Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in slaughtered pigs in the Czech Republic

In the Czech Republic, sera from 551 clinically healthy adult slaughtered pigs (females, 6-8 months old) were collected during the first half of June in 2010. Sera were tested for Toxoplasma gondii-specific IgG antibodies by an enzyme-linked immunosorbent assay; samples with more than 50% S/P were considered as positive. The same samples were also analysed for Neospora caninum antibodies using a commercial competitive-inhibition enzyme-linked immunosorbent assay; samples with more than 30% inhibition were considered as positive. Antibodies against T. gondii were found in 198 pigs (36%) in all districts with prevalences ranging from 18% to 75%. Antibodies against N. caninum were found in 16 pigs (3%); positive animals were found in 4 districts with prevalences ranging from 1% to 20%. Indication of mixed infections (concurrent presence of both N. caninum and T. gondii antibodies) was found in 8 (1·5%) pigs. The results of our study indicate that pigs in the Czech Republic have a relatively high seroprevalence for T. gondii, while they have only a low seroprevalence for N. caninum. Therefore, natural infection with T. gondii seems to be very common in Czech pigs. It is the first evidence of N. caninum antibodies in pigs in the Czech Republic. These results complete data about N. caninum infection in pigs in Europe.     

Vero cell surface proteoglycan interaction with the microneme protein NcMIC(3) mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.     

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.     

The in vitro development of Neospora caninum bradyzoites

Neospora caninum is a recently identified apicomplexan protozoan parasite that is closely related to Toxoplasma gondii. Neospora caninum is of significant economic importance as it causes neurological disease and abortion in numerous animals. Antibodies to BAG1/hsp30 (also known as BAG5), a T. gondii bradyzoite-specific protein, have been demonstrated to react with N. caninum tissue cysts in vivo. Bradyzoite differentiation of N. caninum in vitro was investigated using culture conditions previously utilised for T. gondii in vitro bradyzoite development. Utilising the NC-Liverpool isolate of N. caninum, cyst-like structures developed within 3-4 days of culture of this parasite in human fibroblasts. In addition, an antigen reacting with mAb 74.1.8 (anti-BAG1) and rabbit anti-recombinant BAGI was demonstrable by immunofluorescence, fluorescence-activated cell sorter, and immunoblot analyses. Expression of this antigen was increased by stress conditions, similar to that which has been described for T. gondii bradyzoite induction. Cyst-wall formation in vitro, as assayed by lectin binding, did not occur as readily for N. caninum as it does for T. gondii.     

Serologic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) has well adapted to increasing urbanization, resulting in greater interaction with humans and their domestic pets. Wildlife species in urban areas face a higher risk of exposure to zoonotic pathogens and may be affected by parasites hosted by cats (Toxoplasma gondii) or dogs (Neospora caninum), yet it is unknown to what extent urban T. vulpecula are exposed to these parasites. Antibodies to T. gondii and N. caninum were assayed in sera of 142 adult possums from the city of Sydney, Australia. Using the modified agglutination test, antibodies to T. gondii were found in 9 (6.3%) of the 142 animals in titers of 1:25 (4), 1:50 (1), 1:100 (1), 1:800 (1), 1:3,200 (1), 1:6,400 (1), and 1:12,800 (1). Of some T. vulpecula multiple sera samples within a 2-yr frame could be collected, but seropositive animals in general were not recaptured after initial seroconversion. One possum had a high T. gondii titer on 2 consecutive bleedings, 14 mo apart, and seropositive possums appeared normal when captured. Sex seemed not to have an affect on antibody prevalence, but age and location may play a role. Antibodies to N. caninum were not detected in 1:25 dilution of sera in the N. caninum agglutination test, indicating that T. vulpecula may not have been exposed to this parasite. This is the first serological survey for T. gondii and N. caninum infections in urban T. vulpecula.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.     

Meningoencephalitis associated with an unidentified apicomplexan protozoan in a Pacific harbor seal

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.     

Toxoplasma gondii and Neospora caninum antibodies in dogs from Grenada, West Indies

Toxoplasma gondii and Neospora caninum are structurally similar parasites with many common hosts. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs in Grenada, West Indies. Using a modified agglutination test, antibodies to T. gondii were found in 52 (48.5%) of the 107 dogs, with titers of 1:25 in 17, 1:50 in 19, 1:100 in 7, 1:1,600 in 5, and 1:3,200 or higher in 4. Seroprevalence increased with age from 2.2% in dogs <6 mo old to 18.9% in dogs older than 2 yr, indicating postnatal transmission of T. gondii in this population of canines. There was no correlation between the health of the dogs and the seroprevalence or magnitude of the T. gondii titer. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT). Two of the 107 dogs had N. caninum antibodies (IFAT titers 1:100 and 1:400); these dogs had T. gondii titers of 1:1,600 and 1:50, respectively. Results indicate that these 2 structurally similar protozoa are antigenically different.     

A survey of Neospora caninum and Toxoplasma gondii infection in urban rodents from Brazil

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii , and a few findings indicated that they may be natural intermediate hosts for N. caninum . The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus , Rattus norvegicus , and Mus musculus captured in urban areas of S?o Paulo reveal a striking low frequency of occurrence of these infections.     

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus)

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Prevalence of antibodies to Neospora caninum in wild animals

Antibodies to Neospora caninum were determined in several species of wild animals in the United States by the Neospora agglutination test (NAT). Antibodies (NAT 1:40 or higher) were found in 5 of 249 bison (Bison bison), 5 of 160 caribou (Rangifer tarandus), 4 of 162 moose (Alces alces), 4 of 122 wolves (Canis lupus), and 1 of 224 musk ox (Ovibos moschatus) but not in 197 black bears (Ursus americanus). To our knowledge, this is the first report of antibodies to N. caninum in bison and caribou. The total absence of N. caninum antibodies in black bears indicates that bears are not a host for N. caninum and that there is no cross-reactivity between the NAT and the modified agglutination test (MAT) for Toxoplasma gondii, because more than 80% of black bears in eastern United States have MAT antibodies at a 1:25 serum solution.     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii

The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural differences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5-4 microm, than those of T. gondii which were smooth and 0.5 microm thick.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in the South American opossum (Didelphis marsupialis) from the city of São Paulo, Brazil

Antibodies to Neospora caninum and Toxoplasma gondii were assayed in sera of 396 opossums (Didelphis marsupialis) from the city of S?o Paulo, Brazil. Antibodies to N. caninum were assayed using the indirect immunofluorescent antibody test (IFAT). Antibodies (IFAT, approximately 1:25) to N. caninum were found in 84 opossums (D. marsupialis) in titers of 1:25 in 46, 1:50 in 20, 1:100 in 17, and 1:400 in 1. Antibodies to T. gondii were assayed with the modified agglutination test (MAT) and the IFAT. Antibodies to T. gondii (MAT, approximately 1:25) were found in 82 (20.4%) of the 396 opossums, in titers of 1:25 in 24, 1:50 in 26, 1:100 in 18, 1:200 in 13, and 1:800 in 1. The IFAT antibodies to T. gondii were found in 148 of 396 opossums, in titers of 1:16 in 41, 1:32 in 23, 1:64 in 13, 1:128 in 6, 1:256 in 20, 1:512 in 17, 1:1,024 in 10, 1:2,048 in 10, 1:4,096 in 7, and 1:8,192 in 1. This is the first report of N. caninum and T. gondii infections in D. marsupialis.     

Toxoplasma gondii-like schizonts in the tracheal epithelium of a cat

Toxoplasma gondii-like schizonts were found in tracheal epithelium of an 8-yr-old male cat. The parasites were located in parasitophorous vacuoles within the host cell cytoplasm, divided by schizogony, contained periodic acid-Schiff-positive granules, and reacted with anti-T. gondii serum but not with anti-Neospora caninum serum. Mature schizonts were 7.0 x 5.9 microns (5-10 x 4-10 microns; n = 22) and contained 4-16 merozoites. The merozoites were approximately 5 x 1 microns.     

Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions

Apicomplexan parasites possess an apical complex that is composed of two secretory organelles recognized as micronemes and rhoptries. Rhoptry contents are secreted into the parasitophorous vacuole during the host cell invasion process. Several rhoptry proteins have been identified in Toxoplasma gondii and seem to be involved in host-pathogen interactions and some of them are considered to be important virulence factors. Only one rhoptry protein, NcROP2, has been identified and extensively characterized in the closely related parasite Neospora caninum, and this has showed immunoprotective properties. Thus, with the aim of increasing knowledge of the rhoptry protein repertoire in N. caninum, a subcellular fractionation of tachyzoites was performed to obtain fractions enriched for this secretory organelle. 2-D SDS-PAGE followed by MS and LC/MS-MS were applied for fraction analysis and 8 potential novel rhoptry components (NcROP1, 5, 8, 30 and NcRON2, 3, 4, 8) and several kinases, proteases and phosphatases proteins were identified with a high homology to those previously found in T. gondii. Their existence in N. caninum tachyzoites suggests their involvement in similar events or pathways that occur in T. gondii. These novel proteins may be considered as targets that could be useful in the future development of immunoprophylactic measures.