Veterinary Parasitology 101 (2001) 291310

Advances and prospects for subunit vaccines against protozoa of veterinary importance
Mark C. Jenkins
Immunology and Disease Resistance Laboratory, Agricultural Research Service, US Department of Agriculture (USDA), Beltsville, MA 20705, USA

Abstract Protozoa are responsible for considerable morbidity and mortality in domestic and companion animals. Preventing infection may involve deliberate exposure to virulent or attenuated parasites so that immunity to natural infection is established early in life. This is the basis for vaccines against theilerosis and avian coccidiosis. Vaccination may not be effective or practical with diseases, such as cryptosporidiosis, that primarily afict the immune-compromised or individuals with an incompletely developed immune system. Strategies for combating these diseases often rely on passive immunotherapy using serum or colostrums containing antibodies to parasite surface proteins. Subunit vaccines offer an attractive alternative to virulent or attenuated parasites for several reasons. These include the use of bacteria or lower eukaryotes to produce recombinant proteins in batch culture, the relative stability of recombinant proteins compared to live parasites, and the exibility to incorporate only those antigens that elicit protective immune responses. Although subunit vaccines offer many theoretical advantages, our lack of understanding of immune mechanisms to primary and secondary infection and the capacity of many protozoa to evade host immunity remain obstacles to developing effective vaccines. This review examines the progress made on developing recombinant proteins of Eimeria, Giardia, Cryptosporidium, Toxoplasma, Neospora, Trypanosoma, Babesia, and Theileria and attempts to use these antigens for vaccinating animals against the associated diseases. Published by Elsevier Science B.V.
Keywords: Vaccine; Recombinant antigen; Eimeria; Giardia; Cryptosporidium; Toxoplasma; Neospora; Trypanosoma; Babesia; Theileria

1. Introduction Veterinary parasitic diseases cause enormous annual economic losses to livestock industries worldwide (for review, see Willetts, 1994). Although many of these diseases are
Tel.: +1-301-504-8054; fax: +1-301-504-5306. E-mail address: mjenkins@anri.barc.usda.gov (M.C. Jenkins). 0304-4017/01/$ see front matter. Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 5 7 - X

292

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

drug-treatable, the capacity of protozoan parasites to rapidly develop drug-resistance has prompted the search for alternative means of prevention. The cost of drug treatment can also be prohibitive, especially in developing countries where many of the diseases are commonly found. The nearly complete immunity that is elicited during a primary infection with many protozoa has been the basis for vaccine development. In fact, inoculation of susceptible animals with either virulent or attenuated parasites has been a successful approach to vaccination against several protozoan diseases. There are, however, several drawbacks to using live parasites, not least of which are the need for cold storage, the limited shelf-life of the vaccine, the possibility of causing morbidity and mortality in vaccinates, and the risk of attenuated organisms reverting to a more pathogenic state. Subunit vaccines, derived from native antigens of the parasite or as recombinant proteins from cloned DNA, may overcome these difculties. Rapid advances in our understanding of mechanisms of immunity to many protozoa and the development of molecular tools for generating vaccines comprised of recombinant antigens or cloned DNA itself bodes well for vaccine development in the near future. This expectation must, nonetheless, be tempered by the reality that many protozoa have developed elaborate mechanisms (e.g. antigenic variation) for evading host immunity and that primary infection with one strain may not protect against heterologous strains. The display of highly immunogenic surface antigens that provoke non-protective responses or the secretion of products that cause immunopathology are other examples of protozoan adaptation to host immunity. Thus, the task of researchers seeking to develop vaccines against protozoa is to identify antigens that elicit responses which will destroy the parasite rather than exacerbate the deleterious effects of infection. The difculty is that, generating a protective immune response is dependent on a number of complex interrelated factors. Variables such as antigen composition, processing, and delivery, the need for adjuvant and booster immunization to promote a particular immune response, and the age of host animal all impact the efcacy of vaccination. Also, as the antigenic complexity of a recombinant vaccine increases, so does the cost of production such that it may no longer be competitive with drug treatment. As insight is gained on immunity to protozoa and on the developmental stages and associated antigens targeted by the protective response, then molecular and immunological tools may be harnessed for generating effective vaccines. What follows is a review of efforts to develop subunit vaccines based on recombinant antigens derived from cloned DNA of protozoan parasites of veterinary importance. Also included is a brief description of the immune mechanisms that are thought to play a role in controlling primary and secondary infections because eliciting similar responses with a recombinant antigen may be critical to vaccine efcacy. For purposes of this review, the protozoa are grouped into three categories related primarily to the parasites life cycle. 2. Homoxenous, non-cyst-forming protozoa: Eimeria, Giardia, and Cryptosporidium 2.1. Eimeria Coccidiosis in avian and mammalian hosts is primarily an intestinal disorder characterized by poor weight gain and inefcient feed utilization due to alteration of the intestinal mucosa by protozoa in the genus Eimeria. Although immunovariability has been observed among strains of E. maxima, infection of poultry with Eimeria spp. oocysts usually re-

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

293

sults in complete, albeit species-specic, resistance against challenge (Joyner and Norton, 1973, 1976; Long et al., 1986; Stiff and Bafundo, 1993). Research in mouse models using E. falciformis, E. vermiformis, and E. papillata have shown that CD4+ T cells and associated cytokines, IFN- and IL-12 are important for controlling primary infection, whereas CD8+ T cells are involved in immunity to secondary challenge (Rose et al., 1989a,b; Stiff and Vasilakos, 1990; Rose et al., 1991; Schito and Barta, 1997). Recent studies in chickens indicate that immunity to primary and secondary Eimeria infections is similar to that observed in mice (Lillehoj and Trout, 1996; Jeurissen et al., 1996; Lillehoj and Lillehoj, 2000). Current vaccines consist of a mixture of virulent or precocious strains of coccidia, the latter attenuated by the absence of several merogonic stages (Shirley et al., 1995; Williams, 1998; Williams et al., 1999). While these vaccines are effective in protecting broilers against coccidiosis, the immunizing inoculum must be generated in chickens followed by collection and processing of fecal material for oocysts. Several laboratories over the last two decades have attempted to develop a subunit coccidiosis vaccine using recombinant antigens expressed from DNA of various developmental stages (sporozoites, merozoites, gametes) of Eimeria spp. (for review, see Jenkins, 1998; Vermeulen, 1998). These DNA sequences have been expressed in a variety of prokaryotic and eukaryotic vectors, but immunization has only elicited partial protection against oocyst challenge (Jenkins, 1998; Vermeulen, 1998). The reason for failure to stimulate protective immunity is unknown, but is probably related to either the use of a non-protective antigen or to inappropriate delivery of the vaccine to the mucosal immune system. Studies of resistance elicited by infection with virulent, irradiated, or drug-arrested Eimeria spp. suggests that sporozoite-infected cells are necessary and sufcient for the development of protective immunity (Jeffers and Long, 1985; Jenkins et al., 1991a,b, 1993a,b). There is also evidence for merogonic stages inducing and being targeted by protective immunity (Rose and Hesketh, 1976; McDonald et al., 1988). Recombinant antigens of avian coccidia have generally been derived from cDNA originating from mRNA of extracellular stages of the parasite (sporozoites, merozoites). It is likely that CD4+ and CD8+T cell responses during primary and secondary infections are directed against antigens expressed by parasite-infected cells. Thus, intracellular processing would be required for the presentation of recombinant antigen to the avian immune system. Supporting this idea is a recent nding that recombinant E. tenella sporozoite antigen was protective only if administered to chickens by direct DNA vaccination (Kopko et al., 2000). Delivery of recombinant Eimeria antigens using Salmonella, fowlpox virus (FPV), or herpesvirus of turkeys (HVT) expressing Eimeria DNA sequences may be a viable approach to protect chickens against coccidiosis (Vermeulen, 1998; Cronenberg et al., 1999). These vectors have been shown to elicit CD4+ and CD8+ T cell responses in infected cells (Wang et al., 1998; Ramsay et al., 1999). Protective immunity against coccidiosis will probably require the processing and presentation of recombinant antigen in conjunction with MHC molecules on the surface of host cells for inducing appropriate effector mechanisms that prime the host against subsequent parasite challenge. 2.2. Giardia There is ample evidence that humans and animals develop immunity to Giardia duodenalis (synonyms: Giardia lamblia, Giardia intestinalis) by prior exposure to the parasite

294

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

(reviewed in Heyworth, 1992; Faubert, 1996). Giardiosis is a self-limiting infection in immunocompetent persons, whereas chronic disease, marked by unremitting diarrhea and shedding of Giardia cysts, is commonly observed in the young or persons with hypogammaglobulinemia (Heyworth, 1992). MHC-restricted CD4+ and CD8+ T lymphocyte responses do not appear to be a primary effector mechanism in immunity, possibly because trophozoites do not actually invade cells, but attach to the host intestinal epithelium. It is believed that humoral components, in particular secretory IgA, are involved in resolution of G. duodenalis infection by preventing the attachment of trophozoites to host cells (Kaplan and Altmanshofer, 1985; Inge et al., 1988). There is also in vitro evidence that G. duodenalis-specic IgG can directly lyse the parasite by both complement-dependent and -independent means (Belosevic and Faubert, 1987; Deguchi et al., 1987). Studies in mice indicate that CD4+ T cells play an accessory role in B cell secretion of IgA and protection against G. muris (Heyworth et al., 1987; Heyworth, 1989). Although animals can be vaccinated against giardiosis by immunization with G. duodenalis trophozoite extracts (Vinayak et al., 1992; Olson et al., 2000) or supernatant from G. duodenalis-cell culture (Olson et al., 2000), the cloning and expression of antigen genes for vaccine testing has only recently begun. DNA encoding a 32 kDa agellar protein (Upcroft et al., 1987) or a variable surface protein (VSP) (Aggarwal et al., 1989; Nash and Mowatt, 1992) have been described. Other less well-characterized clones encoding a surface coat protein, or antigens associated with the ventral disc, kinetosomes and funis, or anterolateral axonemes have also been reported (Upcroft et al., 1987). VSP was recently expressed in a vaccine strain of Salmonella typhimurium and used to elicit VSP-specic serum IgG and intestinal IgA responses in mice (Stager et al., 1997). However, the protective efcacy of recombinant VSP antigen or other expressed G. duodenalis sequences against giardiosis has not been tested. 2.3. Cryptosporidium Cryptosporidiosis causes a self-limiting diarrhea in humans and animals that resolves within 12 weeks post-infection (ODonoghue, 1995; Fayer et al., 1997). The disease is prevalent in the young and in immunocompromised persons due to immature or impaired T cell immunity (Casemore et al., 1997). Prior exposure to Cryptosporidium parvum leads to increased, but not complete resistance, to subsequent infection (Chappell et al., 1999). Protective immunity appears, based on studies in humans and mice, to involve CD4+ T lymphocytes and associated the cytokines, IFN- and IL-12 (for review, see Riggs, 1998). Although C. parvum-specic IgG, IgA, and IgM responses have been demonstrated, the role of these antibodies in resolution of cryptosporidiosis remains unclear (for review, see Riggs, 1998). Several studies have shown that passive immunotherapy with hyperimmune serum or colostrum against C. parvum antigens can ameliorate clinical signs of disease (reviewed in Jenkins et al., 1995). Vaccination against cryptosporidiosis has focused on antigens that may be used to generate colostrum for passive immunotherapy rather than stimulating protective immunity (for review, see deGraaf et al., 1999). There are several reasons for this approach. First, vaccination of the most vulnerable individuals (the young) would probably not be effective during the period of peak susceptibility. Second, due to the sporadic occurrence of cryptosporidiosis, the age-related resistance to infection, and since

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

295

disease symptoms are rarely life-threatening, there is little perceived need for widespread vaccination. Thus, cloning of C. parvum DNA has concentrated on expressing epitopes on the surface of invasive stages (sporozoites, merozoites) which may be involved in attachment to host cells of the intestinal epithelium. Although a number of recombinant C. parvum antigens have been described (Lally et al., 1992; Spano et al., 1997; Barnes et al., 1998; Tosini et al., 1999; Priest et al., 2000), the greatest effort has been directed at sequences coding for antigens related to sporozoite 15 or 23 kDa surface proteins (Jenkins et al., 1993a,b; Perryman et al., 1996; Khramtsov et al., 1997; Cevallos et al., 2000). Plasmid DNA expressing the CP15/60 protein has been used to elicit serum and colostrum antibodies in sheep (Jenkins et al., 1995) and cows (Jenkins et al., 1998) against recombinant and native CP15 antigens. Treatment of immunosuppressed adult inbred mice with this anti-CP15/60 hyperimmune bovine colostrum (HBC) prior to and during experimental C. parvum oocyst infection hindered parasite development in vivo (Jenkins et al., 1998). Although it is unknown whether direct plasmid DNA injection is required, a recent study showed that expression of CP15/60 in an eukaryotic vector (baculovirus) was superior to a prokaryotic system (E. coli) in terms of humoral and cellular responses elicited (Iochmann et al., 1999). Nasal immunization of goats and mice with a second CP15 DNA sequence also led to anti-CP15 antibodies in serum that, in the latter host species, remained high for at least 1 year post-immunization (Sagodira et al., 1999a). In mice, CP15-specic T cell proliferative responses were also demonstrated in both the spleen and the mesenteric lymph nodes (Sagodira et al., 1999b). Furthermore, kids challenged with a high number of C. parvum oocysts (106 oocysts per kid) and fed colostrum from CP15-immunized goats shed fewer oocysts and displayed less severe clinical effects compared to kids fed normal colostrum (Sagodira et al., 1999a). Another study showed that administration of monoclonal antibodies (mAbs) specic for a 23 kDa C. parvum sporozoite protein reduced the severity of C. parvum oocyst infection in mice (Perryman et al., 1996). HBC prepared against recombinant CP23 antigen prevented diarrhea and reduced oocyst shedding in calves challenged with 106 C. parvum oocysts (Perryman et al., 1999). Thus, immunization of ruminants with either recombinant C. parvum sporozoite surface antigens or plasmid DNA encoding the CP15 or CP23 antigens appears to be a viable approach to producing colostrum for the passive immunotherapy of cryptosporidiosis.

3. Heteroxenous, tissue cyst-forming coccidia: Toxoplasma, Neospora 3.1. Toxoplasma gondii Due to the impact of toxoplasmosis on human health (e.g. congenital toxoplasmosis, fatal CNS disease in immunosuppressed persons) as well as causing abortion in sheep and goats, there has been considerable interest in developing vaccines against productive T. gondii infection (Uggla and Buxton, 1990; Dubey, 1996). Vaccine development is based on the observation that primary exposure to T. gondii results in complete resistance to secondary challenge (Araujo, 1994; Alexander et al., 1996). This resistance is due in part to the presence of bradyzoites/cysts in many tissues, particularly in muscle and nerve cells. A role for tachyzoites in immunity is indicated by the success of commercial vaccines

296

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

(e.g. Toxovax) that are derived from non-cyst-forming mutant strains of T. gondii (Buxton, 1993). While the innate immune response to T. gondii is mediated by IFN- and TNF -activated macrophages, the adaptive immune response appears to depend on CD8+ T cells activated by IL-2 secretion from CD4+ Th1 cells that have been activated by costimulatory molecules IFN- and IL-12 (reviewed by Alexander et al., 1996). Relevant to the successful generation of attenuated or subunit vaccines against toxoplasmosis, is that immunization with one developmental stage (or antigens associated with this developmental stage) does not always confer protection against other stages (Alexander et al., 1996; Araujo, 1994). For instance, oocyst shedding is prevented in cats inoculated per os with mutant T263 cysts/bradyzoites (Frenkel et al., 1991), but not by parenteral immunization with any stage of the same parasite (Freyre et al., 1993). Immunization with p30 (SAG1) and p22 (SAG2) tachyzoite antigens incorporated into ISCOMs protects mice against tachyzoite or oocyst challenge, but not against cyst challenge (Lunden et al., 1993). These ndings imply that breaking the T. gondii life cycle will require vaccinating different hosts with antigens associated with the stages they are likely to encounter. Several studies have shown that mice and rats immunized with whole T. gondii tachyzoite extracts or specic native antigens such as SAG1 (p30), GRA2 (gp28.5), GRA5 (p21), or excretory-secretory antigens (ESA) conferred protection against tissue cyst or tachyzoite challenge as assessed by reduction in a number of brain cysts (Araujo and Remington, 1984; Duguesne et al., 1990; Darcy et al., 1992; Brinkmann et al., 1993; Lunden et al., 1993; Debard et al., 1996; Zenner et al., 1999; Velge-Roussel et al., 2000). Based on these observations, researchers have utilized either recombinant tachyzoite antigens or plasmid DNA encoding tachyzoite antigens to immunize mice or rats against T. gondii. Although it is difcult to make comparisons between studies that utilized different strains of inbred mice or different strains and challenge inocula of T. gondii or different measures of protection, the data indicate that rSAG1 expressed in either E. coli or BCG confers partial protection against tissue cyst or tachyzoite challenge (Letscher-Bru et al., 1998; Petersen et al., 1998; Aosai et al., 1999; Nielsen et al., 1999; Supply et al., 1999; Angus et al., 2000). In one study, mice immunized with rSAG1 in conjunction with alum showed higher survival rate against tachyzoite challenge (Petersen et al., 1998). Immunization of mice with a mixture of rSAG1 and IL-12 appeared to elicit excellent protection against brain cyst formation relative to control animals or those immunized with rSAG1 alone (Letscher-Bru et al., 1998). Attempts to protect mice against T. gondii oocyst or tissue cyst challenge by immunization with E. coli-derived rSAG2 were unsuccessful (Lunden et al., 1996). In general, protective effects were enhanced by direct immunization with plasmid DNA containing sequences for tachyzoite surface (SAG1), dense granule (GRA1, 4, 7) or rhoptry (ROP4) antigens (Nielsen et al., 1999; Angus et al., 2000; Desolme et al., 2000; Vercammen et al., 2000). In one study, mice immunized with pSAG1 showed higher survival rates and contained fewer brain cysts after cyst challenge (Angus et al., 2000). In the same study, pSAG1 mice were not protected against tachyzoite challenge, which conicts with another study that found 80100% survival of pSAG1-immunized mice (Nielsen et al., 1999). The discrepancy in outcome between the two studies may be due to differences in the mouse strains and plasmid constructs used. Mice inoculated with SAG1 DNA-transfected lymphoma cells also showed good protection as measured by survival against tachyzoite challenge (Aosai et al., 1999). Higher rates of survival and fewer brain cysts were observed in mice immunized with

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

297

plasmid DNA containing sequences for GRA1, GRA7, and ROP2 (Vercammen et al., 2000) or GRA4 (Desolme et al., 2000). In the latter study, the co-injection of mice with pGRA4 and pGMCSF enhanced protective effects. Similar to ndings in DNA vaccination against malaria (Doolan et al., 1998), there appears to be a genetic effect on responses to T. gondii plasmid DNA injection. Although pGRA1-, pGRA7-, and pROP2-immunized C3H mice show enhanced survival and fewer brain cysts after tissue cyst challenge, no protection was observed in either BALB/c or C57/Bl6 mice (Vercammen et al., 2000). These data indicate that development of a subunit vaccine against toxoplasmosis for protecting outbred animals, such as cats or pigs, will require delivery of several antigens from T. gondii life cycle stages using vectors that mimic processing by antigen-presenting cells in primary and secondary infections. 3.2. Neospora Although neosporosis has been described as causing neurological signs in canines, horses, and other animals, the primary impact of this protozoan disease is on the reproductive health of cattle (Dubey, 1999). Studies of natural and experimental Neospora caninum infections indicate that abortions are caused by tachyzoites that arise either from reactivation of bradyzoites/tissue cysts or from oocysts that have been ingested during pregnancy. Rapidly multiplying tachyzoites cross the placenta and infect the fetus, which, depending on gestational age, may lead to abortion (Anderson et al., 1991, 1997; Bjorkman et al., 1996; Par et al., 1996; Williams et al., 2000). Th1 responses and associated cytokines IFN- and IL-12 appear to be involved in immunity to acute N. caninum infection in mice (Kahn et al., 1997; Baszler et al., 1999). Further evidence for Th1 cytokines, is a recent study which showed that mice injected prior to pregnancy with avirulent N. caninum tachyzoites concomitant with neutralizing mAbs to IL-4 transmitted negligible numbers of tachyzoites to their pups after virulent N. caninum tachyzoites challenge during pregnancy (Long and Baszler, 2000). IFN- also appears to be important in N. caninum infections of sheep (Innes et al., 1995) and cattle (Lunden et al., 1998). The prospect for vaccinating cattle against neosporosis appears good. Complete protection against vertical transfer of N. caninum tachyzoites was achieved in mice that had been immunized with whole tachyzoite antigen prior to mating and parasite challenge (Liddell et al., 1999). Conicting results have been found in cattle immunized with whole tachyzoite antigen. Although humoral and cellular immune responses were elicited (Andrianarivo et al., 1999), congenital transfer of N. caninum tachyzoites was not prevented (Andrianarivo et al., 2000) by this immunization regimen. Identifying which antigens are targeted by protective immunity and which are involved in parasite survival within the host cell is the goal of several laboratories. A role for these dense granule and surface proteins in host cell invasion is the inhibitory effect of antibodies against SAG1, SRS2, GRA6, or GRA7 on the invasion of host cells in vitro by N. caninum tachyzoites (Hemphill, 1996; Augustine et al., 1999; Nishikawa et al., 2000a). DNA clones of dense granule proteins, GRA2 (p29) (Ellis et al., 2000), GRA6 (p37, NCDG2) (Liddell et al., 1998), and GRA7 (p33, NCDG1) (Lally et al., 1997; Hemphill et al., 1998) have been described as well as clones for surface antigens SRS2 (Nc-p43, Nc-p35) (Hemphill et al., 1997; Howe et al., 1998), SAG1 (Nc-p29, Nc-p36) (Howe et al., 1998; Sonda et al., 1998).

298

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

In addition, genes for a 40 kDa microneme antigen (Sonda et al., 2000), a TRAP homologue (NcMIC2) (Lovett et al., 2000), and a subtilisin-like serine protease (Nc-p65) (Louie and Conrad, 1999) have been reported. Recent studies showed that mice immunized with recombinant vaccinia virus (VV) or dogs immunized with canine Herpes virus expressing SRS2 elicited N. caninum-specic immune responses (Nishikawa et al., 2000b,c). In addition, immunization of female mice with recombinant VV-SRS2 prior to mating conferred signicant protection against congenital N. caninum infection (Nishikawa et al., 2001). Preliminary trials in our laboratory wherein adult BALB/c mice were immunized with either Nc-GRA6 (rNCDG2) or Nc-GRA7 (rNCDG1) expressed in E. coli, date-mated, and then challenged with N. caninum tachyzoites resulted in only partial protection against congenital transfer of the parasite to offspring (unpublished observations). Studies are underway to test direct injection of plasmid DNA expressing Nc-GRA6 and Nc-GRA7 for eliciting higher levels of protection against congenital neosporosis.

4. Heteroxenous, haemoprotozoa: Trypanosoma, Babesia, Theileria 4.1. Trypanosoma Trypanosomes of veterinary importance include T. brucei brucei, T. congolense, T. vivax, T. cruzi, and T. evansi. The rst three are transmitted by the bite of tsetse ies and are the causative agents of a disease complex in ruminants known in Sub-Saharan Africa as nagana. T. cruzi, the causative agent of Chagas disease (American trypanosomiasis) in Central and South America, is transmitted to both humans and animals by triatomin bugs. Immunity to T. cruzi appears to involve the cytokines IFN- and TNF secreted by CD4+ Th1 and CD8+ Tc1 lymphocytes (Silva et al., 1992; Tarleton, 1995; Dos Reis, 1997; Wizel et al., 1997). Antibodies binding to the surface antigens of T. cruzi trypomastigotes and, in particular, to a membrane-bound transialidase (TS) appear to play a role in protective immunity by possibly blocking parasite invasion of host cells (Krettli and Brenner, 1976; Umekita et al., 1988; Franchin et al., 1997; Costa et al., 1998). In recent studies, mice that were immunized with either recombinant TS protein or plasmid DNA expressing TS showed reduced parasitemia in blood compared to control mice (Costa et al., 1998; Pereira-Chioccola et al., 1999). Mice injected with recombinant TS plasmid DNA mounted a Th1-type response, but eventually succumbed to T. cruzi challenge (Pereira-Chioccola et al., 1999). However, mice immunized with recombinant TS protein showed a typical Th2-type response and 60% survival after T. cruzi challenge. Others have elicited protective immunity against T. cruzi challenge by injection of plasmid DNA expressing the sequence for trypomastigote surface antigen 1 (TSA-1) (Wizel et al., 1998). In vitro studies using sera and cells from pTSA-1 immunized mice showed specic responses to T. cruzi. Paraagellar rod (PFR) proteins derived from the agellum of T. cruzi trypomastigotes and mixed with either Freunds adjuvant, recombinant IL-12, or adenovirus expressing IL-12, have been used to elicit cellular immune responses in mice and 100% survival against T. cruzi challenge infection (Miller et al., 1996; Wrightsman and Manning, 2000). DNA encoding other protective antigens of T. cruzi may be identied by novel techniques such as immunization with whole genomic or cDNA libraries prepared in eukaryotic plasmid vectors (Alberti et al., 1998).

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

299

In contrast to T. cruzi, the African trypanosomes are strictly extracellular parasites. While T. congolense remains conned to the vasculature, the other species are able to invade tissues. Progress on developing vaccines against African trypanosomes has been hindered by the reality that these parasites have developed mechanisms for not only evading immunity through antigenic variation, but also depressing cellular immune responses (Taylor and Mertens, 1999). A major obstacle in identifying candidate vaccine antigens is that the surface of T. brucei trypomastigotes is covered with a highly immunogenic protein, termed variable surface glycoprotein (VSG). T. brucei evades immunity by using at least three different mechanisms for switching VSG expression (Borst and Rudenko, 1994). It has been estimated that the T. brucei genome contains as many as 1000 different VSG gene sequences and that VSG expression can be turned on or off coincident with the production of antibodies by the host against a specic antigenic type (termed variable antigenic type or VAT). The search for vaccine targets on the surface of trypomastigotes should, therefore, be focused on invariant molecules that are essential for parasite survival (Borst and Rudenko, 1994). An overt depression of T cell proliferation and macrophage/monocyte responsiveness has been found in experimental T. brucei and T. congolense infections (Sileghem et al., 1987; Schleifer and Manseld, 1993; Beschin et al., 1998; Mabbott et al., 1998; Taylor, 1998). This immune suppression may be due to counter regulation by the Th2 cytokine, IL-10. As indicated by studies in cattle, IL-10 mRNA is elevated in leukocytes in peripheral blood, lymph nodes, and spleen (Taylor, 1998). Studies in T. congolense-infected cattle showed that mRNA for a second Th2 cytokine, IL-4, is increased in trypanotolerant cattle, but not in trypanosensitive cattle (Mertens et al., 1999). This difference may account for the higher levels of T. congolense-specic IgG1 in resistant cattle (Taylor et al., 1996). Trypanotolerant cattle control both anemia and parasitemia better than trypanosensitive breeds of cattle. Identifying antigens that elicit complement-xing antibodies, such as IgG1 , should be a goal of vaccine development against T. brucei and T. congolense. 4.2. Babesia Although Babesia species have been described in dogs (B. canis) and horses (B. equi), the vast majority of research has been conducted on B. bigemina and B. bovis of cattle. B. bigemina, the principal causative agent of babesiosis in US (also termed Texas red water fever), and B. bovis, which causes bovine piroplasmosis in other parts of the world, are transmitted to cattle by the bite of Boophilus ticks. In the past, babesiosis has been controlled by application of acaricides or by infection of cattle with an attenuated Babesia vaccine. The repeated acaricide resistance in ticks and the costs associated with its use has limited acaricides as a control measure (Palmer and McElwain, 1995). Several ndings support the development of a vaccine against babesiosis. First, cattle that recover from a primary Babesia infection or that have been immunized with attenuated parasites are resistant to challenge infection (Mahoney et al., 1973, 1979; Timms, 1989; Brown and Palmer, 1999). Second, immunization of cattle with native Babesia antigen extracts or culture-derived supernatants containing secreted Babesia antigens elicit protective immunity against both homologous and heterologous challenge (Smith et al., 1981; Timms et al., 1983; Montenegro-James et al., 1987; Timms, 1989). Immunity to acute infection appears to involve macrophages

300

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

and NK cells, while the immune response that controls persistent infection involves activated macrophages and cytophilic Ab (Palmer and McElwain, 1995; Brown and Palmer, 1999). It is generally thought that an effective babesiosis vaccine should activate CD4+ T cells to secrete IFN- that in turn elicits a neutralizing and complement-xing IgG response and activates macrophages to produce the inammatory cytokines IL-1, IL-12, and TNF . A number of different approaches have been taken to identify protective antigens associated with merozoites or merozoite-infected erythrocytes of B. bigemina and B. bovis. A high Mr antigen (70200 kDa) and a 38 kDa cysteine-rich protein, which appear to be neither abundant nor immundominant, elicited 90% protection against B. bovis challenge (Wright et al., 1992). A third recombinant protein, that may have protease activity, also appears to confer signicant protection against B. bovis (Wright et al., 1992). Protective immunity has also been achieved with native B. bovis Mr77-80 (Bv80), which is a spherical body protein (SBP-1) (Goodger et al., 1992; Brown et al., 1993; Dalrymple et al., 1993; Hines et al., 1995) and with native and recombinant Mr60 rhoptry protein (RAP-1, Bv60) (Wright et al., 1992). Although RAP-1 gene sequences are polymorphic, there is sufcient epitope conservation between various B. bovis strains to suggest that this recombinant protein is a good candidate for inclusion in a babesiosis vaccine (Suarez et al., 1991; Dalrymple et al., 1993). Another recombinant B. bovis merozoite antigen, named Mr225 (BvVA1), which is associated with rhoptries, exhibits high levels of cross-reactivity and confers protection against babesiosis in cattle (Dalrymple et al., 1992; Jasmer et al., 1992). Conversely, immunodominant native and recombinant B. bovis antigens of 70, 200, and >1500 kDa have shown no capacity for eliciting protective immunity against B. bovis challenge (Timms et al., 1988; Wright et al., 1992; Brown and Palmer, 1999). Likewise, other immunodominant proteins, termed variable major surface antigens (VMSA), of merozoites have shown no protective efcacy in vaccine trials (reviewed in Dalrymple, 1993). It is possible that VMSAs such as MSA-1 (42 kDa) and MSA-2 (44 kDa) are ineffective because they are encoded by a polymorphic gene family. B. bigemina merozoite antigens Mr72, Mr55, Mr45, and Mr36 have shown protective efcacy in cattle (McElwain et al., 1991). In spite of belonging to a multi-gene family, an Mr58-60 antigen, which is a homologue of B. bovis RAP-1, appears to cross-react between different strains of B. bigemina (Dalrymple, 1993) and confers protection against babesiosis. 4.3. Theileria Piroplasms in the genus Theileria cause signicant impact on cattle production in developing countries throughout the world (Uilenberg et al., 1993). T. parva, the causative agent of East Coast Fever (ECF) is found in Eastern and Central Africa and is transmitted to cattle through the bite of the three-host tick Rhipicephalus appendiculatus. A second important species, T. annulata, is the agent of tropical theileriosis which is found primarily in North Africa, southern Europe, the Middle East, and Central Asia including India. The principal vector of T. annulata is ixodid ticks of the genus Hyalomma. At the injection site, T. annulata sporozoites enter monocytes, undergo schizogonous replication to generate large number of merozoites which, after lysing the host cell, invade erythrocytes and become piroplasms, the developmental stage infectious for ticks (for review, see McKeever and Morrison, 1998; Boulter and Hall, 2000). A similar series of events occur in T. parva,

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

301

except that T and B cells (principally CD4+ T cells) rather than monocytes are invaded by sporozoites (see McKeever and Morrison, 1998; Boulter and Hall, 2000). The pathology of T. parva is associated with the extensive lymphoproliferation of schizont-infected cells. Although both T. parva and T. annulata have similar life cycles, the host immune response to each and the current control measures against these two species are appreciably different. The live vaccine against tropical theileriosis is comprised of attenuated, culture-derived T. annulata, whereas an infection and treatment regimen using a T. parva sporozoite stabilate in conjunction with long acting tetracylines is used against ECF schizonts (Boulter and Hall, 2000). Similar to acaricide tick control, these approaches have a number of drawbacks, not least of which is the expense of vaccination and the requirement for cold storage to preserve vaccine integrity. The sterile immunity that develops after a primary infection is the basis for live and subunit vaccine development against T. parva (Musoke et al., 1997; McKeever et al., 1999), and T. annulata (Musoke et al., 1997; Preston et al., 1999). The innate response to a primary T. annulata infection is carried out by macrophages that produce TNF and NO which suppress parasite proliferation and by NK cells which directly lyse schizont-infected cells (Preston et al., 1999). Adaptive immunity to T. annulata appears to involve both innate and memory immune responses (Preston et al., 1999). Schizont-infected cells are lysed by CD8+ T lymphocytes and killed by macrophages that have been activated by cytokines released by memory CD4+ T cells (Preston et al., 1999). Acquired immunity to T. parva appears to principally involve CD8+ T cell lysis of schizont-infected cells, although CD4+ T cells may be involved (Emery et al., 1981; Baldwin et al., 1992; McKeever et al., 1994; Taracha et al., 1997). While antibodies to T. parva and T. annulata are not thought to play a major role in resistance, high Ab titers that neutralize sporozoites in vitro occur after primary infection (Musoke et al., 1982, 1984). Preventing sporozoite infection of host cells is the aim of vaccines based on the 67 kDa surface antigen of T. parva and the SPAG-1 (sporozoite antigen 1) of T. annulata. Immunization of cattle with SPAG-1 expressed as a fusion protein with a hepatitis virus B core antigen elicited antigen-specic antibodies and CD4+ responses as well as partial protection against T. annulata sporozoite challenge (Boulter et al., 1995). Higher levels of protection were achieved by vaccination with SPAG-1 in ISCOMs or mixed with recombinant p67 T. parva antigen (Boulter et al., 1988). Initial studies using p67 expressed in E. coli as a fusion protein to inuenza virus NS1 showed partial protection against sporozoite challenge (Musoke et al., 1992). Vaccine trials against T. parva using recombinant p67 expressed in a variety of delivery vectors have also shown promising results. For instance, inoculation of cattle with Salmonella dublin expressing recombinant p67 antigen elicited p67-specic humoral and cellular responses and conferred protection against T. parva sporozoite challenge (Heussler et al., 1998). Immunization with a mixture of VV expressing p67 and VV expressing IL-2 elicited signicant protection in cattle against sporozoite challenge (Honda et al., 1998). The use of VV-p67 alone or VV-p67 with VV expressing IL-4 did not provide immunity against T. parva infection (Honda et al., 1998). Immunization of cattle with a second T. parva sporozoite antigen (p32) and a merozoite/piroplasm antigen (mMPSA) failed to elicit a protective response (Skilton et al., 2000). Considerable research has also been conducted on the T. annulata merozoite antigen TAMS-1. Cattle immunized with two allelic forms of this antigen, TAMS1-1 and TAMS1-2, incorporated into ISCOMs showed excellent protection against T. annulata piroplasm challenge (dOliveira et al., 1997). Signicant protection was

302

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

also achieved by direct immunization with recombinant plasmid DNA pTAMS1, but not with S. typhimurium recombinants expressing TAMS1 antigens (dOliveira et al., 1997).

5. Concluding remarks Although it is difcult to make generalizations about such a diverse group of organisms, the protozoa described above elicit immune responses in most individuals that eventually control parasite replication and limit disease severity. This immunity is the basis of live and attenuated vaccines for several protozoan diseases. Many of these live vaccines have not gained widespread acceptance because of a requirement for maintaining the cold-chain between production and use in the eld. In theory, recombinant antigens should have a longer shelf-life than vaccines composed of live or attenuated organisms. The difculty is to identify the antigens that are targeted by the hosts protective immune response and express and deliver those proteins to induce the appropriate memory response (Watts and Kennedy, 1999). As pointed out in a previous review, a more feasible goal may be to rst develop vaccines that protect against clinical disease (Cox, 1997). A secondary goal would then be to optimize subunit vaccines, by incorporating antigens from other developmental stages, for instance, to achieve solid immunity. For many protozoan diseases, the severity of infection is directly related to the number of replicating parasites in the host. A subunit vaccine that protects against clinical signs, but allows for limited parasite replication may be an ideal strategy for protecting susceptible individuals. The complex nature of hostparasite interactions, is a formidable, but not an insurmountable obstacle to developing recombinant protozoan vaccines.

Acknowledgements The author wishes to thank Drs. J.P. Dubey, S. Liddell, R. OHandley, and K. Taylor for critical reviews of the manuscript.

References
Aggarwal, A., Merritt, J.W., Nash, T.E., 1989. Cysteine-rich variant surface proteins of Giardia lamblia. Mol. Biochem. Parasitol. 32, 3947. Alberti, E., Acosta, A., Sarmiento, M.E., Hidalgo, C., Vidal, T., Fachado, A., Fonte, L., Izquierdo, L., Infante, J.F., Finlay, C.M., Sierra, G., 1998. Specic cellular and humoral immune response in BALB/c mice immunized with an expression genomic library of Trypanosoma cruzi. Vaccine 16, 608612. Alexander, J., Jebbari, H., Bluethmann, H., Satoskar, A., Roberts, C.W., 1996. Immunological control of Toxoplasma gondii and appropriate vaccine design. Curr. Top. Microbiol. Immunol. 219, 183195. Anderson, M.L., Blanchard, P., Barr, B.C., Dubey, J.P., Hoffman, R.L., Conrad, P.A., 1991. Neospora infection as a major cause of abortion in California dairy cattle. J. Am. Vet. Med. Assoc. 198, 241244. Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 11691172.

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

303

Andrianarivo, A.G., Choromanski, L., McDonough, S.P., Packham, A.E., Conrad, P.A., 1999. Immunogenicity of a killed Neospora caninum tachyzoite preparation formulated with different adjuvants. Int. J. Parasitol. 29, 16131625. Andrianarivo, A.G., Rowe, J.D., Barr, B.C., Anderson, M.L., Packham, A.E., Sverlow, K.W., Choromanski, L., Loui, C., Grace, A., Conrad, P.A., 2000. A POLYGENTM -adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge. Int. J. Parasitol. 30, 985990. Angus, C.W., Klivington-Evans, D., Dubey, J.P., Kovacs, J.A., 2000. Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents. J. Infect. Dis. 181, 317324. Aosai, F., Mun, H.S., Norose, K., Chen, M., Hata, H., Kobayashi, M., Kiuchi, M., Stauss, H.J., Yano, A., 1999. Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice. Microbiol. Immunol. 43, 8791. Araujo, F.G., 1994. Immunization against Toxoplasma gondii. Parasitol. Today 10, 358360. Araujo, F.G., Remington, J.S., 1984. Partially puried antigen preparation of Toxoplasma gondii protect against lethal infection in mice. Infect. Immun. 45, 122126. Augustine, P.C., Jenkins, M.C., Dubey, J.P., 1999. Effect of polyclonal antisera developed against dense granule-associated Neospora caninum protein on cell invasion and development in vitro by N. caninum tachyzoites. Parasitology 119, 441445. Baldwin, C.L., Iams, K.P., Brown, W.C., Grab, D.J., 1992. Theileria parva: CD4+ helper and cytotoxic T cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes. Exp. Parasitol. 75, 1930. Barnes, D.A., Bonnin, A., Huang, J.X., Gousset, L., Wu, J., Gut, J., Doyle, P., Dubremetz, J.F., Ward, H., Petersen, C., 1998. A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Mol. Biochem. Parasitol. 96, 93110. Baszler, T.V., Long, M.T., McElwain, T.F., Mathison, B.A., 1999. Interferon- and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice. Int. J. Parasitol. 29, 16351646. Belosevic, M., Faubert, G.M., 1987. Lysis and immobilization of Giardia muris trophozoites in vitro by immune serum from susceptible and resistant mice. Parasite Immunol. 9, 1119. Beschin, A., Brys, L., Radwanska, M., De Baetselier, P., 1998. Trypanosoma brucei infection elicits nitric oxide-dependent and nitric oxide-independent suppressive mechanisms. J. Leuk. Biol. 63, 429439. Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 14411444. Borst, P., Rudenko, G., 1994. Antigenic variation in African trypanosomes. Science 264, 18721873. Boulter, N., Hall, R., 2000. Immunity and vaccine development in the bovine theilerioses. Adv. Parasitol. 44, 4297. Boulter, N.R., Brown, C.G., Kirvar, E., Glass, E., Campbell, J., Morzaria, S., Nene, V., Musoke, A., dOliveira, C., Gubbels, M.J., Jongejan, F., Hall, F.R., 1988. Different vaccine strategies used to protect against Theileria annulata. Ann. NY Acad. Sci. 849, 234246. Boulter, N.R., Glass, E.J., Knight, P.A., Bell-Sakyi, L., Brown, C.G., Hall, R., 1995. Theileria annulata sporozoite antigen fused to hepatitis B core antigen used in a vaccine trial. Vaccine 13, 11521160. Brinkmann, V., Remington, J.S., Sharma, S.D., 1993. Vaccination of mice with the protective F3G3 antigen of Toxoplasma gondii activates CD4+, but not CD8+ T cells and induces toxoplasma specic IgG antibody. Mol. Immunol. 30, 353358. Brown, W.C., Palmer, G.H., 1999. Designing blood-stage vaccines against Babesia bovis and B. bigemina. Parasitol. Today 15, 275281. Brown, W.C., Woods, V.M., Dobbelaere, D.A., Logan, K.S., 1993. Heterogeneity in cytokine proles of Babesia bovis-specic bovine CD4+ T cell clones activated in vitro. Infect. Immun. 61, 32733281. Buxton, D., 1993. Toxoplasmosis: the rst commercial vaccine. Parasitol. Today 9, 335337. Casemore, D.P., Wright, S.E., Coop, R.L., 1997. Cryptosporidiosishuman and animal epidemiology. In: Fayer, R. (Ed.), Cryptosporidium and cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 6592. Cevallos, A.M., Zhang, X., Waldor, M.K., Jaison, S., Zhou, X., Tzipori, S., Neutra, M.R., Ward, H.D., 2000. Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infect. Immun. 68, 41084116.

304

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

Chappell, C.L., Okhuysen, P.C., Sterling, C.R., Wang, C., Jakubowski, W., Dupont, H.L., 1999. Infectivity of Cryptosporidium parvum in healthy adults with pre-existing anti-C. parvum serum immunoglobulin G. Am. J. Trop. Med. Hyg. 60, 157164. Costa, F., Franchin, G., Pereira-Chioccola, V.L., Ribeirao, M., Schenkman, S., Rodrigues, M., 1998. Immunization with a plasmid DNA containing the gene of transialidase reduces Trypanosoma cruzi infection in mice. Vaccine 16, 768774. Cox, F.E.G., 1997. Designer vaccines for parasitic diseases. Int. J. Parasitol. 27, 11471157. Cronenberg, A.M., Van Geffen, C.E.H., Dorrestein, J., Vermeulen, A.N., Sondermeijer, P.J.A., 1999. Vaccination of broilers with HVT expressing an Eimeria acervulina antigen improves performance after challenge with Eimeria. Acta Virol. 43, 192197. Dalrymple, B.P., 1993. Molecular variation and diversity in candidate vaccine antigens from Babesia. Acta Trop. 53, 227238. Dalrymple, B.P., Jorgensen, W.K., De Vos, A.J., Wright, I.G., 1992. Analysis of the composition of samples of Babesia bovis and the inuence of different environmental conditions on genetically distinct subpopulations. Int. J. Parasitol. 22, 731737. Dalrymple, B.P., Casu, R.E., Peters, J.M., Dimmock, C.M., Gale, K.R., Boese, R., Wright, I.G., 1993. Cloning and characterization of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains. Mol. Biochem. Parasitol. 59, 181189. Darcy, F., Maes, P., Gras-Masse, H., Auriault, C., Bossus, M., Deslee, D., Godard, I., Cesbron, M.-F., Tartar, A., Capron, A., 1992. Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplama gondii p30 antigen. J. Immunol. 149, 36363641. Debard, N., Buzoni-Gatel, D., Bout, D., 1996. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect. Immun. 64, 21582166. deGraaf, D.C., Spano, F., Petry, F., Sagodira, S., Bonnin, A., 1999. Speculation on whether a vaccine against cryptosporidiosis is a reality or a fantasy. Int. J. Parasitol. 29, 12891306. Deguchi, M., Gillin, F.D., Gigli, I., 1987. Mechanism of killing Giardia lamblia trophozoites by complement. J. Clin. Invest. 79, 12961302. Desolme, B., Mevelec, M.-N., Buzoni-Gatel, D., Bout, D., 2000. Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene. Vaccine 18, 25122521. dOliveira, C., Feenstra, A., Vos, H., Osterhaus, A.D., Shiels, B.R., Cornelissen, A.W., Jongejan, F., 1997. Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems. Vaccine 15, 17961804. Doolan, D.L., Hedstrom, R.C., Gardner, M., Sedegah, M., Wang, H., Gramzinski, R.A., Margalith, M., Hobart, P., Hoffman, S.L., 1998. DNA vaccination as an approach to malaria control: current status and strategies. Curr. Top. Microbiol. Immunol. 226, 3756. Dos Reis, G.A., 1997. Cell-mediated immunity in experimental Trypanosoma cruzi infection. Parasitol. Today 13, 335342. Dubey, J.P., 1996. Strategies to reduce transmission of Toxoplasma gondii to animals and humans. Vet. Parasitol. 64, 6570. Dubey, J.P., 1999. Neosporosis in cattle: biology and economic impact. J. Am. Vet. Med. Assoc. 214, 11601163. Duguesne, V., Auriault, C., Darcy, F., Decavel, J.-P., Capron, A., 1990. Protection of nude rats against Toxoplasma infection by excreted-secreted antigen-specic helper T cells. Infect. Immun. 58, 21202126. Ellis, J.T., Ryce, C., Atkinson, R., Balu, S., Jones, P., Harper, P.A.W., 2000. Isolation, characerization, and expression of a GRA2 homologue from Neospora caninum. Parasitology 120, 383390. Emery, D.L., Tenywa, T., Jack, R.M., 1981. Characterization of the effector cell that mediates cytotoxicity against Theileria parva (East Coast Fever) in immune cattle. Infect. Immun. 32, 13011304. Faubert, G.M., 1996. The immune response to Giardia. Parasitol. Today 12, 140145. Fayer, R., Speer, C.A., Dubey, J.P., 1997. The general biology of Cryptosporidium. In: Fayer, R. (Ed.), Cryptosporidium and cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 142. Franchin, G., Pereira-Chioccola, V.L., Schenkman, S., Rodrigues, M.M., 1997. Passive transfer of a monoclonal antibody specic for a sialic acid-dependent epitope on the surface of Trypanosoma cruzi trypomastigotes reduces infection in mice. Infect. Immun. 65, 25482554.

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

305

Frenkel, J.K., Pfefferkorn, E.R., Smith, D.D., Fishback, J.L., 1991. Prospective vaccine prepared from a new mutant of Toxoplasma gondii. Am. J. Vet. Res. 52, 759763. Freyre, A., Choromanski, L., Fishback, J.L., Popiel, I., 1993. Immunization of cats with tissue cysts, bradyzoites, tachyzoites of the T-263 strain of Toxoplasma gondii. J. Parasitol. 79, 716719. Goodger, B.V., Waltisbuhl, D.J., Commins, M.A., Wright, I.G., 1992. Babesia bovis: dextran sulfate as an adjuvant for and precipitant of protective immunogens. Int. J. Parasitol. 22, 465469. Hemphill, A., 1996. Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein. Infect. Immun. 64, 42794287. Hemphill, A., Felleisen, R., Connolly, B., Gottstein, B., Hentrich, B., Muller, N., 1997. Characterization of a cDNA-clone encoding Nc-p43, a major Neospora caninum tachyzoite surface protein. Parasitology 115, 581 590. Hemphill, A., Gajendran, N., Sonda, S., Fuchs, N., Gottstein, B., Hentrich, B., Jenkins, M., 1998. Identication and characterization of a dense granule-associated protein in Neospora caninum tachyzoites. Int. J. Parasitol. 28, 429438. Heussler, V.T., Taracha, E.L.N., Musoke, A., Duchateau, L., McKeever, D.J., 1998. Immunization with live attenuated Salmonella dublin expressing a sporozoite protein confers partial protection against Theileria parva. Vaccine 16, 834841. Heyworth, M.F., 1989. Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice. J. Parasitol. 75, 246251. Heyworth, M.F., 1992. Immunology of Giardia and Cryptosporidium infections. J. Infect. Dis. 166, 465472. Heyworth, M.F., Carlson, J.R., Ermak, T.H., 1987. Clearance of Giardia muris infection requires helper/inducer T lymphocytes. J. Exp. Med. 165, 17431748. Hines, S.A., Palmer, G.H., Jasmer, D.P., Goff, W.L., McElwain, T.F., 1995. Immunization of cattle with recombinant Babesia bovis merozoite surface antigen-1. Infect. Immun. 63, 349352. Honda, Y., Waithaka, M., Taracha, E.L.N., Duchateau, L., Musoke, A.J., McKeever, D.J., 1998. Delivery of the Theileria parva p67 antigen to cattle using recombinant vaccine virus: IL-2 enhances protection. Vaccine 16, 12761282. Howe, D.K., Crawford, A.C., Lindsay, D., Sibley, L.D., 1998. The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infect. Immun. 66, 53225328. Inge, P.M.G., Edson, C.M., Farthing, M.J.G., 1988. Attachment of Giardia lamblia to rat intestinal epithelial cells. Gut 29, 795801. Innes, E.A., Panton, W.R., Marks, J., Trees, A.J., Holmdahl, J., Buxton, D., 1995. Interferon gamma inhibits the intracellular multiplication of Neospora caninum, as shown by incorporation of 3 H uracil. J. Comp. Pathol. 113, 95100. Iochmann, S., Sagodira, S., Mevelec, M.-N., Reperant, J.-M., Naciri, M., Coursaget, P., Bout, D., 1999. Comparison of the humoral and cellular immune responses to two preparations of Cryptosporidium parvum CP15/60 recombinant protein. Microbiol. Pathol. 26, 307315. Jasmer, D.P., Reduker, D.W., Perryman, L.E., McGuire, T.C., 1992. A Babesia bovis 225-kilodalton protein located on the cytoplasmic side of the erythrocyte membrane has sequence similarity with a region of glycogen phosphorylase. Mol. Biochem. Parasitol. 52, 263269. Jeffers, T.K., Long, P.L., 1985. Eimeria tenella: immunogenicity of arrested sporozoites in chickens. Exp. Parasitol. 66, 175180. Jenkins, M.C., 1998. Progress on developing a recombinant coccidiosis vaccine. Int. J. Parasitol. 28, 11111119. Jenkins, M.C., Augustine, P.C., Barta, J.R., Castle, M.D., Danforth, H.D., 1991a. Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts. Exp. Parasitol. 72, 285293. Jenkins, M.C., Augustine, P.C., Danforth, H.D., Barta, J.R., 1991b. X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induce a protective immune response(s). Infect. Immun. 59, 40424048. Jenkins, M.C., Seferian, P.G., Augustine, P.C., Danforth, H.D., 1993a. Protective immunity against coccidiosis elicited by radiation-attenuated Eimeria maxima sporozoites that are incapable of asexual development. Avian Dis. 37, 7482.

306

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

Jenkins, M.C., Fayer, R., Tilley, M., Upton, S., 1993b. Cloning and expression of cDNA encoding epitopes shared by 15- and 60-kDa proteins of Cryptosporidium parvum sporozoites. Infect. Immun. 61, 23772382. Jenkins, M.C., Kerr, D., Fayer, R., Wall, R., 1995. Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen. Vaccine 13, 16581664. Jenkins, M., OBrien, C., Trout, J., Guidry, A., Fayer, R., 1998. Hyperimmune bovine colostrum specic for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice. Vaccine 17, 24532460. Jeurissen, S.H., Janse, E.M., Vermeulen, A.N., Vervelde, L., 1996. Eimeria tenella infections in chickens: aspects of hostparasite interaction. Vet. Immunol. Immunopathol. 54, 231238. Joyner, L.P., Norton, C.C., 1973. The immunity arising from continuous low-level infection with Eimeria tenella. Parasitology 67, 333340. Joyner, L.P., Norton, C.C., 1976. The immunity arising from continuous low-level infection with Eimeria maxima and Eimeria acervulina. Parasitology 72, 115125. Kahn, I.A., Scwartzman, J.D., Fonseka, S., Kasper, L.H., 1997. Neospora caninum: role for immune cytokines in host immunity. Exp. Parasitol. 85, 2434. Kaplan, B., Altmanshofer, D., 1985. Giardia muris adherence to intestinal epitheliumthe role of specic anti-Giardia antibodies. Microecol. Ther. 15, 133140. Khramtsov, N.V., Oppert, B., Montelone, B.A., Upton, S.J., 1997. Sequencing, analysis, and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein. Biochem. Biophys. Res. Commun. 230, 164166. Kopko, S.H., Martin, D.S., Barta, J.R., 2000. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies. Poult. Sci. 79, 336342. Krettli, A.U., Brenner, Z., 1976. Protective effects of specic antibodies in Trypanosoma cruzi infections. J. Immunol. 116, 755760. Lally, N.C., Baird, G.D., McQuay, S.J., Wright, F., Oliver, J.J., 1992. A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein. Mol. Biochem. Parasitol. 56, 6978. Lally, N.C., Jenkins, M.C., Liddell, S., Dubey, J.P., 1997. A dense granule protein (NCDG1) gene from Neospora caninum. Mol. Biochem. Parasitol. 87, 239243. Letscher-Bru, V., Villard, O., Risse, B., Zuake, M., Klein, J.-P., Kien, T.T., 1998. Protective effect of vaccination with a combination of recombinant surface antigen 1 and interleukin-12 against toxoplasmosis in mice. Infect. Immun. 66, 45034506. Liddell, S., Lally, N.C., Jenkins, M.C., Dubey, J.P., 1998. Isolation of the cDNA encoding a dense granule associated antigen (NCDG2) of Neospora caninum. Mol. Biochem. Parasitol. 93, 153158. Liddell, S., Jenkins, M.C., Collica, C.M., Dubey, J.P., 1999. Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination. J. Parasitol. 85, 10721075. Lillehoj, H.S., Lillehoj, E.P., 2000. Avian coccidiosis: a review of acquired intestinal immunity and vaccination strategies. Avian Dis. 44, 408425. Lillehoj, H.S., Trout, J.M., 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9, 349360. Long, M.T., Baszler, T.V., 2000. Neutralization of maternal IL-4 modulates congenital protozoal transmission: comparison of innate versus acquired immune responses. J. Immunol. 164, 47684774. Long, P.L., Johnson, J., McKenzie, M.E., Perry, E., Crane, M.S.J., Murray, P.K., 1986. Immunization of young broiler chickens with low level infections of Eimeria tenella, Eimeria acervulina, or Eimeria maxima. Avian Pathol. 15, 271278. Louie, K., Conrad, P.A., 1999. Characterization of a cDNA encoding a subtilisin-like serine protease (NC-p65) of Neospora caninum. Mol. Biochem. Parasitol. 103, 211223. Lovett, J.L., Howe, D.K., Sibley, L.D., 2000. Molecular characterization of thrombospondin-related anonymous protein homologue in Neospora caninum. Mol. Biochem. Parasitol. 107, 3343. Lunden, A., Lovgren, K., Uggla, A., Araujo, F.G., 1993. Immune responses and resistance to Toxoplasma gondii in mice immunized with antigens of the parasite incorporated into immunostimulating complexes. Infect. Immun. 61, 26392643. Lunden, A., Parmley, S.F., Lovgren-Bengtsson, K., Araujo, F.G., 1996. Use of recombinant antigen, SAG2, expressed as a glutathione-S-transferase fusion protein to immunize mice against Toxoplasma gondii. Parasitol. Res. 83, 69.

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

307

Lunden, A., Marks, J., Maley, S.W., Innes, E.A., 1998. Cellular immune responses in cattle experimentally infected with Neospora caninum. Parasite Immunol. 20, 519526. Mabbott, N.A., Coulson, P.S., Smythies, L.E., Wilson, R.A., Sternberg, J., 1998. African trypanosome infections in mice that lack the interferon- receptor gene: nitric oxide-dependent and independent suppression of T-cell proliferative responses and the development of anaemia. Immunology 94, 476480. Mahoney, D.F., Wright, I.G., Mirre, G.B., 1973. Bovine babesiasis: the persistence of immunity to Babesia argentina and B. bigemina in calves (Bos taurus) after naturally acquired infection. Ann. Trop. Med. Parasitol. 67, 197203. Mahoney, D.F., Wright, I.G., Goodger, B.V., 1979. Immunity in cattle to Babesia bovis after single infections with parasites of various origin. Aust. Vet. J. 55, 1012. McDonald, V., Wisher, M.H., Rose, M.E., Jeffers, T.K., 1988. Eimeria tenella: immunological diversity between asexual generations. Parasite Immunol. 10, 649660. McElwain, T.F., Perryman, L.E., Musoke, A.J., McGuire, T.C., 1991. Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins. Mol. Biochem. Parasitol. 47, 213222. McKeever, D.J., Morrison, W.I., 1998. Novel vaccines against Theileria parva: prospects for sustainability. Int. J. Parasitol. 28, 693706. McKeever, D.J., Taracha, E.L., Innes, E.L., MacHugh, N.D., Awino, E., Goddeeris, B.M., Morrision, W.I., 1994. Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph. Proc. Natl. Acad. Sci. USA 91, 19591963. McKeever, D.J., Taracha, E.L.N., Morrison, W.I., Musoke, A.J., Morzaria, S.P., 1999. Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies. Parasitol. Today 15, 263 267. Mertens, B., Taylor, K., Muriuki, C., Rocchi, M., 1999. Cytokine mRNA proles in trypanotolerant and trypanosusceptible cattle infected with the protozoan parasite Trypanosoma congolense: protective role for interleukin-4? J. Interf. Cytokin. Res. 19, 5965. Miller, M.J., Wrightsman, R.A., Manning, J.E., 1996. Trypanosoma cruzi: protective immunity in mice immunized with paraagellar rod proteins is associated with a T-helper type 1 response. Exp. Parasitol. 84, 156167. Montenegro-James, S., Benitez, M.T., Leon, E., Lopez, R., Ristic, M., 1987. Bovine babesiosis: induction of protective immunity with culture-derived Babesia bovis and Babesia bigemina immunogens. Parasitol. Res. 74, 142150. Musoke, A.J., Nantulya, V.M., Buscher, G., Masake, R.A., Otim, B., 1982. Bovine immune responses to Theileria parva: neutralizing antibodies to sporozoites. Immunology 45, 663668. Musoke, A.J., Nantulya, V.M., Rurangirwa, F.R., Buscher, G., 1984. Evidence for a common protective antigenic determinant on sporozoites of several Theileria parva strains. Immunology 52, 231238. Musoke, A., Morzaria, S., Nkonge, C., Jones, E., Nene, V., 1992. A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle. Proc. Natl. Acad. Sci. USA 89, 514518. Musoke, A.J., McKeever, D., Nene, V., 1997. Subunit vaccines for the control of tick-borne diseases: implications for the future. Parasitologia 39, 131137. Nash, T.E., Mowatt, M.R., 1992. Characterization of a Giardia lamblia variant-specic surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size. Mol. Biochem. Parasitol. 51, 219228. Nielsen, H.V., Lauemoller, S.L., Christiansen, L., Buus, S., Fomsgaard, A., Petersen, E., 1999. Complete protection against lethal Toxoplasma gondii infection in mice immunized with plasmid DNA encoding the SAG1 gene. Infect. Immun. 67, 63586363. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000a. Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. Int. J. Parasitol. 30, 11671171. Nishikawa, Y., Kousaka, Y., Fukumoto, S., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000b. Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus. Parasitol. Res. 86, 934939. Nishikawa, Y., Ikeda, H., Fukumoto, S., Xuan, X., Nagasawa, K., Otsuka, H., Mikami, T., 2000c. Immunization of dogs with a canine herpesvirus vector expressing Neospora caninum surface protein, NcSRS2. Int. J. Parasitol. 30, 11671171. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2001. Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene. Vaccine 19, 17101716.

308

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

ODonoghue, P.J., 1995. Cryptosporidium and cryptosporiosis in man and animals. Int. J. Parasitol. 25, 139195. Olson, M.E., Ceri, H., Morck, D.W., 2000. Giardia vaccination. Parasitol. Today 16, 213217. Palmer, G.H., McElwain, T.F., 1995. Molecular basis for vaccine development against anaplasmosis and babesiosis. Vet. Parasitol. 57, 233253. Par, J., Thurmond, M., Hietala, S., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133139. Pereira-Chioccola, V.L., Costa, F., Ribeirao, M., Soares, I.S., Arena, F., Schenkman, S., Rodrigues, M., 1999. Comparison of antibody and protective immune responses against Trypanosoma cruzi infection elicited by immunization with a parasite antigen delivered as naked DNA or recombinant protein. Parasite Immunol. 21, 103110. Perryman, L.E., Jasmer, D.P., Riggs, M.W., Bohnet, S.G., McGuire, T.C., Arrowood, M.J., 1996. A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes. Mol. Biochem. Parasitol. 80, 137147. Perryman, L.E., Kapil, S.J., Jones, M.L., Hunt, E.L., 1999. Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein. Vaccine 17, 21422149. Petersen, E., Nielsen, H.V., Christiansen, L., Spenter, J., 1998. Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii. Vaccine 16, 12831289. Preston, P.M., Hall, F.R., Glass, E.J., Campbell, J.D.M., Darghouth, M.A., Ahmed, J.S., Shiels, B.R., Spooner, R.L., Jongejan, F., Brown, C.G.D., 1999. Innate and adaptive immune response co-operate to protect cattle against Theileria annulata. Parasitol. Today 15, 268274. Priest, J.W., Kwon, J.P., Arrowood, M.J., Lammie, P.J., 2000. Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum. Mol. Biochem. Parasitol. 106, 261271. Ramsay, A.J., Kent, S.J., Strugnell, R.A., Suhrbier, A., Thomson, S.A., Ramshaw, I.A., 1999. Genetic vaccination strategies for enhanced cellular, humoral, and mucosal immunity. Immunol. Rev. 171, 2744. Riggs, M.W., 1998. Immunology: host response and development of passive immunotherapy and vaccines. In: Fayer, R. (Ed.), Cryptosporidium and cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 129162. Rose, M.E., Hesketh, P., 1976. Immunity to coccidiosis: stages of the life-cycle of Eimeria maxima which induce, and are affected by, the response of the host. Parasitology 73, 2537. Rose, M.E., Wakelin, D., Hesketh, P., 1989a. Interferon-gamma-mediated effects upon immunity to coccidial infections in the mouse. Parasite Immunol. 13, 6374. Rose, M.E., Wakelin, D., Hesketh, P., 1989b. Gamma interferon controls Eimeria vermiformis primary infection in BALB/C mice. Infect. Immun. 57, 15991603. Rose, M.E., Smith, A.L., Wakelin, D., 1991. Gamma interferon-mediated inhibition of Eimeria vermiformis growth in cultured broblasts and epithelial cells. Infect. Immun. 59, 580586. Sagodira, S., Buzoni-Gatel, D., Iochmann, S., Naciri, M., Bout, D., 1999a. Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA. Vaccine 17, 23462355. Sagodira, S., Iochmann, S., Mevelec, M.N., Dimier-Poisson, I., Bout, D., 1999b. Nasal immunization of mice with Cryptosporidium parvum DNA induces systemic and intestinal immune responses. Parasite Immunol. 21, 507516. Schito, M.L., Barta, J.R., 1997. Nonspecic immune responses and mechanisms of resistance to Eimeria papillata infections in mice. Infect. Immun. 65, 31653170. Schleifer, K.M., Manseld, J.M., 1993. Suppressor macrophages in African trypanosomiasis inhibit T cell proliferative responses by nitric oxide and prostaglandins. J. Immunol. 151, 54925503. Shirley, M.W., Bushell, A.C., Bushell, J.E., McDonald, V., Roberts, B., 1995. A live attenuated vaccine for the control of avian coccidiosis: trials in broiler breeders and replacement layer ocks in the United Kingdom. Vet. Rec. 137, 453457. Sileghem, M., Hamers, R., De Baetselier, P., 1987. Experimental Trypanosoma brucei infection selectively suppress both interleukin 2 production and interleukin-2 receptor expression. Eur. J. Immunol. 17, 14171421. Silva, J.S., Morrisey, P.J., Grabstein, K.H., 1992. Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection. J. Exp. Med. 175, 169174. Skilton, R.A., Musoke, A.J., Wells, C.W., Yagi, Y., Nene, V., Spooner, P.R., Gachanja, J., Osaso, J., Bishop, R.P., Morzaria, S.P., 2000. A 32 kDa surface antigen of Theileria parva: characterization and immunization studies. Parasitology 120, 553564.

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

309

Smith, R.D., James, M.A., Ristic, M., Aikawa, M., Vega y Murguia, C.A., 1981. Bovine babesiosis: protection of cattle with culture-derived soluble Babesia bovis antigen. Science 212, 335338. Sonda, S., Fuchs, N., Connolly, B., Fernandez, P., Gottstein, B., Hemphill, A., 1998. The major 36 kDa Neospora caninum tachyzoite surface protein is closely related to the major Toxoplasma gondii surface antigen. Mol. Biochem. Parasitol. 97, 97108. Sonda, S., Fuchs, N., Gottstein, B., Hemphill, A., 2000. Molecular characterization of a novel microneme antigen in Neospora caninum. Mol. Biochem. Parasitol. 108, 3951. Spano, F., Puri, C., Putignani, L., Crisanti, A., 1997. Cloning of the entire COWP gene of Cryptosporidium parvum and ultrastructural localization of the protein during sexual parasite development. Parasitology 114, 427437. Stager, S., Gottstein, B., Muller, N., 1997. Systemic and local antibody response in mice induced by a recombinant peptide fragment from Giarda lamblia variant surface protein (VSP) H7 produced by Salmonella typhimurium vaccine strain. Int. J. Parasitol. 27, 965971. Stiff, M.I., Bafundo, K.W., 1993. Development of immunity in broilers continuously exposed to Eimeria sp. Avian Dis. 37, 295301. Stiff, M.I., Vasilakos, J.P., 1990. Effect of in vivo T-cell depletion on the effector T-cell function of immunity to Eimeria falciformis. Infect. Immun. 58, 14961499. Suarez, C.E., Palmer, G.H., Jasmer, D.P., Hines, S.A., Perryman, L.E., McElwain, T.F., 1991. Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes. Mol. Biochem. Parasitol. 46, 4552. Supply, P., Sutton, P., Coughlan, S.N., Bilo, K., Saman, E., Trees, A.J., Cesbron-Delauw, M.-F., Locht, C., 1999. Immunogenicity of recombinant BCG producing the GRA1 antigen from Toxoplasma gondii. Vaccine 17, 705714. Taracha, E.L.N., Awino, E., McKeever, D.J., 1997. Distinct CD4+ T cell helper requirements in Theileria parva-immune and nave bovine CTL precursors. J. Immunol. 159, 45394545. Tarleton, R.L., 1995. The role of T cells in Trypanosoma cruzi infections. Parasitol. Today 11, 712. Taylor, K.A., 1998. Immune response of cattle to African trypanosomes: protective or pathogenic? Int. J. Parasitol. 28, 219240. Taylor, K.A., Mertens, 1999. Immune responses of cattle infected with African trypanosomes, Mem. Inst. Oswaldo Cruz. 94, 239244. Taylor, K.A., Lutje, V., Kennedy, D., Authie, E., Boulange, A., Logan-Henfrey, L., Gichuki, B., Gettinbly, G., 1996. Trypanosoma congolense: B-lymphocyte responses differ between trypanotolerant and trypanosusceptible cattle. Exp. Parasitol. 83, 106116. Timms, P., 1989. Development of babesial vaccines. Trans. R. Soc. Trop. Med. Hyg. 83, 7379. Timms, P., Dalgliesh, R.J., Barry, D.N., Dimmock, C.K., Rodwell, B.J., 1983. Babesia bovis: comparison of culture-derived parasites, non-living antigen, and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J. 60, 7577. Timms, P., Barry, D.N., Gill, A.C., Sharp, P.J., deVos, A.J., 1988. Failure of a recombinant Babesia bovis antigen to protect cattle against heterologous strain challenge. Res. Vet. Sci. 45, 267269. Tosini, F., Caccio, S., Tamburrini, A., LaRosa, G., Pozio, E., 1999. Identication and characterization of three antigenic proteins from Cryptosporidium parvum sporozoites using a DNA library expressing poly-histidine tagged peptides. Int. J. Parasitol. 29, 19251933. Uggla, A., Buxton, D., 1990. Immune responses against Toxoplasma and Sarcocystis infections in ruminants: diagnosis and prospects for vaccination. Rev. Sci. Tech. Off. Int. Epiz. 9, 441462. Uilenberg, G., Dobbelaere, D.A.E., deGee, A.L.W., Koch, H.T., 1993. Progress in research on tick-borne diseases: theileriosis and heartwater. Vet. Quart. 15, 4854. Umekita, L.F., Takehara, H.A., Mota, I., 1988. Role of antibody Fc in the immune clearance of Trypanosoma cruzi infections. Immunol. Lett. 17, 8589. Upcroft, J.A., Capon, A.G., Dharmkrong-At, A., Healey, A., Boreham, P.F.L., Upcroft, P., 1987. Giardia intestinalis antigens expressed in Escherichia coli. Mol. Biochem. Parasitol. 26, 267276. Velge-Roussel, F., Marcelo, P., Lepage, A.C., Buzoni-Gatel, D., Bout, D.T., 2000. Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments. Infect. Immun. 68, 969972. Vercammen, M., Scorza, T., Huygen, K., De Braekeleer, J., Diet, R., Jacobs, D., Saman, E., Vershchueren, H., 2000. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect. Immun. 68, 3845.

310

M.C. Jenkins / Veterinary Parasitology 101 (2001) 291310

Vermeulen, A.N., 1998. Recent progress in recombinant vaccine development against coccidiosis. A review and prospects into the next millenium. Int. J. Parasitol. 28, 11211130. Vinayak, V.K., Kum, K., Khanna, R., Khuller, M., 1992. Systemic-oral immunization with 56 kDa molecule of Giardia lamblia affords protection in experimental mice. Vaccine 10, 2127. Wang, R., Doolan, D.L., Charoenvit, Y., Hedstrom, R.C., Gardner, M.J., Hobart, P., Tine, J., Sedegah, M., Fallarme, V., Sacci Jr., J.B., Kaur, M., Klinman, D.M., Hoffman, S.L., Weiss, W.R., 1998. Simultaneous induction of multiple antigen-specic cytotoxic T lymphocytes in nonhuman primates by immunization with a mixture of four Plasmodium falciparum DNA plasmids. Infect. Immun. 66, 41934202. Watts, A.M., Kennedy, R.C., 1999. DNA vaccination strategies against infectious diseases. Int. J. Parasitol. 29, 11491163. Willetts, N., 1994. Microorganisms in the development of subunit vaccines against parasites. Crit. Rev. Microbiol. 20, 7985. Williams, R.B., 1998. Epidemiological aspects of the use of live anticoccidial vaccines for chickens. Int. J. Parasitol. 7, 10891098. Williams, R.B., Carlyle, W.W., Bond, D.R., Brown, I.A., 1999. The efcacy and economic benets of Paracox, a live attenuated anticoccidial vaccine, in commercial trials with standard broiler chickens in the United Kingdom. Int. J. Parasitol. 29, 341355. Williams, D.J., Guy, C., McGarry, J., Guy, F., Tasker, L., Smith, R.F., MacEachern, K., Cripps, P.J., Kelly, D.F., Trees, A.J., 2000. Neospora caninum-associated abortion in cattle: the time of experimentally-induced parasitemia during gestation determines fetal survival. Parasitology 121, 347358. Wizel, B., Nunes, M., Tarleton, R., 1997. Identication of Trypanosoma cruzi trans-sialidase family members as targets of protective CD8+ TC1 responses. J. Immunol. 159, 61206130. Wizel, B., Garg, N., Tarleton, R.L., 1998. Vaccination with trypomastigote surface antigen 1-encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection. Infect. Immun. 66, 50735081. Wright, I.G., Casu, R., Commins, M.A., Dalrymple, B.P., Gale, K.R., Goodger, B.V., Riddles, P.W., Waltisbuhl, D.J., Abetz, I., Berrie, D.A., Bowles, Y., Dimmock, C., Hayes, T., Kalnins, H., Leatch, G., Scheiwe, P.C., Smith, W., Rode-Bramanis, K., White, M.A., 1992. The development of a recombinant Babesia vaccine. Vet. Parasitol. 44, 313. Wrightsman, R.A., Manning, J.E., 2000. Paraagellar rod proteins administered with alum and IL-12 or recombinant adenovirus expressing IL-12 generates antigen-specic responses and protective immunity in mice against Trypanosoma cruzi. Vaccine 18, 14191427. Zenner, L., Estaquier, J., Darcy, F., Maes, P., Capron, A., Cesbron-Delauw, M.-F., 1999. Protective immunity in the rat model of congenital toxoplasmosis and the potential of excreted-secreted antigens as vaccine components. Parasite Immunol. 21, 261272.