The antigenic composition of Neospora caninum

International Journal for Parasitology 29 (1999) 1175±1188

The antigenic composition of Neospora caninum A. Hemphill a, *, N. Fuchs a, S. Sonda a, A. Hehl b a

Institute of Parasitology, Faculties of Veterinary Medicine and Medicine, University of Bern, LaÈnggass-Strasse 122, CH-3012 Bern, Switzerland b Institute of Parasitology, University of ZuÈrich, Winterthurerstrasse 266, CH-8057 ZuÈrich, Switzerland Received 18 January 1999; received in revised form 25 May 1999; accepted 25 May 1999

Abstract Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identi®ed. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identi®ed, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are de®nitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and puri®cation of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level speci®c antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

1. Introduction Neospora caninum is an apicomplexan parasite which is structurally very similar to, but antigenically distinct from, the closely related Toxoplasma gondii. Neospora caninum causes neosporosis, namely neuromuscular disorders, * Corresponding author: Tel: 0041 31 6312384; fax: 0041 31 6312622; e-mail: [email protected]

paralysis and death in dogs, and abortion and neonatal morbidity in cattle, sheep, goats, horses and deer. Due to its high prevalence in cattle, N. caninum has now emerged as an important cause of bovine abortion worldwide, and neosporosis has been recognised as an economically important disease with considerable impact on the livestock industry [1, 2]. The pioneering work of Dubey and co-workers has established N. caninum as an independent

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 8 5 - 5

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species [3±5]. However, BjerkaÊs et al. [6] were the ®rst to describe N. caninum as an unidenti®ed cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. In 1988, Dubey and coworkers identi®ed a similar parasite causing neurological disorders, paresis, paralysis, and even death in dogs in the USA. They isolated the parasite in cell culture, and subsequently proposed a new genus, Neospora, and species, Neospora caninum [3, 4]. For the serological detection of anti-N. caninum antibodies, an IFAT was developed, and polyclonal rabbit antisera against N. caninum tachyzoites were generated for immunohistochemical detection of the parasite in paran-embedded tissue sections [7]. Using these diagnostic tools, BjerkaÊs and Dubey [8] demonstrated that the parasites identi®ed in 1984 in Norway were indeed N. caninum.

2. Some aspects of the biology of Neospora caninum Neospora caninum is an obligate intracellular parasite, and tachyzoites have been detected in a variety of tissue and cell types [1, 5]. This suggests that N. caninum exhibits no, or only little, host cell speci®city, being capable of invading a wide range of nucleated cells. The tachyzoites, enclosed in a parasitophorous vacuole, proliferate by endodyogeny, producing several hundred new parasites in a few days p.i. Proliferating tachyzoites form a membrane bound pseudocyst, and as this pseudocyst has reached a critical mass, host cell lysis and release of tachyzoites occurs, which subsequently infect neighbouring cells [2]. In contrast, the slowly dividing N. caninum bradyzoites are, as is the case for T. gondii, capable of forming intracellular tissue cysts surrounded by a solid cyst wall. Tissue cysts can persist within an infected host for several years without causing any signi®cant clinical manifestations [1]. N. caninum tissue cysts containing bradyzoites have been detected almost exclusively in the central nervous system [9]. Neospora caninum is a major cause of abortion of cattle worldwide [1, 2, 10±12]. In 1993, Conrad

et al. [13] isolated N. caninum from aborted bovine foetuses, and using this ®rst bovine isolate, bovine fetal infection and death was reproduced experimentally [14]. Thus, transplacental infection through tachyzoites, as in the case of congenital toxoplasmosis, presents a recognised mode of transmission of neosporosis. This process can occur repeatedly in the same animal [4, 6, 15]. However, prenatal infection of the foetus does not always lead to disease, and the parasite might reside silently within tissues in clinically normal o€springs. Experimental transplacental transmission has been induced in several other species, e.g. dogs [16], cats [17], sheep [18±20], cattle [14], goats [21], mice [22, 23], pigs [24], and even in non-human primates [25]. Moreover, oral inoculation of N. caninum into neonatal calves by adding the parasites to the colostrum revealed that oral infection by tachyzoites through colostrum could be an additional route of vertical transmission in newborn calves [26]. Another mode of transmission takes place through the ingestion of tissue harbouring N. caninum tissue cysts [27, 28]. While earlier studies failed to show conclusively whether cats or dogs would be possible de®nitive hosts of N. caninum, more recent oral inoculation experiments performed by McAllister et al. demonstrated that cats are most likely not [29], but that dogs are a de®nitive host of N. caninum [30]. In terms of its phylogenetic status, N. caninum is currently placed into the family Sarcocystidae and is established as a sister group to T. gondii in the phylum Apicomplexa [31]. Until recently, N. caninum was the only member of the genus Neospora. However, Marsh et al. isolated an apicomplexan parasite from the central nervous system of an adult equine which was ultrastructurally very similar to Neospora. It shows distinct di€erences to N. caninum of bovine and canine origin with respect to immunoreactive proteins, and a seven nucleotide di€erence in the sequence of the internal transcribed spacer 1 (ITS1) region was found. This suggests that this isolated parasite could represent a new Neospora species and it was named Neospora hughesi [32].

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3. In vitro cultivation of Neospora caninum Cultivation of N. caninum tachyzoites has been achieved in many host-cell types, both primary cells and established cell lines. Essentially the same techniques for cultivation and cryopreservation as previously described for T. gondii tachyzoites can be applied [33]. The proliferation rate seems to vary among di€erent Neospora isolates, and is also dependent on which host cells are used. Extracellular maintenance of N. caninum tachyzoites in the presence of growth medium for longer than 4 h results in a rapid loss of infectivity [34]. This is in contrast to what has been reported for T. gondii tachyzoites which remain infective after extracellular maintenance for up to 72 h (De Braganca K, Peschka B, Peters B, Seitz HM. How long does the obligate intracellular parasite Toxoplasma gondii survive in cell free medium? Proceedings of the 10th Japanese±German Cooperative Symposium on Protozoan Diseases, September 11±15 1996, Hannover, Germany). In vitro cultures were employed for assaying the susceptibility of N. caninum tachyzoites to over 40 chemotherapeutic agents [35±38]. The tissue culture system has also been used for investigating in more detail the processes which occur during adhesion and invasion of host cells [34], and has also enabled genetic manipulation of the parasite. Using the existing DNA vectors originally developed for T. gondii, it has been shown that T. gondii proteins are faithfully expressed and correctly targeted in N. caninum tachyzoites [39, 40]. Surely, investigations dealing with the basic biology and pathology of neosporosis will bene®t from the development of molecular genetic tools for N. caninum. Not only the tachyzoite stage, but also the bradyzoite stage of the parasite can be induced to develop in vitro for a limited time (Weiss LM, Ma Y. In vitro development of bradyzoites and cysts of Neospora caninum. Molecular Parasitology Meeting VIII, September 24±28 1997, Marine Biological Laboratory: Woods Hole, MA, USA) employing the protocol previously described for in vitro tissue cyst formation of T. gondii [41]. However, these in vitro

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generated tissue cysts remain to be thoroughly characterised. In vitro cultivation has enabled the development of diagnostic tools for detection of Neospora infections, and especially for discriminating neosporosis from infections with the closely related Toxoplasma and Sarcocystis species. These include indirect methods such as demonstration of antibodies in blood, or direct methods such as immunohistopathological and histopathological visualisation of the parasite or its immunopathological e€ects, in vitro isolation of parasites, or the detection of parasite DNA by PCR (reviewed in [1, 2, 42]). Indirect diagnosis of N. caninum infections rely on the detection of antibodies speci®cally directed against N. caninum antigens. Serological assays are important tools for demonstrating exposure to the parasite and assessing the risks of acquiring neosporosis. Speci®c ELISAs [43±46] and agglutination tests [47, 48] have been developed. Bacterially expressed recombinant antigens have been produced which are useful for serodiagnosis in bovine neosporosis [49, 50]. 4. Host±parasite interactions and antigen detection In terms of identifying Neospora molecules which could be used as vaccines, and with respect to developing tools for further improving immunodiagnosis, the characterisation of parasite proteins and glycoproteins which take part in the complex relationship between parasite and host is of prime importance. On the one hand, it is obvious that the host±parasite relationship is strongly dependent on the host immune reaction, which will be mainly targeted towards those parasites or parasite components, which are directly accessible to the actions of the host immune system. This process largely determines the outcome of the infection, and contributes to either the elimination or survival of the parasite. Similar to what has been found in T. gondii infections, the immune response will primarily target and eliminate fast-growing tachyzoites, and drive the development towards the persistent and immunologically much less reactive, encysted

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bradyzoites. The interconversion between the two stages during the chronic phase of the N. caninum infection, however, appears to be less restricted in certain tissues at least, as demonstrated by the repeated vertical transmission to o€spring, whereas vertical transmission of T. gondii is always associated with seroconversion during pregnancy. Thus, the immunobiology of N. caninum infections needs to be investigated, and antigenic components which contribute to humoral and cellular immune responses are likewise of importance. On the other hand, host± parasite interactions take place at the cellular level, with parasite and the target cell establishing direct physical, most likely receptor-mediated, contact. This level of interaction is most important for intracellular parasites, since recognition of suitable receptors on the host cell surface could be crucially involved in triggering subsequent host cell invasion mechanisms. An antigen suitable for immunodiagnosis must exhibit immunogenic properties in that it induces a strong humoral immune response which allows discrimination between closely related species. In addition, this antigen should be suciently abundant, accessible for the immune system, and if considered for vaccination, the immune response (humoral and/or cellular) must induce protection against challenge with highly virulent strains. It has been shown in the past, that many of the antigens identi®ed in related apicomplexan parasites are either associated with surface structures, or they are found localised within the secretory organelles, namely micronemes, rhoptries and dense granules. These localisations imply that some of the corresponding molecules might carry out important functions during host cell entry or intracellular development of these parasites (Fig. 1). A considerable amount of work has been invested in identifying and characterising N. caninum tachyzoite proteins which take part in the complex interactions between parasite and host. In addition, associated comparative studies, especially in relation to T. gondii, were performed to ®nd out whether antigenic di€erences between these parasites would be useful in developing a diagnostic assay for N. caninum. Several

Fig. 1. Scanning (SEM) and transmission (TEM) electron microscopy of in vitro cultivated Neospora caninum tachyzoites (NC-1-isolate). (A) Tachyzoites interacting with a bovine macrophage. The arrows indicate those parasites which are in the process of achieving host cell entry. Bar =5.7 mm. (B) Longitudinal section. DG = dense granules, R = rhoptries, M = micronemes. Bar = 1 mm.

approaches, such as raising mAbs or generating polyclonal antisera directed against whole parasites, immunoscreening of cDNA expression libraries with sera from infected cattle, or subcellular fractionation of parasites and preparation of anity-puri®ed, monospeci®c antibodies or mAbs directed against particular parasite antigens, were used. 4.1. The use of monoclonal antibodies (mAbs) Several mAbs directed against N. caninum tachyzoites or subcellular fractions have been raised so far. The ®rst one to be useful for the

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diagnosis of N. caninum infections by immunohistochemistry was originally reported by Cole in 1993 [51]. Subsequent immunoblotting studies, however, demonstrated that this mAb bound to an epitope on eight major and several minor N. caninum antigens with Mr s ranging between 31 and 97.4 kDa [52]. In addition, reactivity with a T. gondii tachyzoite antigen with an Mr of 107 kDa was also observed. Immunogold labelling showed that the antibody recognised an epitope associated with micronemes, dense granules, basal portions of the rhoptries, and the tubulo± vesicular network within the parasitophorous vacuole. In T. gondii tachyzoites, micronemes and the basal portions of the rhoptries were also stained [52]. Baszler et al. [53] generated a more speci®c mAb (named mAb 4A4±2) against N. caninum tachyzoites. This mAb recognised a 65 kDa surface antigen. Binding of this mAb was inhibited by mild periodate treatment of N. caninum antigen, indicating that the epitope involved was of carbohydrate nature. They also demonstrated that antisera obtained from cows with con®rmed Neospora-induced abortions reacted consistently with antigens of 25, 65 and 116 kDa Mr on immunoblots, and binding of the mAb 4A4±2 to N. caninum antigen was consistently inhibited by these sera. Based on these ®ndings they developed a competitive inhibition ELISA (CI-ELISA) for the serological diagnosis of Neospora infections. Sera from cattle experimentally infected with T. gondii, Sarcocystis cruzi, Sarcocystis hominis and Sarcocystis hirsuta do not inhibit binding of mAb 4A4±2 to N. caninum antigen in the CI-ELISA, demonstrating the speci®city of this assay [53]. A chicken mAb raised against Eimeria acervulina sporozoites had previously been shown to recognise the conoid of these parasites and to inhibit sporozoite invasion of lymphocytes in vitro. This antibody also stained the conoid from six other species of Eimeria, as well as the apical complexes of both T. gondii and N. caninum tachyzoites. This indicates that this mAb identi®ed a conserved epitope on the conoid which is important in host cell invasion by apicomplexan parasites [54].

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Using speci®c surface labelling, seven distinct surface antigens of N. caninum tachyzoites of Mr ranging between 17 and 56 kDa were identi®ed (Schares G, Conraths FJ. Characterization of surface antigens of Neospora caninum tachyzoites. Proceedings COST 820, Vaccines against Animal Coccidiosis, October 10±12 1996, Copenhagen, Denmark). All but one (a 56 kDa antigen) could be immunoprecipitated using sera from N. caninum infected animals. Surface protease digestion by chymotrypsin and trypsin showed that the 42 and 43 kDa antigens were sensitive to these proteolytic enzymes, while surface antigens of 29, 32 and 35 kDa exhibited only limited sensitivity. Most of the epitopes of the surface antigens showed conformational dependency, and the 32 kDa antigen appeared to be a glycoprotein. Subsequently, mAbs directed against N. caninum tachyzoites were raised in order to analyse these proteins with respect to their function in host cell invasion (Schares G, Dubremetz JF, Loyens A, BaÈrwald A, Conraths FJ. Characterization of Neospora caninum antigens by monoclonal antibodies. VIIth International Coccidiosis Conference and European Union COST 820 Workshop, September 1±5 1997, Keble College: Oxford, England). These antibodies were characterised using immunoblotting, immunoprecipitation, indirect immuno¯uorescence and immunogold EM. Several of these antibodies were directed against three of the abovementioned surface antigens of N. caninum with Mr (as determined by SDS±PAGE under non-reducing conditions) of 17, 42 and 43 kDa. Another antibody directed against a dense granule antigen of 35 kDa was also generated. A set of mAbs, directed against N. caninum iscom antigen, were characterised by BjoÈrkman and Hemphill [55]. Iscoms are immunostimulating complexes with a cage-like structure of about 40 nm in diameter. They are composed of QuilA, cholesterol, phospholipids and antigen. Their main applications have been as adjuvants and as carriers of immunogens in vaccines. Neospora iscoms have been used earlier as antigens in ELISA systems for demonstrating the presence of antibodies to N. caninum in sera from dogs and cattle [43, 56]. The method used for construction

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of the iscoms was designed to select for membrane antigens. However, it was not known if the incorporated antigens were of extra- or intracellular origin. Six mAbs raised to N. caninum iscoms were used in western blots in order to test their reactivity with non-reduced and reduced N. caninum extracts, as well as with N. caninum iscoms. These antibodies recognised antigens of 18, 30/32, 41 and 65 kDa. Antibodies directed against the 30/32 kDa doublet, the 18 kDa antigen and against the 41 kDa band bound to the tachyzoite surface. As methanol-permeabilised tachyzoites were labelled by immuno¯uorescence, additional punctated cytoplasmic staining, indicative for dense granule organelles, was demonstrated. By immunogold on-section labelling of N. caninum tachyzoites the presence of the 30/ 32 kDa doublet on both the surface and within the dense granules of the parasite was con®rmed. Western-blotting showed that the epitope recognised on the 18 kDa antigen was at least partially composed of carbohydrate residues. The mAbs reacting with the 65 kDa protein failed to detect any epitopes in western blots with non-reduced antigen of both crude N. caninum extracts and iscoms. However, this mAb stained the apical pole by immuno¯uorescence after permeabilisation of tachyzoites. The mAbs directed against N. caninum iscoms did not react with T. gondii tachyzoites, neither by immunoblotting, nor by immuno¯uorescence. Their usefulness for the speci®c immunohistochemical detection of N. caninum in paran-embedded tissue is currently being assessed. Another set of mAbs directed against a N. caninum membrane protein fraction was generated by Howe et al. [57]. By employing these mAbs they characterised the p29 and p35 immunodominant antigens of N. caninum tachyzoites. These antigens were identi®ed by probing immunoblots of tachyzoite extracts with antisera from mice, canines and bovines which were experimentally or naturally infected with N. caninum tachyzoites (see below).

4.2. Approaches involving polyclonal antisera In 1992, Barta and Dubey [58] were the ®rst to characterise a rabbit anti-N. caninum hyperimmuneserum raised against intact N. caninum tachyzoites by western blot analysis and immuno-EM. They identi®ed approximately 20 immunodominant antigens with Mr ranging between 16 and 80 kDa. Parasite antigens separated by non-reducing SDS±PAGE were recognised more intensely than antigens separated under reducing conditions, indicating the presence of conformational epitopes. These antigens were localised predominantly within the dense granules, in the micronemes, the posterior end of the rhoptries, and on the parasitophorous vacuole membrane. No labelling of the N. caninum tachyzoite surface could be detected. Bjerkas et al. [59] showed that immune sera from a wide range of animal species exhibited a similar recognition pattern when visualised by immunoblotting, with ®ve major (17, 27, 29, 30 and 46 kDa) and several minor N. caninum antigenic bands. As determined by immuno-EM using monospeci®c antibodies, the 17 kDa antigen was found to be localised within the body part of the rhoptries, while the 29 and 30 kDa antigens were distributed over the parasitophorous vacuole network, the dense granules and on the parasitophorous vacuole membrane. Again, no tachyzoite surface staining was found. In addition, it was demonstrated that a polyclonal rabbit hyperimmuneserum directed against N. caninum tachyzoites did not exhibit labelling of external membranes. In contrast, a rabbit antiT. gondii hyperimmuneserum exhibited a marked surface staining of T. gondii tachyzoites [59]. Another approach, leading to the identi®cation of two dense granule proteins in N. caninum tachyzoites, involved immunoscreening of a N. caninum tachyzoite cDNA library with sera from N. caninum infected cows. Subcloning of the two cDNAs and expression of respective recombinant proteins in Escherichia coli lead to the identi®cation of these antigens as useful candidates to detect anti-N. caninum antibodies by ELISA [50]. Polyclonal antisera against recombinant NCDG1 and NCDG2 were generated and were used for

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further characterisation of these antigens. The respective cDNA clones were further analysed. They both encode dense granule proteins of 33 kDa (NCDG1) [60] and 36 kDa (NCDG2) [61]. Sequence analysis of a full length cDNA clone encoding NCDG1 revealed that it contained three hydrophobic regions, namely a putative signal sequence at the N-terminus, and two additional ones. The third hydrophobic region represented a putative transmembrane region [60]. Taken together, the predicted aa sequence of NCDG1 shared structural similarity with other dense granule proteins from T. gondii [62]. NCDG2 appears to be closely related to the T. gondii dense granule protein GRA6, with 47% nucleotide sequence identity [61]. In order to identify potential surface membrane components of N. caninum tachyzoites, the following approach was used in our laboratory. Puri®ed tachyzoites were fractionated using the non-ionic detergent Triton-X-114. Analysis by SDS±PAGE of this membrane protein fraction and subsequent immunoblotting using a polyclonal rabbit anti-N. caninum antiserum demonstrated major reactive bands of approximately 43, 36 and 33 kDa. The polyclonal antiserum was anity-puri®ed on these three bands, and the resulting antibodies were subsequently shown to be uniquely directed against their corresponding antigens named Nc-P43, Nc-P36 and Nc-P33. The anity-puri®ed antibodies were used to further characterise these three proteins. Immunoblotting, immuno¯uorescence staining and immunogold EM of N. caninum tachyzoites demonstrated that Nc-P43 and Nc-P36 were indeed major tachyzoite surface proteins [63±66], while the 33 kDa protein was a dense granule-associated protein and identical to the previously identi®ed NCDG1 [67]. Shortly after invasion, Nc-P33/NCDG1 was targeted to the parasitophorous vacuole membrane, and, at later timepoints after infection, was also found on the parasitophorous vacuolar network. This suggested that this protein could play a functional role in the modi®cation of the parasitophorous vacuole and its membrane. Nc-P33/ NCDG1 and Nc-P43 were detected in both the tachyzoite and the bradyzoite stage of N. cani-

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num, while Nc-P36 was exclusively expressed in tachyzoites [68]. Both N. caninum surface proteins were antigenically distinct from the surface proteins present on T. gondii tachyzoites and bradyzoites. However, Nc-P36 exhibits a striking aa sequence similarity (76.3% similarity with 51.3% identities) to P30 (SAG1) the major T. gondii tachyzoite surface protein which has previously been implied in attachment to host cells [66, 69] (see Fig. 2). The deduced aa sequence of Nc-P43 was also determined (GenBank accession No. U93870). Comparison of the Nc-P43 sequence with Toxoplasma surface antigens shows a high degree of similarity to the members of the family of SAG1-related antigens, with the highest similarity (44% identical aa) to the SAG1-related sequence 2 (SRS2) protein (GenBank accession number AF012276). The degree of conservation is less than for the SAG1/Nc-P36 pair, but more careful analysis unambiguously shows the preservation of an overall architecture which is a hallmark for the members of this family of GPIanchored surface antigens (Fig. 2). All 12 cysteine residues in the mature proteins after cleavage of the putative N-terminal signal sequence can be aligned without introduction of signi®cant gaps. In addition to cysteines which form disul®de bridges and are involved in secondary structure formation, other structural aas such as prolines are strongly conserved, suggesting that the orthologs are under selective pressure to preserve their overall topology despite considerable accumulated sequence divergence. These ®ndings are identical to the ones obtained by Howe et al. [57] who have independently identi®ed these antigens using antisera from mice, canines and bovines which were experimentally or naturally infected with Neospora. The two most immunodominant ones were of 29 and 35 kDa Mr , and aa sequence analysis showed that they were identical to Nc-p43 and Nc-p36. Di€erences in Mr are attributed to di€erent SDS±PAGE separation conditions. According to the sequence similarities with SAG1 and SRS2, Howe et al. proposed to name these antigens NcSAG1 (for Nc-p36) and Nc-SRS2 (for Nc-p43), respectively [57]. MAbs

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Fig. 2. Pairwise alignments of Nc-P36 (A) and NcP43 (B) with their respective Toxoplasma gondii orthologues. Identical positions are in black, similar amino acids are shaded. Conserved cysteines are marked with an asterisk. The alignments were generated with CLUSTALW [82] via the world wide web at http://ferrari.ibcp.fr/cgi-bin/Mail_clustalw.pl using the blosum weight matrix and default settings, with minor manual editing. For further information refer to [65] and [69].

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directed against these two proteins were used to characterise the biochemical features and investigate the localisation of these antigens. Surface labelling with biotin and immunogold EM showed that these two proteins were displayed on the surface of the parasites. Parasite lysates were analysed by SDS±PAGE following phospholipase C-treatment, and western blotting using antibodies directed against the crossreacting determinant of GPI anchors revealed the presence of six glycolipid anchored surface proteins, including NcSAG1 (Nc-p36) and NcSRS2 (Nc-p43) [57]. Phylogenetic analysis of the two major Neospora surface antigens in the context of the 10 known members of the SAG1 family emphasises the striking degree of conservation between Nc-P43/SRS2 and Nc-P36/SAG1 orthologs (Fig. 3). Considering the full-length aa sequences, it was found that the corresponding proteins from the two genera are more closely related than the members of the T. gondii SAG1 family to each other, with the exception of the SAG5 group which possibly represents two relatively

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recent gene duplication events (Spano F, Ricci I, Di Crisanti M, Puri C, Crisanti A. The growing family of SAG1-related antigens of Toxoplasma gondii: molecular characterization and expression analysis of the SAG5 gene cluster. Proceedings European Union COST 820 Annual Workshop, October 22±25 1998, Toledo, Spain). This observation together with the apparent lack of crossreactive epitopes is especially interesting in terms of the biology, evolution, host-range, and pathogenicity of these phenotypically related coccidian parasites. Thus, although the surface proteins SAG1, SRS2, Nc-P43 and Nc-36 exhibited distinct antigenic and biochemical di€erences, their localisation and their similarities in a putative secondary structure strongly suggest a common function. Recombinant Nc-P43 and Nc-P36 expressed in E. coli are currently evaluated as potential vaccine candidates in a murine model (Eperon S, Fuchs N, Sonda S, Hemphill A, Gottstein B. Vaccination of mice with recombinant proteins against Neospora caninum. Proceedings European Union COST 820 Annual Workshop, October 22±25 1998, Toledo, Spain). Polyclonal antibodies directed against the T. gondii bradyzoite antigen 5 (BAG5, also designated BAG1/hsp30) expressed as a recombinant protein in E. coli were generated [70]. Staining of paran sections of mouse brain tissue harbouring N. caninum tissue cysts demonstrated the presence of crossreactive epitopes between N. caninum and T. gondii bradyzoites. This antiserum represents a valuable tool for the study of stage conversion events occurring during di€erent phases of N. caninum infections. 4.3. Antigens mediating cellular immune responses

Fig. 3. Phylogenetic relationship of the 10 Toxoplasma gondii SAG1 family members and Neospora caninum homologues. The distance matrix was generated with the GCG program suite (Genetics Computer Group, Madison Wisc., USA). The rooted phylogram represented here was constructed using `GrowTree' and the UPGMA method.

Since N. caninum, like T. gondii, is an obligate intracellular parasite, not only antibody synthesis, but also the cell mediated immune mechanisms, are important elements of the immune response against N. caninum. In fact, a vaccine against neosporosis might be only successful if both humoral and cell mediated immune responses are stimulated. Kasper and Kahn [71] demonstrated that vaccination of mice with intact N. caninum tachyzoites protects these mice

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against a lethal challenge from T. gondii, and that this protection is mediated by antigen-crossreactive CD8 + T-cells obtained from spleens of N. caninum vaccinated mice. These observations di€er signi®cantly from those in earlier studies which indicated that immunisation of mice did not protect against lethal challenge from T. gondii [72]. However, those studies had employed the very virulent T. gondii RH strain, and it appears that protection is highly dependent on the strain used for challenge. This was con®rmed by Lindsay et al. [73] who investigated the e€ect of vaccination of mice with N. caninum with respect to oral challenge with T. gondii oocysts. This indicated that infection with N. caninum provides some protection against fatal infection with T. gondii oocysts of a moderately pathogenic strain, but not against tachyzoites of a highly pathogenic strain. The protection achieved by N. caninum vaccination is much less than that provided by previous exposure to T. gondii [73]. Aspects of the cellular immune responses in cattle experimentally infected with N. caninum have now been studied more recently [74]. Signi®cant proliferative responses against crude N. caninum lysate was observed in peripheral blood mononuclear cells (PBMC) from infected calves from days 4±6 p.i. In addition, high levels of interferon gamma were recorded. In addition PBMC did also proliferate in response to crude T. gondii lysate, but only moderate interferon gamma levels were recorded, and no antibodies directed against T. gondii could be detected in respective sera. This suggested that the two parasites may possess crossreacting T-cell epitopes, but that the T-cells speci®c for N. caninum may have a di€erent functional capacity [74]. A group of low Mr (<30 kDa) N. caninum antigens was identi®ed which, separated by SDS±PAGE and bound to nitrocellulose, stimulated CD4 T-cell proliferation in vitro from calves experimentally infected with N. caninum [75]. Again proliferation was accompanied by high levels of interferon gamma. Several of these antigens were also recognised by antibodies produced by these animals. Taken together these studies provide an important basis for further research on antigens capable of stimulating cellular immune responses.

4.4. Molecular genetic approaches In the last two years novel approaches to accelerate gene discovery have been introduced in T. gondii which have a direct impact on antigen research in N. caninum in several ways. In a multicentre collaboration more than 10 000 expressed sequence tags (ESTs) were generated from cDNA libraries from T. gondii tachyzoites and bradyzoites [76±79]. Of those, approximately 30% were assigned putative identities based on homology with sequences in GenBank using relatively stringent criteria. Among the identi®ed ESTs were many known Toxoplasma genes which are highly expressed, but also a considerable number of phylogenetically restricted genes from the Apicomplexa including a previously described gene coding for a Neospora dense granule antigen [77]. These would be of signi®cant interest, both in terms of the biology of parasites belonging to this phylum, which are characterised by the presence of specialised apical organelles, and also as potential targets for development of new drugs and vaccines. Even though T. gondii and N. caninum share antigens with a considerable degree of sequence conservation at the aa level, such as the SAG1 related surface antigens, it will in most cases not be sucient to isolate related antigens from Neospora by degenerate PCR based on T. gondii sequence information. The current bene®t of this database for Neospora research (until a similar project can be initiated) lies in the much increased possibility to rapidly identify novel Neospora antigens based on homology with apicomplexan sequences in the rapidly growing EST database, and thus facilitates functional and immunological analysis. A N. caninum antigen identi®ed through the presence of conserved sequences is the homologue of the T. gondii microneme protein MIC2 (Lovett JL, Howe DK, Carruthers VB, Sibley LD. Molecular identi®cation and characterisation of Neospora caninum MIC2. Molecular Parasitology Meeting IX, September 13±17 1998, Marine Biology Laboratory: Woods Hole, MA, USA). MIC2 is a member of the TRAP protein family found in apicomplexan parasites (reviewed in [80]). These proteins are composed of a N-

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terminal integrin (I)-like domain, a variable number of thrombospondin (TSP)-like repeats, a transmembrane domain, and a C-terminal cytoplasmic extension. Degenerate PCR primers to conserved regions in the I domain and TSP repeats of known TRAP homologues were used to identify the MIC2 homologue in Neospora. Neospora MIC2 shares 61% identity with T. gondii MIC2 with respect to its aa sequence, and also localises to the micronemes. The highly developed molecular genetics and transformation techniques in T. gondii, together with the scarcity of epitopes which crossreact with anti-Neospora antisera, make this model apicomplexan a useful null-background to express and identify novel antigens. Heterologous expression of Toxoplasma proteins in N. caninum has already been achieved. Two independent studies investigating expression and/or tracking of the Toxoplasma proteins SAG1, GRA2, NTPase3 and ROP2, have been performed on the NC-1 strain by using transfection techniques and vectors developed for T. gondii [39, 40], demonstrating the feasibility of transfection-based approaches for heterologous expression and functional analysis of antigens. In view of these results it seems possible to use similar techniques in gene discovery experiments, for example by expressing a N. caninum cDNA library in T. gondii and using FACS techniques in combination with speci®c antisera to screen for novel surface antigens. In particular, the recent development of shuttle vectors for overexpression of proteins [81] would greatly facilitate the isolation of identi®ed genes. The main impact of heterologous expression, however, will be in the functional analysis of vaccine candidates, virulence factors and potential drug targets. 5. Concluding remarks During this decade, N. caninum has attracted considerable attention as an important causative agent of abortion in cattle and neuromuscular disease in dogs. Major e€orts have been invested in order to develop sensitive and speci®c molecular tools for assessing the presence of this para-

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site within tissues and body ¯uids. These tools often rely on the detection of speci®c antigens through direct (immunohistochemistry) or indirect (serological) diagnostic methods. Thus, diagnostic antigens play a major role in assessing the relevance of neosporosis as a potential risk factor for animal, but also for human, health and they are prerequisites for carrying out sero-epidemiological investigations. However, N. caninum also provides an ideal model organism, complementary to T. gondii, for the investigation of the basic biology of intracellular parasitism. Diagnostically useful antigens have been found to be often localised either on the parasite surface, or in those secretory organelles which apparently play a role during and after the host cell entry process (micronemes, rhoptries and dense granules). Molecular approaches, such as transfection of parasites employing similar techniques as those previously developed for Toxoplasma, will be increasingly used to elucidate the pathogenetic events, as well as the functional signi®cance of these antigens during the course of neosporosis, but these techniques will also be important to prepare potential new immunotherapeutic tools for future vaccination against infection or disease mediated by N. caninum.

Acknowledgements We acknowledge Toni Wyler and Maja Suter (Institute for Zoology and Institute for Veterinary Pathology, respectively) and Rudolf Giovanoli (Department of Chemistry and Biochemistry) for allowing the use of their electron microscopy facilities, and Norbert MuÈller for many suggestions and critical reading of the manuscript. Moreover, it is a great pleasure to thank Bruno Gottstein (Institute of Parasitology, University of Bern) for his constant encouragement and enthusiastic support. This work was ®nanced through a grant from the Swiss National Science Foundation (project grant No. 31.46846.96), the COST 820 grant (No. C96.0068) provided by the `Bundesamt fuÈr Bildung und Wissenschaft', and the `Stiftung zur

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Foerderung der wissenschaftlichen Forschung der UniversitaÈt Bern'.

References [1] Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis. Vet Parasitol 1996;67:1±59. [2] Hemphill A. The host±parasite relationship in neosporosis. Adv Parasitol 1999; 43, 47±104. [3] Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988;192:1269±85. [4] Dubey JP, Hanel AL, Lindsay DS, Topper MJ. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J Am Vet Med Assoc 1988;193:12591263. [5] Dubey JP, Lindsay DS. Neosporosis. Parasitol Today 1993;9:452±8. [6] BjerkaÊs I, Mohn SF, Presthus J. Unidenti®ed cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Zeitschr Parasitenkunde 1984;70:271±4. [7] Lindsay DS, Dubey JP. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am J Vet Res 1989;50:1981±3. [8] BjerkaÊs I, Dubey JP. Evidence that Neospora caninum is identical to the Toxoplasma-like parasite of Norwegian dogs. Acta Vet Scand 1991;32:407±10. [9] Lindsay DS, Speer CA, Toivio-Kinaucan MA, Dubey JP, Blagburn BL. Use of infected cultured cells to compare ultrastructural features of Neospora caninum from dogs and Toxoplasma gondii. Am J Vet Res 1993;54:103± 6. [10] Thilsted JP, Dubey JP. Neosporosis-like abortions in a herd of dairy cattle. J Vet Diagn Invest 1989;1:205±9. [11] Anderson ML, Blanchard PC, Barr BC, Dubey JP, Ho€man RL, Conrad PA. Neosporalike protozoan infection as a major cause of abortion in California dairy cattle. J Am Vet Med Assoc 1991;198:241±4. [12] Barr BC, Anderson ML, Dubey JP, Conrad PA. Neospora-like protozoal infection associated with bovine abortions. Vet Pathol 1991;28:110±6. [13] Conrad PA, Barr BC, Sverlow KW et al. In vitro isolation and characterization of a Neospora sp. from aborted bovine fetuses. Parasitol 1993;106:239±49. [14] Barr BC, Rowe JD, Sverlow KW et al. Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate. J Vet Diagn Invest 1994;6:207±15. [15] Barr BC, Conrad PA, Breitmeyer R et al. Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: four cases (1990±1992). J Am Vet Med Assoc 1993;202:113±7. [16] Cole RA, Lindsay DS, Blagburn BL, Sorjonen DC, Dubey JP. Vertical transmission of Neospora caninum in dogs. J Parasitol 1995;81:208±11.

[17] Dubey JP, Lindsay DS. Transplacental Neospora caninum infection in cats. J Parasitol 1989;75:765±71. [18] McAllister MM, McGuire AM, Jolley WR, Lindsay DS, Trees AJ, Stobart RH. Experimental neosporosis in pregnant ewes and their o€spring. Vet Pathol 1996;33:647± 55. [19] Buxton D, Maley SW, Thomson KM, Trees AJ, Innes EA. Experimental infection of non-pregnant and pregnant sheep with Neospora caninum. J Comp Pathol 1997;117:1±16. [20] Buxton D, Maley SW, Wright S, Thomson KM, Rae AG, Innes AE. The pathogenesis of experimental neosporosis in pregnant sheep. J Comp Pathol 1998;118:267± 79. [21] Lindsay DS, Rippey NS, Powe TA, Sartin EA, Dubey JP, Blagburn BL. Abortion, fetal death, and stillbirth in pregnant pygmy goats inoculated with tachyzoites of Neospora caninum. American J Vet Res 1995;56:1176±80. [22] Cole RA, Lindsay DS, Blagburn BL, Dubey JP. Vertical transmission of Neospora caninum in mice. J Parasitol 1995;81:730±2. [23] Long MT, Baszler TV. Fetal loss in Balb/c mice infected with Neospora caninum. J Parasitol 1996;82:608±11. [24] Jensen L, Jensen TK, Lind P, Hendriksen SA, Uggla A, Bille-Hansen V. Experimental porcine neosporosis. APMIS 1998;106:475±82. [25] Barr BC, Conrad PA, Sverlow KW, Tarantal AF, Hendrickx AG. Experimental fetal and transplacental Neospora infection in the nonhuman primate. Lab Invest 1994;71:236±42. [26] Uggla A, Stenlund S, Holmdahl OJ et al. Oral Neospora caninum inoculation of neonatal calves. Int J Parasitol 1998;28:1467±72. [27] Dubey JP, Lindsay DS. Transplacental Neospora caninum infection in dogs. Am J Vet Res 1989;50:1578±9. [28] Dubey JP, Lindsay DS, Lipscomb T P. Neosporosis in cats. Vet Pathol 1990;27:335±9. [29] McAllister MM, Jolley WR, Wills RA, Lindsay DS, McGuire AM, Tranas JD. Oral inoculation of cats with tissue cysts of Neospora caninum. Am J Vet Res 1998;59:441±4. [30] McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are de®nitive hosts of Neospora caninum. Int J Parasitol 1998;28:1473±8. [31] Ellis J, Luton K, Baverstock PR, Brindley PJ, Nimmo KA, Johnson AM. The phylogeny of Neospora caninum. Mol Biochem Parasitol 1994;64:303±11. [32] Marsh AE, Barr BC, Packham AE, Conrad PA. Description of a new Neospora species. J Parasitol 1998;84:983±91. [33] Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton, FL: CRC Press, 1988. [34] Hemphill A, Gottstein B, Kaufmann H. Adhesion and invasion of bovine endothelial cells by Neospora caninum. Parasitol 1996;112:183±97.

A. Hemphill et al. / International Journal for Parasitology 29 (1999) 1175±1188 [35] Lindsay DS, Dubey JP. Evaluation of anti-coccidial drugs' inhibition of Neospora caninum development in cell cultures. J Parasitol 1989;75:990±2. [36] Lindsay DS, Rippey NS, Cole RA et al. Examination of the activities of 43 chemotherapeutic agents against Neospora caninum tachyzoites in cultured cells. Am J Vet Res 1994;55:976±81. [37] Lindsay DS, Butler JM, Rippey NS, Blagburn BL. Demonstration of synergistic e€ects of sulfonamides and dihydrofolate reductase/thymidylate synthase inhibitors against Neospora caninum tachyzoites in cultured cells, and characterization of mutants resistant to pyrimethamine. Am J Vet Res 1996;57:68±72. [38] Lindsay DS, Butler JM, Blagburn BL. Ecacy of decoquinate against Neospora caninum tachyzoites in cell culture. Vet Parasitol 1997;68:35±40. [39] Howe DK, Mercier C, Messina M, Sibley LD. Expression of Toxoplasma gondii genes in the closely related apicomplexan parasite Neospora caninum. Mol Biochem Parasitol 1997;86:29±36. [40] Beckers CJ, Wake®eld T, Joiner KA. The expression of Toxoplasma proteins in Neospora caninum and the identi®cation of a gene encoding a novel rhoptry protein. Mol Biochem Parasitol 1997;89:209±23. [41] Boothroyd JC, Black M, Bonnefoy S et al. Genetic and biochemical analysis of development in Toxoplasma gondii. Philosophical Transactions of the Royal Society of London Series BÐBiological Sciences 1997;352:1347±54. [42] Ellis JT. Polymerase chain reaction approaches for the detection of Neospora caninum and Toxoplasma gondii. Int J Parasitol 1998;28:1053±60. [43] BjoÈrkman C, Lunden A, Holmdahl OJM, Barber J, Trees AJ, Uggla A. Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen. Parasite Immunol 1994;16:643±8. [44] Pare J, Hietala SK, Thurmond MC. An enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora sp. infection in cattle . J Vet Diagn Invest 1995;7:352±59. [45] Dubey JP, Lindsay DS, Adams DS et al. Serologic responses of cattle and other animals infected with Neospora caninum. Am J Vet Res 1996;57:320±36. [46] Osawa T, Wastling J, Maley S, Buxton D, Innes E A. A multiple antigen ELISA to detect Neospora-speci®c antibodies in bovine sera, bovine fetal ¯uids, ovine and caprine sera. Vet Parasitol 1998;79:19±34. [47] Packham AE, Sverlow KW, Conrad PA et al. A modi®ed agglutination test for Neospora caninum: development, optimization, and comparison to the indirect ¯uorescent-antibody test and enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 1998;5:467±73. [48] Romand S, Thulliez P, Dubey JP. Direct agglutination test for serologic diagnosis of Neospora caninum infection. Parasitol Res 1998;60:50±3. [49] Louie K, Sverlow KW, Barr BC, Anderson M L, Conrad PA. Cloning and characterization of two recombinant Neospora protein fragments and their use in sero-

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

1187

diagnosis of bovine neosporosis. Clinic Diagn Lab Immunol 1997;4:692±9. Lally NC, Jenkins MC, Dubey JP. Evaluation of two Neospora caninum recombinant antigens for use in an ELISA for the diagnosis of bovine neosporosis. Clinic Diagn Lab Immunol 1996;3:275±9. Cole RA, Lindsay DS, Dubey JP, Blagburn BL. Detection of Neospora caninum in tissue sections using a murine monoclonal antibody. J Vet Diagn Invest 1993;5:579±84. Cole RA, Lindsay DS, Dubey JP, Toivio-Kinnucan MA, Blagburn BL. Characterization of a murine monoclonal antibody generated against Neospora caninum by western blot analysis and immunoelectron microscopy. Am J Vet Res 1994;55:1717±22. Baszler TV, Knowles DP, Dubey JP, Gay JM, Mathison BA, McElwain TF. Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition ELISA. J Clin Microbiol 1996;34:1423±8. Sasai K, Lillehoj HS, Hemphill A et al. A chicken monoclonal antibody identi®es a common epitope which is present on the apicomplexan coccidian parasites. J Parasitol 1998;84:654±6. BjoÈrkman C, Hemphill A. Characterization of Neospora caninum iscom antigens using monoclonal antibodies. Parasite Immunol 1998;20:73±80. BjoÈrkman C, Holmdahl J, Uggla A. An indirect enzyme linked immunoassay (ELISA) for demonstration of antibodies to Neospora caninum in serum and milk of cattle. Vet Parasitol 1997;68:251±6. Howe DK, Crawford AC, Lindsay D, Sibley LD. The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infect Immun 1998;66:5322±8. Barta JR, Dubey JP. Characterization of anti-Neospora caninum hyperimmune rabbit serum by Western blot analysis and immunoelectron microscopy. Parasitol Res 1992;78:689±94. Bjerkas I, Jenkins MC, Dubey JP. Identi®cation and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis. Clinic Diagn Lab Immunol 1994;1:214±21. Lally NC, Jenkins M, Lidell S, Dubey JP. A dense granule protein (NCDG1) gene from Neospora caninum. Mol Biochem Parasitol 1997;87:239±43. Liddell S, Lally NC, Jenkins MC, Dubey JP. Isolation of the cDNA encoding a dense granule associated antigen (NCDG2) of Neospora caninum. Mol Biochem Parasitol 1998;93:153±8. Cesbron-Delauw MF, Lecordier L, Mercier C. Role of secretory dense granule organelles in the pathogenesis of toxoplasmosis. Curr Top Microbiol Immunol 1996;219:59±65.

1188

A. Hemphill et al. / International Journal for Parasitology 29 (1999) 1175±1188

[63] Hemphill A, Gottstein B. Identi®cation of a major surface protein on Neospora caninum tachyzoites. Parasitol Res 1996;82:497±504. [64] Hemphill A, Fuchs N, Sonda S, Gottstein B, Hentrich B. Identi®cation and partial characterization of a 36 kD surface protein on Neospora caninum tachyzoites. Parasitol 1996;115:371±80. [65] Hemphill A, Felleisen R, Connolly B, Gottstein B, Hentrich B, MuÈller N. Characterization of a cDNA clone encoding Nc-p43, a major Neospora caninum surface protein. Parasitol 1997;115:581±90. [66] Sonda S, Fuchs N, Connolly B, Fernandez P, Gottstein B, Hemphill A. The major 36 kDa Neospora caninum tachyzoite surface protein is closely related to the major Toxoplasma gondii surface antigen 1. Mol Biochem Parasitol 1998;97:97±108. [67] Hemphill A, Gajendran N, Sonda S et al. Identi®cation and characterization of a dense granule-associated protein in Neospora caninum tachyzoites. Int J Parasitol 1998;28:429±38. [68] Fuchs N, Sonda S, Gottstein B, Hemphill A. Di€erential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites. J Parasitol 1998;84:753±8. [69] Tomavo S. The major surface proteins of Toxoplasma gondii: structures and functions. Curr Top Microbiol Immunol 1996;219:45±54. [70] McAllister MM, Parmley SF, Weiss LM, Welch VJ, McGuire AM. An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen. J Parasitol 1996;82:354±5. [71] Kasper LH, Kahn IA. Antigen-speci®c CD8 + T-cells protect against lethal toxoplasmosis in mice infected with Neospora caninum. Infect Immun 1998;66:1554±60. [72] Lindsay DS, Blagburn BL, Dubey JP. Infection of mice with Neospora caninum (Protozoa: Apicomplexa) does not protect against challenge with Toxoplasma gondii. Infect Immun 1990;58:2699±700.

[73] Lindsay DS, Lenz SD, Dykstra CC, Blagburn BL, Dubey JP. Vaccination of mice with Neospora caninum: response to oral challenge with Toxoplasma gondii oocysts. J Parasitol 1998;84:311±5. [74] Lunden A, Marks J, Maley SW, Innes EA. Cellular immune responses in cattle experimentally infected with Neospora caninum. Parasite Immunol 1998;20:519±26. [75] Marks J, Lunden A, Harkins D, Innes E. Identi®cation of Neospora antigens recognized by CD4 T-cells and immune sera from experimentally infected cattle. Parasite Immunol 1998;20:303±9. [76] Ajioka JW, Boothroyd JC, Brunk BP et al. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res 1998;8:18±28. [77] Ajioka JW. Toxoplasma gondii: ESTs and gene discovery. Int J Parasitol 1998;28:1025±31. [78] Hehl A, Manger ID, Boothroyd JC. Genetic analysis in Toxoplasma: gene discovery with expressed sequence tags and rapid mapping of natural polymorphisms. METHODS: A companion to Methods Enzymol 1997;13:89±102. [79] Manger ID, Hehl A, Parmley S et al. Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identi®cation of developmentally regulated genes. Infect Immun 1998;66:1632±7. [80] Naitza S, Spano F, Robson KJH, Crisanti A. The thrombospondin-related protein family of apicomplexan parasites: the gears of the cell invasion machinery. Parasitol Today 1998;14:479±84. [81] Black MW, Boothroyd JC. Development of a stable episomal shuttle vector for Toxoplasma gondii. J Biol Chem 1998;273:3972±9. [82] Thompson JD, Higgins DG, Gibson TJ. CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-speci®c gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673±80.