Cross-immunity studies between T. gondii and H. hammondi

Mice and hamsters that were experimentally infected with H. hammondi (CR-4 strain) oocysts developed immunity and did not die after challenged with lethal doses of oocysts from a mouse-virulent T. gondii strain (M-7741 strain) (Frenkel and Dubey, Reference Frenkel and Dubey1975). In contrast, cats that were experimentally infected with H. hammondi and shed oocysts were not immunized against excretion of T. gondii oocysts (Frenkel and Dubey, Reference Frenkel and Dubey1975). An additional study approached cross-immunity between T. gondii and H. hammondi, by using six H. hammondi strains in an infection model of mice and hamsters (Christie and Dubey, Reference Christie and Dubey1977). The authors observed that 103 of 108 mice that were orally inoculated with H. hammondi oocysts survived a lethal challenge dose (10⁵ oocysts) of T. gondii (strain M-7741). The H. hammondi-inoculated hamsters also developed immunity against a lethal dose of T. gondii oocysts, but this immunity was variable depending on the H. hammondi strain. The two most immunogenic H. hammondi strains conferred protection to fatal toxoplasmosis in 100 and 83% of the hamsters, respectively, after challenging with T. gondii oocysts (Christie and Dubey, Reference Christie and Dubey1977).

Immunization of goats with H. hammondi had a protective effect against abortion induced by T. gondii; however, the immunization did not prevent transplacental transmission of T. gondii in pregnant does (Munday and Dubey, Reference Munday and Dubey1988). Partial immunity against toxoplasmosis was also obtained in Tamar wallabies (Macropus eugenii), that were orally infected with 1 × 10⁵ oocysts of H. hammondi (Reddacliff et al. Reference Reddacliff, Parker, Dubey, Nicholls, Johnson and Cooper1993).

Serologic cross-reactivity between T. gondii and H. hammondi

When H. hammondi was first described, some rodent species experimentally infected with this parasite developed cross-reacting antibodies against T. gondii antigens in the DT (Frenkel and Dubey, Reference Frenkel and Dubey1975). Further studies were carried out and confirmed that serologic cross-reactivity between T. gondii and H. hammondi occurred with sera from other animals, besides rodents. Weiland et al. (Reference Weiland, Rommel and von Seyerl1979) investigated cross-reactivity between T. gondii and H. hammondi in four animal species (120 mice, six dogs, six rabbits and six pigs) by using five serological tests (DT, CFT, ELISA, IFAT and IHA). Half of the animals were orally inoculated with T. gondii oocysts and the other half received H. hammondi oocysts by the same route. Sera from H. hammondi-infected mice reacted with T. gondii antigens in three tests (DT, ELISA and CFT). Sera from dogs infected with H. hammondi recognized T. gondii antigens by DT and ELISA. Sera from rabbits exhibited cross-reaction between the two parasites by ELISA. The sera of pigs infected with H. hammondi did not cross-react with T. gondii in any of the five serological tests. In this study, the IFAT was considered the most Toxoplasma-specific method. During the course of infection, the animals infected with T. gondii presented higher titres in the tests when compared with those infected with H. hammondi (Weiland et al. Reference Weiland, Rommel and von Seyerl1979). Munday and Dubey (Reference Munday and Dubey1986) observed that sheep that were inoculated with H. hammondi oocysts presented cross-reactivity with T. gondii antigen by IFAT. Before infection with H. hammondi, the sheep had no detectable antibodies to T. gondii. After oral inoculation with H. hammondi oocysts, the animals presented antibody titres of 1:16 to T. gondii by IFAT. Goats infected with H. hammondi were shown to produce antibodies against T. gondii tested by DT, with titres up to 1:64 (Dubey, Reference Dubey1981).

The antigenic similarity between T. gondii and H. hammondi was investigated using sera from experimentally infected mice (Araujo et al. Reference Araujo, Dubey and Remington1984). The authors employed T. gondii tachyzoites (RH strain) for two antigen detection procedures: (1) the antigen was labelled with ¹²⁵I, immune-precipitated with sera from T. gondii or H. hammondi-infected mice, run by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiographed; (2) the antigen was lysed, separated by SDS–PAGE and tested by immunoblot. The sera from mice that were orally inoculated with H. hammondi oocysts recognized T. gondii antigens of MW 92·5 kDa (two antigens), 66·2 kDa, between 66·2 and 45 kDa, and between 31 and 21·5 kDa; compared with the T. gondii-positive reaction only a T. gondii antigen of 21·5 kDa was not recognized by the H. hammondi-positive mouse serum (Araujo et al. Reference Araujo, Dubey and Remington1984). This study confirmed that T. gondii and H. hammondi have similar antigenic components in their tachyzoites.

To facilitate scientific communication among research groups and laboratories, Sibley et al. (Reference Sibley, Pfefferkorn and Boothroyd1991) proposed a nomenclature system for naming mutants, genes and gene products of T. gondii, which was based on the system used for the yeast Saccharomyces cerevisiae. As an example, the surface protein (P30) was designated SAG1, which is the product of the SAG1 gene. However, as it was observed more recently that SAG1 genes belong to a superfamily of related genes, named SRS (SAG1-related sequences), which encode a superfamily of structurally related surface proteins from T. gondii, the name of SAG1 (P30) has been changed to SRS29B (Wasmuth et al. Reference Wasmuth, Pszenny, Haile, Jansen, Gast, Sher, Boyle, Boulanger, Parkinson and Grigg2012).

Riahi et al. (Reference Riahi, Bouteille and Darde1998) studied the antigenic similarity of T. gondii and H. hammondi employing five monoclonal antibodies (MAbs) against T. gondii surface antigens and a polyclonal mouse serum to H. hammondi. In order to produce enough H. hammondi antigen, the authors used an in vitro model that allows the production of H. hammondi cysts up to 3 months in cell culture (Riahi et al. Reference Riahi, Darde, Bouteille, Leboutet and Pestre-Alexandre1995). The cyst formation, confirmed by ultrastructural characteristics of the organism, started from 6 days after sporozoites were inoculated into feline kidney cells (CRFK). At 4 days of infection, the authors assumed that the multiplying H. hammondi zoites were tachyzoites (Riahi et al. Reference Riahi, Bouteille and Darde1998).

Tachyzoite antigens from T. gondii and H. hammondi were tested by IFAT and WB. By combining the two serologic techniques (IFAT and WB), five T. gondii antigens (MW of 30, 32, 35, 66 and 90 kDa) were recognized using polyclonal anti-H. hammondi serum. An interesting finding obtained by Riahi et al. (Reference Riahi, Bouteille and Darde1998) was the recognition of the SAG1 (SRS29B) antigen by the anti-H. hammondi serum, as this protein is a major antigen of T. gondii (Burg et al. Reference Burg, Perelman, Kasper, Ware and Boothroyd1988) and had been considered to be a specific marker for the parasite (Mineo et al. Reference Mineo, McLeod, Mack, Smith, Khan, Ely and Kasper1993). Despite the antigenic similarity between T. gondii and H. hammondi, MAbs targeted against H. hammondi antigens were produced, and five of them did not cross-react with T. gondii antigens (Riahi et al. Reference Riahi, Leboutet, Labrousse, Bouteille and Darde2000). These findings are promising for the characterization of H. hammondi-specific antigens or epitopes, which could enable development of serologic tests for this parasite that would not cross-react with T. gondii.

The genome of a German strain of H. hammondi was sequenced and the genomic synteny between this parasite and T. gondii was higher than 95% (Walzer et al. Reference Walzer, Adomako-Ankomah, Dam, Herrmann, Schares, Dubey and Boyle2013). It was found that orthologues of key T. gondii mouse virulence genes are functionally conserved in H. hammondi, but these data were not enough to explain the phenotypic differences observed between both parasites (Walzer et al. Reference Walzer, Adomako-Ankomah, Dam, Herrmann, Schares, Dubey and Boyle2013). In a recent work, the genomes of 62 strains of T. gondii were compared with those from H. hammondi, N. caninum and Sarcocystis neurona (Lorenzi et al. Reference Lorenzi, Khan, Behnke, Namasivayam, Swapna, Hadjithomas, Karamycheva, Pinney, Brunk, Ajioka, Ajzenberg, Boothroyd, Boyle, Darde, Diaz-Miranda, Dubey, Fritz, Gennari, Gregory, Kim, Saeij, Su, White, Zhu, Howe, Rosenthal, Grigg, Parkinson, Liu, Kissinger, Roos and David Sibley2016); these authors demonstrated that T. gondii possesses an expansion of parasite-specific secretory pathogenesis determinants (SPDs) when compared with the three latter parasites. The SPDs encompass genes encoding secretory proteins from micronemes, dense granules, rhoptries and surface antigens, whose expansion and diversity are associated with the patterns of transmission, host range and pathogenicity of T. gondii (Lorenzi et al. Reference Lorenzi, Khan, Behnke, Namasivayam, Swapna, Hadjithomas, Karamycheva, Pinney, Brunk, Ajioka, Ajzenberg, Boothroyd, Boyle, Darde, Diaz-Miranda, Dubey, Fritz, Gennari, Gregory, Kim, Saeij, Su, White, Zhu, Howe, Rosenthal, Grigg, Parkinson, Liu, Kissinger, Roos and David Sibley2016). It was reported that T. gondii shares a high number of orthologues with H. hammondi (7095) and N. caninum (6308) (Lorenzi et al. Reference Lorenzi, Khan, Behnke, Namasivayam, Swapna, Hadjithomas, Karamycheva, Pinney, Brunk, Ajioka, Ajzenberg, Boothroyd, Boyle, Darde, Diaz-Miranda, Dubey, Fritz, Gennari, Gregory, Kim, Saeij, Su, White, Zhu, Howe, Rosenthal, Grigg, Parkinson, Liu, Kissinger, Roos and David Sibley2016).

The search for more practical and efficient methods to detect specific antibodies against T. gondii pushed the establishment of tests based on antigenic fractions, which are gradually replacing traditional tests based on whole organisms (agglutination, DT and IFAT) or total extracts of parasite antigens. Recognized immunodominant antigens of T. gondii, including the tachyzoite surface antigens SAG1 (p30) (Kasper, Reference Kasper1987; Mineo et al. Reference Mineo, McLeod, Mack, Smith, Khan, Ely and Kasper1993), SAG2 (p22) (Prince et al. Reference Prince, Auer, Huskinson, Parmley, Araujo and Remington1990), SAG3 (P43) (Cesbron-Delauw et al. Reference Cesbron-Delauw, Tomavo, Beauchamps, Fourmaux, Camus, Capron and Dubremetz1994), SAG4 (p18) (Odberg-Ferragut et al. Reference Odberg-Ferragut, Soete, Engels, Samyn, Loyens, Van Beeumen, Camus and Dubremetz1996) and several other proteins from dense granules, rhoptries and micronemes are being produced as recombinant proteins (reviewed by Holec-Gasior, Reference Holec-Gasior2013).

So far, it is not known whether humans may be infected with H. hammondi, and in case it happens, the possibility of serologic cross-reactivity with T. gondii antigens cannot be ruled out. Cats seem to shed T. gondii and H. hammondi in similar proportions as reported in a recent study from Germany (Schares et al. Reference Schares, Ziller, Herrmann, Globokar, Pantchev and Conraths2016), and humans and animals of many species are potentially exposed to H. hammondi. The establishment of H. hammondi infections in most animal species has not been rigorously investigated; however, the life cycle of H. hammondi seems to lack avian hosts (Dubey and Sreekumar, Reference Dubey and Sreekumar2003).

Cross-immunity studies between T. gondii and N. caninum

Cross-protection of mice immunized with N. caninum and challenged with T. gondii is probably T. gondii strain- and dose-dependent. In one study, mice were immunized with N. caninum and died after challenging with the highly virulent RH strain of T. gondii (Lindsay et al. Reference Lindsay, Blagburn and Dubey1990). When mice were immunized with N. caninum and challenged with a less-virulent T. gondii strain (PLK, a clone from the ME49 strain), the animals had 100% protection against death; immunization with a higher dose of N. caninum tachyzoites improved protection against T. gondii-induced disease (Kasper and Khan, Reference Kasper and Khan1998). Similar levels of protection were observed when mice were immunized with N. caninum tachyzoites and then challenged with oocysts of T. gondii from a moderately virulent strain (Lindsay et al. Reference Lindsay, Lenz, Dykstra, Blagburn and Dubey1998). In another study, pregnant sheep were immunized with a T. gondii sheep vaccine (Toxovax^®, Intervet, Cambridge, UK) and challenged with a high dose of N. caninum tachyzoites (10⁷ tachyzoites per animal) at 90 days of gestation (Innes et al. Reference Innes, Lundén, Esteban, Marks, Maley, Wright, Rae, Harkins, Vermeulen, McKendrick and Buxton2001). No protection against fetal death was observed. The authors speculated that if the sheep were challenged with a lower dose of N. caninum tachyzoites, there perhaps would be some degree of cross-immunity (Innes et al. Reference Innes, Lundén, Esteban, Marks, Maley, Wright, Rae, Harkins, Vermeulen, McKendrick and Buxton2001). The confirmation of cross-immunity between T. gondii and N. caninum in some studies suggests that these parasites share antigens which may also be involved in serologic cross-reactivity.

Serologic cross-reactivity between T. gondii and N. caninum

In the initial observation of N. caninum in dogs, sera from five animals that were naturally infected with the parasite tested negative for T. gondii by the DT (Bjerkas et al. Reference Bjerkas, Mohn and Presthus1984). In addition, dogs that were naturally or experimentally infected with N. caninum did not cross-react to T. gondii by IFAT, when using 1:50 dilutions as cutoff (Dubey et al. Reference Dubey, Hattel, Lindsay and Topper1988b ). Accordingly, the same or higher dilution cutoffs by IFAT have been found to be appropriate to avoid cross-reactivity between N. caninum and T. gondii in serum samples from different hosts (Lobato et al. Reference Lobato, Silva, Mineo, Amaral, Segundo, Costa-Cruz, Ferreira, Borges and Mineo2006; Silva et al. Reference Silva, Lobato, Mineo and Mineo2007; Benetti et al. Reference Benetti, Schein, dos Santos, Toniollo, da Costa, Mineo, Lobato, de Oliveira Silva and Gennari2009). Apical reactions, i.e. reactions limited to the apex of the parasite were regarded as non-specific in N. caninum IFAT. Cross-reactivity with apical antigens is potentially caused by the high conservation of antigens in the apical organelles of a variety of Apicomplexan parasites, including T. gondii. In contrast, a complete peripheral fluorescence of the parasite was considered as a positive response (Pare et al. Reference Pare, Hietala and Thurmond1995b ).

Cross-reactive antigens between N. caninum and T. gondii have been observed by immunohistochemistry using tissues of naturally- or experimentally infected animals. A rabbit anti-N. caninum serum cross-reacted with T. gondii in tissue sections from mice (Barr et al. Reference Barr, Conrad, Dubey and Anderson1991). A bradyzoite antigen from T. gondii, designated as BAG1 (synonymous to BAG5) (Weiss et al. Reference Weiss, LaPlace, Tanowitz and Wittner1992; Parmley et al. Reference Parmley, Weiss and Yang1995), was used to produce hyperimmune serum in rabbit, which cross-reacted with bradyzoites of N. caninum (McAllister et al. Reference McAllister, Parmley, Weiss, Welch and McGuire1996). Polyclonal sera against T. gondii tachyzoites induced strong cross-reactivity with N. caninum tachyzoites by immunohistochemistry (Sundermann et al. Reference Sundermann, Estridge, Branton, Bridgman and Lindsay1997). The use of MAbs specific to T. gondii (Sundermann et al. Reference Sundermann, Estridge, Branton, Bridgman and Lindsay1997) or a combination of two MAbs specific to N. caninum (Uzêda et al. Reference Uzêda, Schares, Ortega-Mora, Madruga, Aguado-Martinez, Corbellini, Driemeier and Gondim2013), were demonstrated to avoid immunohistological cross-reactivity between these protozoa.

Although species-specific MAbs have been developed, serologic cross-reactions between T. gondii and N. caninum have been shown to occur also by means of MAbs. Sundermann et al. (Reference Sundermann, Estridge, Branton, Bridgman and Lindsay1997) generated MAbs against T. gondii tachyzoites and observed among 26 MAbs tested by IFAT, five antibodies that cross-reacted with N. caninum tachyzoites. Kobayashi et al. (Reference Kobayashi, Narabu, Yanai, Hatano, Ito, Imai and Ike2013) cloned the NcBAG1 gene and generated MAbs against its recombinant protein, which recognized TgBAG1. Liao et al. (Reference Liao, Xuan, Huang, Shirafuji, Fukumoto, Hirata, Suzuki and Fujisaki2005a ) produced 384 MAbs against N. caninum by immunizing mice with N. caninum tachyzoites; 10 of the 384 MAbs were also reactive against T. gondii tachyzoites. Similarly, Sohn et al. (Reference Sohn, Cheng, Drummond, Peng, Vermont, Xia, Cheng, Wastling and Bradley2011) developed 46 MAbs using a mouse immunized with a mixed fraction of N. caninum organelles and some of the MAbs cross-reacted with T. gondii. MAbs generated to oocyst antigens of T. gondii cross-reacted by immunofluorescence with the sporocyst wall (Dumetre and Darde, Reference Dumetre and Darde2007) and tissue the cyst wall of N. caninum (Gondim et al. Reference Gondim, Wolf, Vrhovec, Pantchev, Bauer, Langenmayer, Bohne, Teifke, Dubey, Conraths and Schares2016). These findings show that cross-reactive antigens between T. gondii and N. caninum are present in tachyzoites, tissue cysts and oocysts of these parasites.

Affinity-purified antibodies raised against a 38 kDa microneme-associated protein of N. caninum (NcMIC3) also recognized a 45 kDa protein in tachyzoite extracts of T. gondii (Sonda et al. Reference Sonda, Fuchs, Gottstein and Hemphill2000). MAbs raised against T. gondii reacted with N. caninum tachyzoites by IFAT, but only at low titres (10–40) (Latif and Jakubek, Reference Latif and Jakubek2008). A number of reports demonstrated the usefulness of MAbs against T. gondii, which do not cross-react with N. caninum (Baszler et al. Reference Baszler, Adams, Vander-Schalie, Mathison and Kostovic2001; Uchida et al. Reference Uchida, Ike, Kurotaki, Ito and Imai2004; Srinivasan et al. Reference Srinivasan, Baszler, Vonlaufen, Leepin, Sanderson, Wastling and Hemphill2006; Cunha-Junior et al. Reference Cunha-Junior, Silva, Silva, Souza, Souza, Prudencio, Pirovani, Cezar, Barbosa, Goulart and Mineo2010).

Certain surface antigens of N. caninum, although not identical to those from T. gondii, were homologous to them (Hemphill et al. Reference Hemphill, Felleisen, Connolly, Gottstein, Hentrich and Muller1997; Howe et al. Reference Howe, Crawford, Lindsay and Sibley1998; Howe and Sibley, Reference Howe and Sibley1999). Two surface antigens of N. caninum, similar to SAG1 and SRS2 from T. gondii, were called NcSAG1 and NcSRS2. MAbs against NcSAG1 (6C11, Ncmab-4) did not cross react with T. gondii (Björkman and Hemphill, Reference Björkman and Hemphill1998; Howe et al. Reference Howe, Crawford, Lindsay and Sibley1998). Evaluation of several MAbs against NcSRS2 (5H5, Ncmab-10, 5·2·15) revealed no cross-reactions with T. gondii antigens in immunoblot (Björkman and Hemphill, Reference Björkman and Hemphill1998; Howe et al. Reference Howe, Crawford, Lindsay and Sibley1998; Schares et al. Reference Schares, Dubremetz, Dubey, Barwald, Loyens and Conraths1999a ).

Antigens of N. caninum tested by WB using monoclonal or polyclonal antibodies, resulted in the identification of a limited number of specific immunodominant bands (often referred to as ‘immunodominant antigens’) and other less reactive bands (Table 1). The comparison of these antigens based on their molecular weights is difficult, as differences on the SDS-PAGE conditions, especially the use of reducing or non-reducing conditions, cause variation in the estimated molecular weights. Most likely, some of these immunodominant bands observed in WBs may represent more than a single protein.

Table 1.

Immunodominant bands recognized by Neospora caninum-infected or immunized animals in tachyzoite antigen

^a Cross-reacting immunodominant antigen bands underlined.

In WB, non-reduced antigens exhibit much stronger reactivity than reduced antigens (Barta and Dubey, Reference Barta and Dubey1992) which is a clear indication that most of the epitopes recognized on these antigens are conformational epitopes. Under non-reduced conditions a large number of researchers observed mainly four areas with specific immunodominant bands in N. caninum tachyzoite antigen: 14–19 kDa, 29–32 kDa, 30–36 kDa, and 36–40 kDA. However, other areas with major bands of reactions were also observed with non-reduced antigens, but differed widely between studies (Table 1).

Sera from animals immunized with recombinant forms of the major N. caninum antigens NcSAG1 and NcSRS2 provided evidence that these antigens are among those recognized in the range 29–32 kDa and 36–40 kDA bands (Howe et al. Reference Howe, Crawford, Lindsay and Sibley1998) (Table 2).

Table 2.

Neospora caninum-recombinant antigens and cross-reactions tested against Toxoplasma gondii and related protozoan parasites

Under reduced conditions there is a dominant band between 17–19 kDa which most likely represent reactions against NcGRA7 (Alvarez-Garcia et al. Reference Alvarez-Garcia, Pitarch, Zaballos, Fernandez-Garcia, Gil, Gomez-Bautista, Aguado-Martinez and Ortega-Mora2007) and other antigens (Table 2). However, as demonstrated by MAbs (4·7·12; Ncmab-7) in combination with immunoprecipitation, surface biotinylation and immuno-electron microscopy, a surface antigen might also be among those migrating in WB at 17–19 kDa under reduced conditions (Björkman and Hemphill, Reference Björkman and Hemphill1998; Schares et al. Reference Schares, Dubremetz, Dubey, Barwald, Loyens and Conraths1999a ). Further immunodominant banding areas are at 34–36 and 37–46 kDa, eventually also representing reactions to NcSAG1, NcGRA6, NcGRA7 and NcSRS2 (Table 2). When tested with polyclonal antibodies against T. gondii, only minor reactions were observed with antigen bands regarded as specific for N. caninum in WB (Barta and Dubey, Reference Barta and Dubey1992; Björkman et al. Reference Björkman, Lunden, Holmdahl, Barber, Trees and Uggla1994, Reference Björkman, Holmdahl and Uggla1997).

For the diagnosis of T. gondii infection in veterinary investigations, the use of recombinant and synthetic antigens, developed using novel molecular techniques, have expanded diagnostic options as alternatives to native antigens directly isolated from cultivated parasites. For diagnosis of T. gondii infection in cats, sheep and pigs, some species-specific ELISAs are available that have performed well when compared with previous reference serological techniques, as reviewed by Wyrosdick and Schaefer (Reference Wyrosdick and Schaefer2015).

ELISAs for N. caninum antibodies in sera from dogs and cattle have been developed in several studies (Table 3). Conventional ELISAs using crude soluble antigen showed higher levels of serologic cross-reactivity to T. gondii when compared with IFAT (Björkman et al. Reference Björkman, Lunden, Holmdahl, Barber, Trees and Uggla1994; Silva et al. Reference Silva, Lobato, Mineo and Mineo2007). Serologic cross-reactivity with T. gondii was also observed when polyclonal mouse and cat sera were tested by a N. caninum ELISA using crude antigen (Nishikawa et al. Reference Nishikawa, Claveria, Fujisaki and Nagasawa2002). In contrast, the same mouse and cat sera did not cross-react by an ELISA based on N. caninum recombinant antigen (NcSRS2) (Nishikawa et al. Reference Nishikawa, Claveria, Fujisaki and Nagasawa2002). ELISAs prepared with N. caninum tachyzoite antigen associated with immunostimulating complexes (ISCOM), and using MAbs as secondary antibodies, presented better specificity than conventional ELISAs (Björkman et al. Reference Björkman, Lunden, Holmdahl, Barber, Trees and Uggla1994, Reference Björkman, Holmdahl and Uggla1997). The ISCOM particles have affinity for surface proteins, which minimizes interference by internal non-specific antigens (Björkman et al. Reference Björkman, Lunden, Holmdahl, Barber, Trees and Uggla1994). Moreover, none of the MAbs developed against N. caninum ISCOM incorporated antigens (including also Ncmab-4, and Ncmab-10 mentioned above) cross-reacted with T. gondii (Björkman and Lunden, Reference Björkman and Lunden1998). An ELISA based on immuno-affinity-purified native NcSRS2 showed no significant cross-reactions when tested with sera from cattle experimentally infected with a variety of protozoan parasites including also ten cattle infected with T. gondii (Schares et al. Reference Schares, Rauser, Sondgen, Rehberg, Barwald, Dubey, Edelhofer and Conraths2000). In addition, the TgSAG2A molecule has been demonstrated to be specific to T. gondii, considering that no cross-reactivity has been shown with N. caninum when using recombinant protein or even mimotopes derived from this molecular marker, as characterized by A4D12 MAb (Bela et al. Reference Bela, Oliveira Silva, Cunha-Junior, Pirovani, Chaves-Borges, Reis de Carvalho, Carrijo de Oliveira and Mineo2008; Carvalho et al. Reference Carvalho, Silva, Cunha-Junior, Souza, Oliveira, Bela, Faria, Lopes and Mineo2008; Cunha-Junior et al. Reference Cunha-Junior, Silva, Silva, Souza, Souza, Prudencio, Pirovani, Cezar, Barbosa, Goulart and Mineo2010; Santana et al. Reference Santana, Silva, Vaz, Pirovani, Barros, Lemos, Dietze, Mineo and Cunha-Junior2012; Macedo et al. Reference Macedo, Cunha, Cardoso, Silva, Santiago, Silva, Pirovani, Silva, Mineo and Mineo2013).

Table 3.

Cross-reactions tested for Neospora caninum in published in-house ELISAs

^a Cross-reactions observed in serum dilutions lower than the cutoff.

^b Rabbit sera against T. gondii and S. cruzi do not react in WB with ISCOM antigen.

^c MAbs against ISCOM antigens do not recognize T. gondii in WB.

^d Mouse serum immunized with ISCOM antigen does not recognize T. gondii.

^e Some sera showed elevated levels of cross-reactions in WB.

Nowadays it is becoming clear that there is a need to characterize new molecular markers that are species-specific for T. gondii and N. caninum for the development of new diagnostic tools (Zhang et al. Reference Zhang, Lee, Yu, Kawano, Huang, Liao, Kawase, Zhang, Zhou, Fujisaki, Nishikawa and Xuan2011; Regidor-Cerrillo et al. Reference Regidor-Cerrillo, Garcia-Lunar, Pastor-Fernandez, Alvarez-Garcia, Collantes-Fernandez, Gomez-Bautista and Ortega-Mora2015). In this context, the identification of cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites is mandatory and the proteomics approach constitutes an appropriate strategy for this purpose (Zhang et al. Reference Zhang, Lee, Yu, Kawano, Huang, Liao, Kawase, Zhang, Zhou, Fujisaki, Nishikawa and Xuan2011). These authors demonstrated the usefulness of proteomics to immuno-screen for cross-reactive or species-specific antigens from both parasites. Moreover, they showed that there was significant homology in the antigenic proteome profiles between the two parasites (Zhang et al. Reference Zhang, Lee, Yu, Kawano, Huang, Liao, Kawase, Zhang, Zhou, Fujisaki, Nishikawa and Xuan2011). Taking together, these findings shed light on the process to design new diagnostic tools in order to avoid cross-reactivity between N. caninum and T. gondii diagnostic tests.

The characterization of cross-reactive antigens between T. gondii and N. caninum has been achieved in some studies. An NTPase identified in N. caninum tachyzoites was antigenically cross-reactive to the NTPases of T. gondii (Asai et al. Reference Asai, Howe, Nakajima, Nozaki, Takeuchi and Sibley1998). Protein disulphide isomerase (PDI), heat-shock protein 70 (HSP70) and ribosomal protein P1 (RP1), were identified as cross-reactive antigens between the two parasites even when using MAbs, due to the high degree of homology among these parasite components (Liao et al. Reference Liao, Xuan, Huang, Shirafuji, Fukumoto, Hirata, Suzuki and Fujisaki2005a ). Zhang et al. (Reference Zhang, Compaore, Lee, Liao, Zhang, Sugimoto, Fujisaki, Nishikawa and Xuan2007a ) demonstrated that antibodies raised against the apical membrane antigen 1 of T. gondii (TgAMA 1) also recognize recombinant NcAMA 1. The ribosomal phosphoprotein (P0) was shown to be a cross-reactive antigen between T. gondii and N. caninum (Zhang et al. Reference Zhang, Lee, Liao, Compaore, Zhang, Kawase, Fujisaki, Sugimoto, Nishikawa and Xuan2007b ); antibodies raised against rNcPO inhibited the growth of both T. gondii and N. caninum tachyzoites. A protease with 42 kDa was localized in the rhoptry of T. gondii by means of a MAb; this MAb also reacted to a 42 kDa protein in N. caninum, which was also localized in the rhoptry of this parasite (Ahn et al. Reference Ahn, Song, Son, Shin and Nam2001).

The genome of N. caninum (NC-Liverpool strain) was compared with the available genome of the ME-49 strain of T. gondii (Reid et al. Reference Reid, Vermont, Cotton, Harris, Hill-Cawthorne, Konen-Waisman, Latham, Mourier, Norton, Quail, Sanders, Shanmugam, Sohal, Wasmuth, Brunk, Grigg, Howard, Parkinson, Roos, Trees, Berriman, Pain and Wastling2012). The authors found a high synteny between the two genomes and pointed out that most divergences occurred within the SRS antigens. Transcriptome analysis suggested that N. caninum uses fewer SRS antigens than T. gondii (Reid et al. Reference Reid, Vermont, Cotton, Harris, Hill-Cawthorne, Konen-Waisman, Latham, Mourier, Norton, Quail, Sanders, Shanmugam, Sohal, Wasmuth, Brunk, Grigg, Howard, Parkinson, Roos, Trees, Berriman, Pain and Wastling2012). Therefore, selecting those species-specific parasitic surface antigens for the establishment of serologic tests, such as SRSs, may favour the specificity of these tests.

Chimeric antigens

Almost 30 years ago, the production of recombinant polypeptides derived from genes encoding T. gondii antigens revolutionized the search for more efficient serologic methods (Johnson et al. Reference Johnson, Illana, McDonald and Asai1989; Johnson and Illana, Reference Johnson and Illana1991). The development of ELISAs based on a mixture of recombinant antigens, rather than the use of a single recombinant protein, has been presumed to increase the sensitivity of the ELISA for human sera, while maintaining the desired specificity (Johnson et al. Reference Johnson, Roberts and Tenter1992; Aubert et al. Reference Aubert, Maine, Villena, Hunt, Howard, Sheu, Brojanac, Chovan, Nowlan and Pinon2000). ELISAs containing a mixture of recombinant antigens were also tested for antibodies against T. gondii in sheep and cats (Tenter et al. Reference Tenter, Vietmeyer and Johnson1992).

A chimeric antigen is the fusion of gene fragments constructed as a single gene and expressed to form a hybrid protein (Yang et al. Reference Yang, Chang and Chao2004). The use of chimeric antigens for T. gondii serology is promising. Chimeric proteins are usually larger than single recombinant antigens resulting in a better binding to microtitre plates. In addition, as a chimeric antigen preparation consists of a single antigen, it may be easier to standardize than mixtures of recombinant antigens (Beghetto et al. Reference Beghetto, Spadoni, Bruno, Buffolano and Gargano2006; Lau et al. Reference Lau, Thiruvengadam, Lee and Fong2011; Holec-Gasior et al. Reference Holec-Gasior, Ferra and Drapala2012a ).

Several chimeric antigens have been tested by WB or ELISA for the detection of human antibodies against T. gondii. Among the developed chimeric antigens tested by serology, are preparations including parts of well-characterized immunodominant proteins, such as SAG1, SAG2, MIC1, MIC2, MIC3, MAG1, M2AP, GRA1, GRA2, GRA3 and ROP1 (Beghetto et al. Reference Beghetto, Spadoni, Bruno, Buffolano and Gargano2006; Lau et al. Reference Lau, Thiruvengadam, Lee and Fong2011; Holec-Gasior et al. Reference Holec-Gasior, Ferra and Drapala2012a , Reference Holec-Gasior, Ferra, Drapala, Lautenbach and Kur b ; Ferra et al. Reference Ferra, Holec-Gasior and Kur2015a ). The standardization of a chimeric-antigen-based test for T. gondii antibodies depends on various factors, including an optimal selection of antigens and proper expression of all desired epitopes. In addition to humans, a recent paper reports the first trial of chimeric antigens employed for T. gondii serology in farm animals (horses, swine and sheep) (Ferra et al. Reference Ferra, Holec-Gasior and Kur2015b ); the authors validated their antigen with more than 400 sera and also included 15 sera, which were serologically positive for N. caninum but negative for T. gondii. It is interesting to note that each animal species responded differently to the chimeric-antigen preparations used in the ELISAs, but the SAG2–GRA1–ROP1_L construct reached the best overall specificity and sensitivity for the three tested species (Ferra et al. Reference Ferra, Holec-Gasior and Kur2015b ). Despite progress in the development of chimeric antigens for T. gondii serology, in particular for humans, inadequate information is available about the serologic cross-reactivity potential of those tests with sera from animals infected with other Toxoplasmatinae parasites.

Synthetic peptides, serotyping and stage-specific antigens

The combination of molecular engineering and chemical synthesis of antigens has been applied for the development of serological techniques with improved sensitivity and specificity. Synthetic peptides representing several epitopes of numerous antigens have been used in microarray assays for serotyping of viral and bacterial diseases (Neuman de Vegvar et al. Reference Neuman de Vegvar, Amara, Steinman, Utz, Robinson and Robinson2003; Nahtman et al. Reference Nahtman, Jernberg, Mahdavifar, Zerweck, Schutkowski, Maeurer and Reilly2007).

In T. gondii infections, the humoral response has been demonstrated to be partially strain-specific. In one study, MAbs produced against SAG2A from naturally infected mice recognized the surface antigens encoded by the SAG2 allele of type I and III strains, but not of type II strains (Parmley et al. Reference Parmley, Gross, Sucharczuk, Windeck, Sgarlato and Remington1994). Another study identified a MAb showing differences in the recognition of type II and III strains (Bohne et al. Reference Bohne, Gross and Heesemann1993). Kong et al. (Reference Kong, Grigg, Uyetake, Parmley and Boothroyd2003) screened nucleotide sequences from types I, II and III of T. gondii for the identification of polymorphic regions from genes coding selected antigens. Allele-specific peptides were synthetized and screened by ELISA using sera from mice and humans. Synthetic peptides based on SAG2A, GRA3, GRA6 and GRA7 were able to discriminate type II from non-type II infections (Kong et al. Reference Kong, Grigg, Uyetake, Parmley and Boothroyd2003). In subsequent studies, serotyping for T. gondii infections using new recombinant polypeptides or new or improved synthetic peptides, as well as target populations from different regions, have been performed by ELISAs (Peyron et al. Reference Peyron, Lobry, Musset, Ferrandiz, Gomez-Marin, Petersen, Meroni, Rausher, Mercier, Picot and Cesbron-Delauw2006; Sousa et al. Reference Sousa, Ajzenberg, Vilanova, Costa and Darde2008, Reference Sousa, Ajzenberg, Marle, Aubert, Villena, da Costa and Darde2009). In two studies, peptide-microarrays were validated to discriminate the serological responses against clonal-type T. gondii strains in samples from humans (Maksimov et al. Reference Maksimov, Zerweck, Maksimov, Hotop, Gross, Spekker, Daubener, Werdermann, Niederstrasser, Petri, Mertens, Ulrich, Conraths and Schares2012b ) and cats (Maksimov et al. Reference Maksimov, Zerweck, Dubey, Pantchev, Frey, Maksimov, Reimer, Schutkowski, Hosseininejad, Ziller, Conraths and Schares2013). The latter study used sera from experimentally infected cats for validation and showed significant type-specific differences in the IgG response against the tested peptide panel. However, in many peptides, reactions were not clonal type-specific (Maksimov et al. Reference Maksimov, Zerweck, Dubey, Pantchev, Frey, Maksimov, Reimer, Schutkowski, Hosseininejad, Ziller, Conraths and Schares2013).

A peptide microarray was developed and validated for serotyping T. gondii infections in humans, aiming to differentiate between different manifestations of T. gondii infection (Maksimov et al. Reference Maksimov, Zerweck, Maksimov, Hotop, Gross, Pleyer, Spekker, Daubener, Werdermann, Niederstrasser, Petri, Mertens, Ulrich, Conraths and Schares2012a ). Thirty eight T. gondii synthetic peptides, consisting of 18 peptides characterized in previous studies, and 20 novel peptides, predicted by bioinformatics approach, were tested to differentiate acute, latent and ocular infections. Some peptides based on dense granule and microneme antigens (GRA2-28, MIC3-282 and MIC3-191) showed promising results for differentiation between acute and latent infections (Maksimov et al. Reference Maksimov, Zerweck, Maksimov, Hotop, Gross, Pleyer, Spekker, Daubener, Werdermann, Niederstrasser, Petri, Mertens, Ulrich, Conraths and Schares2012a ).

The use of synthetic peptides for T. gondii serotyping may have a great potential for discriminating between T. gondii infections and infections caused by other parasite species expected to induce some degree of serologic cross-reactivity. Of course, one important prerequisite is that peptides applied in such tests are not specific for particular clonal-types or genotypes of T. gondii or related parasite species. In addition, potential cross-reactivities need to be addressed; for instance, synthetic peptides considered to react specifically with antibodies generated by T. gondii infections need to be tested with sera from animals exposed to H. hammondi, as this parasite has a very close genetic relationship to the former (Walzer et al. Reference Walzer, Adomako-Ankomah, Dam, Herrmann, Schares, Dubey and Boyle2013, Reference Walzer, Wier, Dam, Srinivasan, Borges, English, Herrmann, Schares, Dubey and Boyle2014). The use of T. gondii-specific peptides for diagnosis in animals should also be tested with N. caninum, which may cross-react, as shown in Table 3. To our knowledge, there are no published studies using synthetic peptides to differentiate infections in animals cause by T. gondii, H. hammondi or N. caninum.

Stage-specific antigens (native or recombinant) from T. gondii have been investigated for decades by several research groups, including oocysts and sporozoites which can be produced in cats (Kasper et al. Reference Kasper, Bradley and Pfefferkorn1984; Ferguson et al. Reference Ferguson, Brecht and Soldati2000; Dumetre and Darde, Reference Dumetre and Darde2007; Possenti et al. Reference Possenti, Cherchi, Bertuccini, Pozio, Dubey and Spano2010, Reference Possenti, Fratini, Fantozzi, Pozio, Dubey, Ponzi, Pizzi and Spano2013; Bushkin et al. Reference Bushkin, Motari, Magnelli, Gubbels, Dubey, Miska, Bullitt, Costello, Robbins and Samuelson2012; Fritz et al. Reference Fritz, Bowyer, Bogyo, Conrad and Boothroyd2012). In contrast, antigens from Neospora spp. are mostly obtained from tachyzoites. Only small numbers of studies have produced N. caninum tissue cysts in cell culture or in animals (Weiss et al. Reference Weiss, Ma, Halonen, McAllister and Zhang1999; Tunev et al. Reference Tunev, McAlilster, Anderson-Sprecher and Weiss2002; Risco-Castillo et al. Reference Risco-Castillo, Fernandez-Garcia and Ortega-Mora2004; Vonlaufen et al. Reference Vonlaufen, Guetg, Naguleswaran, Muller, Björkman, Schares, von Blumroeder, Ellis and Hemphill2004), or have induced oocyst production in canid definitive hosts (McAllister et al. Reference McAllister, Dubey, Lindsay, Jolley, Wills and McGuire1998; Schares et al. Reference Schares, Heydorn, Cuppers, Conraths and Mehlhorn2001; Gondim et al. Reference Gondim, Gao and McAllister2002).

The identification of the first bradyzoite-specific gene of N. caninum (NcSAG4), an orthologue to TgSAG4, allowed the production and characterization of the recombinant protein rNcSAG4 (Fernandez-Garcia et al. Reference Fernandez-Garcia, Risco-Castillo, Zaballos, Alvarez-Garcia and Ortega-Mora2006). This antigen has been applied for the development of a stage-specific ELISA for bovine neosporosis (Aguado-Martinez et al. Reference Aguado-Martinez, Alvarez-Garcia, Fernandez-Garcia, Risco-Castillo, Arnaiz-Seco, Rebordosa-Trigueros, Navarro-Lozano and Ortega-Mora2008).

The production of Hammondia spp. antigens is even more difficult, because there is no permanent culture for these parasites (Riahi et al. Reference Riahi, Darde, Bouteille, Leboutet and Pestre-Alexandre1995; Schares et al. Reference Schares, Meyer, Barwald, Conraths, Riebe, Bohne, Rohn and Peters2003; Gondim et al. Reference Gondim, Meyer, Peters, Rezende-Gondim, Vrhovec, Pantchev, Bauer, Conraths and Schares2015). Therefore, most antigens from Hammondia spp. are derived from oocysts or from parasite cysts from intermediate hosts (Riahi et al. Reference Riahi, Bouteille and Darde1998, Reference Riahi, Leboutet, Bouteille, Dubremetz and Darde1999, Reference Riahi, Leboutet, Labrousse, Bouteille and Darde2000; Walzer et al. Reference Walzer, Adomako-Ankomah, Dam, Herrmann, Schares, Dubey and Boyle2013). Generation of recombinant antigens from Hammondia spp. is lacking. It would enable studies on exposure of animals and humans to these parasites, as well as on serologic cross-reactivity with related protozoa.

A serologic test for humans based on a T. gondii sporozoite-specific protein has been evaluated and seems to represent a promising method to discriminate between oocyst-induced infections from those transmitted by ingestion of infected meat (Hill et al. Reference Hill, Coss, Dubey, Wroblewski, Sautter, Hosten, Munoz-Zanzi, Mui, Withers, Boyer, Hermes, Coyne, Jagdis, Burnett, McLeod, Morton, Robinson and McLeod2011), which is an important consideration in epidemiological investigations. This study was the first to use a sporozoite protein, called T. gondii embryogenesis-related protein (TgERP) in a serologic test for T. gondii. In a recent study another sporozoite-derived protein called CCp5A was successfully used in an ELISA to identify oocyst-infected animals (Santana et al. Reference Santana, Gebrim, Carvalho, Barros, Barros, Pajuaba, Messina, Possenti, Cherchi, Reiche, Navarro, Garcia, Pozio, Mineo, Spano and Mineo2015); this test was able to identify antibodies in sera from humans, pigs, mice and chickens that were naturally or experimentally infected with T. gondii oocysts and discriminate between animals infected from ingestion of oocysts or by carnivorism. Further investigations are necessary for validation and to confirm whether the sporozoite proteins TgERP (Hill et al. Reference Hill, Coss, Dubey, Wroblewski, Sautter, Hosten, Munoz-Zanzi, Mui, Withers, Boyer, Hermes, Coyne, Jagdis, Burnett, McLeod, Morton, Robinson and McLeod2011) and CCp5A (Santana et al. Reference Santana, Gebrim, Carvalho, Barros, Barros, Pajuaba, Messina, Possenti, Cherchi, Reiche, Navarro, Garcia, Pozio, Mineo, Spano and Mineo2015) cross-react with sporozoite proteins derived from Hammondia spp. and Neospora spp. It is also important to test whether low infectious dose with oocysts will elicit detectable antibodies to TgERP or CCp5A.

Type or stage-specific antigens have a wide spectrum of applications, including investigations of infection outbreaks and identification of risk factors in selected populations (e.g. ingestion of oocyst or tissue cysts). Those antigens should be used with caution and only after rigorous validation in epidemiological studies.