats early after F. hepatica infection showed that inflammatory responses in the livers of infected animals are transiently depressed or delayed. A type 0 cytokine profile was initially observed in the liver and hepatic lymph nodes, followed by a type 2 profile 2 weeks after infection in the liver. In contrast, cytokine downregulation was sustained in the spleen throughout this period, suggesting that the effects of parasite infection differ between local and systemic sites (Tliba et al. 2002a, 2000b). A type 0 cytokine profile was also observed by Cervi et al. (2001) early after F. hepatica infection in rats. As discussed above, down-regulation of type 1 responses in cattle during chronic F. hepatica infection was described earlier by Brown et al. (1994). Brady et al. (1999) showed that an F. hepatica infection suppressed a type 1 response to Bordetella pertussis in concurrently infected mice, delayed clearance of B. pertussis from the lungs of mice, and suppressed the IFNγ response by splenocytes in mice vaccinated with a B. pertussis vaccine. Fasciola hepatica infection was shown to down-regulate type 1 responses in mice (O’Neill et al. 2000). 5.2.3.2. Effect of Fasciola spp. ESPs Our laboratory is interested in understanding how infections with Fasciola spp. mediate this apparent host immune

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response. One of the most obvious interactions between the invading Fasciola parasite and the host is via the continuous release of ESPs into the host environment. Several immune evasion strategies have been attributed to F. hepatica and F. gigantica ESPs. Fasciola hepatica ESPs have been shown to modulate accessory cell function, firstly through lowering phagocytic activity and the antigen presentation ability of rat peritoneal cells in vitro (Masih et al. 1996). Secondly, F. hepatica ESPs contain four non-lipid molecules that can cause chemokinesis in sheep neutrophils (Jefferies et al. 1996). Finally, an IL5-like molecule among F. hepatica ESPs stimulates eosinophil maturation, which leads to a rise in eosinophil peroxidase activity in mice (Milbourne and Howell 1993). This has added weight to the suggestion that F. hepatica induces an inappropriate immune response. Fasciola hepatica also suppresses lymphocyte proliferation during infection in rats, and F. hepatica ESPs may play a role in dampening rat immune responses (Cervi et al. 1996, 1998; Cervi and Masih 1997). 12–23 kDa glycoproteins among F. hepatica ESPs were shown to stimulate a rat mononuclear cell population that, upon adoptive transfer, suppressed the delayed type hypersensitivity response to parasite and nonrelated antigens (Cervi et al. 1996). Fasciola hepatica ESPs could also suppress ConA- or lipopolysaccharide-stimulated proliferation of rat spleen mononuclear cells in vitro (Cervi and Masih 1997). In other hosts, increasing concentrations of F. hepatica ESPs resulted in decreasing levels of proliferation of ConA- or PHAstimulated sheep and human lymphocytes (Jefferies et al. 1996). Fasciola hepatica ESPs have also been shown to contribute to the modulation of host free radical levels (Baeza et al. 1993; Jefferies et al. 1997; Cervi et al. 1999). Firstly, F. hepatica ESPs decrease RNS production by activated rat peritoneal lavage cells in vitro (Cervi et al. 1999) and inhibit RNS production by activated sheep neutrophils while stimulating RNS production by activated human neutrophils in vitro (Jefferies et al. 1997). Secondly, F. hepatica decreases O•2− production by activated sheep and human neutrophils (Jefferies et al. 1997). Baeza et al. (1993) similarly observed that F. hepatica infection decreased the metabolic burst of cattle neutrophils. Together, these findings suggest that F. hepatica has evolved strategies to avoid damage from host cell free radical production. Significantly less work has investigated the role of F. gigantica ESPs in immunomodulation. The majority of the research into F. gigantica ESPs has focussed on identifying immunodiagnostic antigens and on the development of monoclonal antibodies specific for these antigens (Yadav and Gupta 1995; Fagbemi et al. 1997; Maleewong et al. 1997, 1999; Viyanant et al. 1997; Intapan et al. 1998; Krailas et al. 1999). One report has shown that F. gigantica ESPs can suppress the respiratory burst of sheep neutrophils, as measured by a decrease in the release of ROS. At least two molecules were responsible for this suppression; both were nonproteinaceous and were either less than 10 kDa or greater than 50 kDa (El Ghaysh et al. 1999). Neutrophils are an important component of the acute inflammatory cascade (Haslett et al. 1989). By dampening the neutrophil’s ability to produce ROS, F. gigantica may evade oxidative damage. © 2004 NRC Canada

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5.2.3.3. Cathepsin proteases and immune modulation Cysteine protease activity (corresponding to cathepsin L (EC 3.4.22.15) and cathepsin B (EC 3.4.22.1) proteins) has been identified in somatic extracts of juvenile flukes (Tkalcevic et al. 1995). Recombinant cDNA sequences encoding both cathepsin L and cathepsin B proteases have been isolated from adult fluke cDNA libraries (Heussler and Dobbelaere 1994; reviewed in Irving et al. 2003; Dalton et al. 2003). Juvenile, immature, and mature Fasciola spp. ESPs contain cysteine proteases (cathepsin L or cathepsin B) (Dalton and Heffernan 1989; Carmona et al. 1993; Smith et al. 1993; Wilson et al. 1998; reviewed in Dalton et al. 2003). Only a cathepsin B-like sequence was detected in ESPs from REJ liver flukes (Wilson et al. 1998), but ESPs from immature flukes exhibit at least two cathepsin L proteases, while adult ESPs have at least seven isoenzymes (Wijffels et al. 1994; Dalton et al. 2003). Cathepsin L proteases are major abundant constituents of adult Fasciola spp. ESPs. The nonredundant BLAST database contains 23 cathepsin L-like cDNA sequences all derived from parasite mRNA; 17 of the sequences encode F. hepatica proteins and 6 encode F. gigantica proteins (Irving et al. 2003). Cathepsin L activity increases as the parasite matures (Carmona et al. 1993), which may suggest an increased reliance on cathepsin L in immune evasion or a general role in fluke development and maturation. Cysteine proteases were identified in F. gigantica whole worm extracts (Fagbemi and Hillyer 1991, 1992) and cDNAs encoding cathepsin L were reported (Grams et al. 2001; Dalton et al. 2003). Fasciola hepatica cathepsin L proteases are purported to play key roles in tissue invasion and immune evasion (reviewed in Mulcahy and Dalton 2001). Cathepsin L proteases can degrade both the extracellular matrix (fibrillar collagen, types I and II) and the basement membrane (type IV collagen) (Berasain et al. 1997). The authors proposed that this may aid Fasciola spp. invasion of the tissues. Cathepsin L proteases also degrade haemoglobulins in vitro, pointing to the possible digestion of host haemoglobin for nutritional purposes (Dalton and Heffernan 1989; Wilson et al. 1998). Cathepsin L proteases have been shown to cleave immunoglobulins from mice and humans (all IgG subclasses) in the hinge region of the heavy chain in vitro (Carmona et al. 1993; Smith et al. 1993; Berasain et al. 2000). The potential importance of this cleavage was demonstrated when the addition of adult F. hepatica ESPs along with Fasciola spp. immune sera prevented the antibody-mediated attachment of eosinophils to REJ liver flukes in vitro (Carmona et al. 1993). This was reversed when the cysteine protease inhibitor leupeptin was added to the incubation. This is an important potential evasion mechanism, considering in vitro work that showed the high toxicity of the major basic protein (at micromolar concentrations) released by bovine eosinophils to REJ F. hepatica (Duffus et al. 1980). However, there is no direct evidence that eosinophils actually kill REJ F. hepatica. It should be noted that in this work by Carmona et al. (1993) there was no cytotoxicity mechanism shown in the absence of cathepsin L and the ESPs used were from adult flukes; since the antibody-mediated effector responses against Fasciola spp. appear to be directed against juvenile flukes, a direct role of adult ESPs in perturbing an effector

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response against juvenile parasites is unclear given that these parasites occupy different niches during infection. Such a mechanism is relevant if cathepsin L proteases from immature parasites have properties similar to those of adult cathepsin L proteases. Cysteine proteases isolated from F. gigantica worm extracts have been shown to digest both bovine IgG and bovine globin (derived from bovine haemoglobin) in vitro (Fagbemi and Hillyer 1991, 1992). These findings imply that F. gigantica may evoke evasion strategies similar to those evoked by F. hepatica. Recent work has provided direct evidence for a role for cathepsin L proteases in immunomodulation of the host during F. hepatica infection. Down-regulation of type 1 responses in F. hepatica-infected mice was shown to be mediated by cathepsin L proteases present in ESPs (O’Neill et al. 2001). Injection of mice with purified cathepsin L suppressed the type 1 response to the B. pertussis vaccine and to keyhole limpet hemocyanin. These results show that fluke cathepsin L proteases exert a profound effect on the ability of mice to mount type 1-like responses, implying that the proteases have a direct effect on the pathways determining type 1 responses. Whether these effects also operate in ruminants during fluke infection awaits determination. Other work from our laboratory has also demonstrated a role for cathepsin L in immunomodulation of host T cell responses (Prowse et al. 2002). Cathepsin L was shown to be a major component of ESPs that suppressed T cell proliferation in sheep in vitro. Analysis of the effect of ESPs and recombinant F. hepatica cathepsin L on the expression of 22 different sheep T cell surface markers by flow cytometry showed that ESPs or two recombinant cathepsin L proteases significantly reduced surface CD4 expression. A similar effect on CD4 of human T lymphocytes was also observed. These results show that F. hepatica cathepsin L has a direct effect on at least one major component of the T cell response (CD4), at least in vitro, and that this effect could be involved in the immunomodulation of sheep T cell proliferation. Similar effects were also observed with cathepsin L from F. gigantica (R. Prowse et al., unpublished data). However, further in vivo studies are required to determine whether such effects occur during the course of a fluke infection. The studies by Brady et al. (1999), O’Neill et al. (2000, 2001), and Prowse et al. (2002) provide strong evidence for the notions that F. hepatica actively modulates the host immune response and that cathepsin L proteases released by the parasite play a key role in this immunomodulatory activity.

6. Conclusions Recent studies, briefly summarized here, have begun to elucidate the various immunological mechanisms invoked by the host against Fasciola spp. that lead to a protective resolution of this disease (Table 2). We are only now beginning to understand the various molecular immune mechanisms that result in the actual death of Fasciola parasites in various mammalian hosts. The finding that ITT sheep effectively kill F. gigantica by an immunological mechanism that is apparently completely ineffective against repeated F. hepatica infections indicates © 2004 NRC Canada

Piedrafita et al.

that resistance to Fasciola spp. infection is determined, in part, by biochemical differences between species of Fasciola (Table 2). This review has alluded to at least two potentially important areas of current research (defence enzyme expression and immunosuppressive factors) that may explain these inherent differences between parasite species. A recent phylogenetic survey of cathepsin L and GST sequences in the genus Fasciola has shown that F. hepatica and F. gigantica diverged about 19 ± 2.8 million years ago, providing sufficient time for variations in gene expression between the species to become fixed (Irving et al. 2003). It is of interest that rats can effectively control infections by both Fasciola species (Tables 1 and 2). Thus, biochemical differences between F. hepatica and F. gigantica (which, we propose, lead to the preferential survival of F. hepatica against immunological attack by ITT sheep) clearly do not affect the susceptibility of these species to the rat immune response. This suggests that host-dependent factors play a pivotal role in disease progression or resolution in fasciolosis. In this review, we have suggested that the actual ability of the host to control Fasciola spp. infections involves the induction of appropriate effector molecules (such as nitric oxide) leading to death of the parasite. Whether this inability of the susceptible host to induce an effective immune response against Fasciola spp. and thus control disease progression is inherent to the host or is mediated by a factor released by the parasite (or more likely both) is currently unknown. However, the underlying molecular pathways controlling protective immunity or chronic disease are ultimately determined by differences in gene expression, either between the two parasite species (e.g., when F. hepatica and F. gigantica infect the same ITT sheep host) or between the two hosts (e.g., F. hepatica infection of susceptible ITT sheep compared with the resistant rat host). There has been a recent advent of technologies to quickly identify these inherent differences in gene expression between host and parasite species, including microarray analysis, proteomics, and representational difference analysis. Application of these new technologies promises to further advance our understanding of the molecular pathways leading to protection against Fasciola spp. infections and will ultimately lead to the identification of new targets for vaccine or drug control of fasciolosis. The evidence presented here also shows that F. hepatica and F. gigantica actively modulate the host immune response. It will be of interest to determine whether such modulation of the immune response by Fasciola spp. directly leads to enhanced parasite survival in the various hosts. If F. hepatica and F. gigantica differ in their effectiveness at modulating immune responses in different hosts, this may lead to host-dependent parasite survival and may explain host specificity. It is curious that the estimated time for divergence of the two Fasciola species (19 ± 2.8 million years ago) coincides with a period of radiation for the Pecoran lineages (including ancestors of species such as cattle and sheep) predicted to have occurred 16–28 million years ago (Allard et al. 1992; Irving et al. 2003). This may reflect cospeciation of the Fasciola genus as the host lineages diverged (Hafner and Nadler 1988). Examination of this curious question is a clear focus for future studies.

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7. Acknowledgements Our research is supported by the Australian Centre for International Agricultural Research (ACIAR), Canberra, Australia; Monash University; University of Sydney; Cooperative Research Center for Vaccine Technology, Brisbane, Australia; McGill University; the McGill Institute of Parasitology; Fonds Québécois de la recherche sur la nature and les technologies; the Natural Sciences and Engineering Research Council of Canada; and the Canada Research Chair program. R. Prowse is a recipient of an Australian Postgraduate Award scholarship and a scholarship from the Cooperative Research Center for Vaccine Technology. T. Spithill holds a Canada Research Chair in Immunoparasitology.

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