Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.     

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.     

Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum

The internal transcribed spacer (ITS1) region and the 5′ part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondii isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.     

Diagnosis of Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections in cattle

The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals. Financial support for this study was provided by SeCyT through BID 1728 PICT No. 10858/8.     

Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay.

A recently described PCR test for the identification of Neospora caninum and Toxoplasma gondii has been further developed and optimized in view of its practicability for routine diagnostic application. The N. caninum-specific PCR was adapted to the diagnostic operating standard of the T. gondii-specific PCR in that the uracil DNA glycosidase system was introduced, which eliminates potential carry-over contaminations of amplified target DNA from previous reactions. Furthermore, both PCR tests were optimized by including a DNA hybridization immunoassay based on the use of the commercially available Gen-eti-k DEIA kit. This assay allowed highly sensitive and specific detection of respective DNA amplification products and thus substantially facilitated the reading and interpretation of the test results.     

Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly,karyotype differences,and chromosomal rearrangements

Neospora caninum primarily infects cattle, causing abortions, with an estimated impact of a billion dollars on the worldwide economy annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen Toxoplasma gondii. By revisiting the genome sequence, assembly, and annotation using third-generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii. We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally named Chr VIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing more than 500 new genes. We sequence and annotate the nonphotosynthetic plastid and mitochondrial genomes and show that although apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exist between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii and, more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.

The Apicomplexa comprise a large phylum of parasitic alveolates of medical and veterinary importance, causing deadly diseases such as malaria, cryptosporidiosis, neosporosis, and toxoplasmosis, among others. With the exception of a few commonalities such as their obligatory intracellular lifestyle and the presence of specialized secretory organelles and of secondary endosymbionts, the apicomplexans differ greatly in morphology, host range specificity, pathogenicity, reproductive strategy, and transmission. Understanding the molecular basis of these differences has been the focus of much research. Comparative genomic analyses revealed that, albeit all small, apicomplexans genomes vary greatly in size, ranging from 9 to 130 Mb (Debarry and Kissinger 2011; Blazejewski et al. 2015). Having diverged from a common ancestor 350–824 myr ago (Escalante and Ayala 1995), shy of 900 genes are conserved among them, whereby major genomic rearrangements can be observed (Debarry and Kissinger 2011).High synteny, defined as conserved content and order of a given genomic locus, is rarely observed (Debarry and Kissinger 2011). A seemingly stark exception to this is the genomes of Toxoplasma gondii and Neospora caninum. Morphologically, these parasites are virtually indistinguishable, so much so, that N. caninum was only recognized as a separate species in 1988 (Dubey 2003; Dubey et al. 2002). Moreover, both species show similar tropism within their hosts, where they can infect virtually any nucleated cell. They both show a fast-replicating form (tachyzoite), causing acute disease, that transitions into a slow-dividing form (bradyzoite), which persists in immune-privileged sites, such as the brain, establishing chronic infection. In line with this, initial comparative analysis concluded that these species have largely conserved genomic content and are largely syntenic (Reid et al. 2012). Despite their commonalities, however, the biology of these pathogens also differs significantly. T. gondii infects a wide range of intermediate hosts, including humans, causing deadly disease in immunocompromised individuals or by congenital transmission. In contrast, N. caninum infects primarily cattle, causing abortions with an estimated impact of a billion dollars on the worldwide economy annually (Reichel et al. 2013). Feline species act as definitive hosts of T. gondii, whereas sexual replication of N. caninum occurs only in canids (McAllister et al. 1998; Gondim et al. 2004; King et al. 2010). These biological differences have been largely ascribed to absence, point mutations, and pseudogenization of T. gondii virulence factors in N. caninum and the comparative amplification of surface protein-coding gene families in N. caninum (Khan et al. 2009; Reid et al. 2012).Advancements in genome sequencing technologies have accompanied the fast-paced genomics era. Particularly, third-generation sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies sequencing, outperform prior technologies by providing very long reads that can span regions containing repetitive sequences. This has led to improvements in the assembly of previously unattainable genomes, such as those presenting high proportions of repetitive sequences, allowing whole new genomes to be assembled with high accuracy. Here, we set out to sequence and de novo assemble two N. caninum strain genomes and the T. gondii genome, using PacBio and Oxford Nanopore.     

Endothelial cell invasion by Toxoplasma gondii: differences between cell types and parasite strains

Toxoplasma gondii disseminates and causes congenital infection by invasion of the endothelial cells. The aim of this study was to analyze the ability of two strains to invade two endothelial cell types. Tachyzoites of the RH and ME49 strains were expanded in Balb/c and C57BL6-RAG2?/? mice, respectively. Tachyzoites were harvested from 72 h Vero cell cultures and incubated for 30 min to 4 h at 10:1 parasite/cell ratio in 24-well plates, containing monolayers of either HMEC-1 line or human umbilical cells (HUVECs). The number of infected cells and parasitic vacuoles per infected cell were counted in Wright stained slides. A slow increase in the proportion of infected cells occurred but varied according to cell type–parasite strain combination: ME49 tachyzoites invaded up to 63 % HMEC-1 cells, while RH parasites infected up to 19 % HUVECs. ME49 and RH tachyzoites invaded 49 and 46 % HUVECs and HMEC-1 cells, respectively. Reinvasion and formation of new parasitophorous vacuoles of infected cells was more frequent than invasion of noninfected cells. The results support that the factors influencing invasion, and thus dissemination and vertical transmission, are parasite type, host cell type/subtype, and activation state. Interestingly, T. gondii virulence does not seem to relay on its invasion efficiency, but probably on replication speed.

Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran

One hundred twenty camels were blood-sampled and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by indirect fluorescent antibody test (IFAT) in Mashhad, Iran, during years 2004–2005. Of the 120 camels, antibodies to N. caninum were found in three in titers of 1:20 and in four in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in three camels in titers 1:20 and in two camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide (BIOGENE, Iran).     

Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii

Protein palmitoylation is the reversible covalent attachment of palmitic acid onto proteins. This post-translational modification has been shown to play a part in diverse processes such as signal transduction, cellular localization and regulation of protein activity. Although many aspects of protein palmitoylation have been identified in mammalian and yeast cells, little is known of this modification in Toxoplasma gondii. In order to determine the functional role of protein palmitoylation in T. gondii, tachyzoites were treated with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Parasites treated with 2-BP displayed a significant increase in non-circular trails which were longer than those trails left by non-treated parasites. Furthermore, 2-BP treatment reduced the invasion process to the host cells. Long-term treatment of intracellular tachyzoites resulted in major changes in parasite morphology and shape in a dose-dependent manner. These results suggest that palmitoylation could be modifying proteins that are key players in gliding, invasion and cytoskeletal proteins in T. gondii.     

Kennel dogs as sentinels of Leishmania infantum, Toxoplasma gondii, and Neospora caninum in Majorca Island, Spain

Kennel dogs can serve as sentinels and/or reservoirs of diseases of veterinary and zoonotic interest because they have often roamed free and lived outdoors, thus being exposed to pathogens. We tested dogs from the kennel of Inca (Majorca/Mallorca, Balearic Islands, Spain) for evidence of infection with three protozoan parasites: Leishmania infantum, Toxoplasma gondii, and Neospora caninum. Exposure to L. infantum was found in 56.3% of 48 dogs (37.5% by Western blot, 43.8% by PCR). Only 30% of infected dogs had leishmaniosis-like lesions. Seroprevalence to T. gondii was 58.7% of 46 dogs using the modified agglutination test (MAT, titer 1:25). None of the 44 dogs tested had N. caninum antibodies using a commercial competitive ELISA, probably because the surveyed dogs did not roam in the proximity of cattle farms. Results confirm the endemicity of L. infantum and also the widespread presence of T. gondii in the Mediterranean island of Majorca.     

Protection against Lethal Neospora caninum Infection in Mice Induced by Heterologous Vaccination with a mic1 mic3 Knockout Toxoplasma gondii Strain

Neospora caninum and Toxoplasma gondii are closely related, obligate intracellular parasites infecting a wide range of vertebrate hosts and causing abortion and neonatal morbidity and mortality. Several lines of evidence suggest that cross immunity between these two pathogens could be exploited in the design of strategies for heterologous vaccination. We assessed the ability of an attenuated strain of T. gondii (“mic1-3KO strain”) conferring strong protection against chronic and congenital toxoplasmosis to protect mice against lethal N. caninum infection. Mice immunized with mic1-3KO tachyzoites by the oral and intraperitoneal routes developed a strong cellular Th1 response and displayed significant protection against lethal heterologous N. caninum infection, with survival rates of 70% and 80%, respectively, whereas only 30% of the nonimmunized mice survived. We report here the acquisition of heterologous protective immunity against N. caninum following immunization with a live attenuated mic1-3KO strain of T. gondii.Neospora caninum and Toxoplasma gondii are closely related apicomplexans displaying extensive morphological and genetical similarity (37). These cyst-forming coccidians of the family Sarcocystidae (11) cause similar disorders in different animals (15). Despite controversial documentation on their phylogenetic relationship (30), molecular (31) and biological studies have shown that these species have followed different evolutionary paths and have different life cycles and host preferences. Canids are the definitive hosts of N. caninum, which causes neosporosis, a major disease of cattle, whereas felids are the definitive hosts of T. gondii, which causes toxoplasmosis, a major disease of sheep, goats, and humans (10). Both parasites are responsible for important economic losses in livestock production through neonatal mortality and abortion. T. gondii also causes congenital neuropathology and opportunistic infections in immunocompromised humans (41), but there is no conclusive evidence to suggest that N. caninum can infect humans (29).Previous clinical and diagnostic studies have shown that specific antibodies directed against N. caninum or T. gondii cross-react in serological and immunohistochemical tests, suggesting a possible convergence of immune responses during infections with T. gondii and N. caninum (32, 38). It has recently been shown that antibodies directed against N. caninum antigens inhibit host cell invasion by both these parasites (22, 43). Similarly, the specific cellular responses stimulated upon experimental infections with N. caninum are also stimulated in vitro by T. gondii antigenic lysate (21, 26). Consistent with these findings, CD8⁺ T cells specific for N. caninum have been shown to protect mice against lethal T. gondii infection (19). The existence of cross-reactive epitopes between N. caninum and T. gondii antigens is supported by the high level of sequence identity between conserved proteins (13). A number of cross-reactive antigens have been identified in the micronemes, rhoptries, and dense granules of tachyzoites and in bradyzoites (2, 3, 28, 43). All these observations suggest that the conserved antigenicity between N. caninum and T. gondii might represent a rational basis for the development of efficient vaccines for the control of both parasitic diseases.A vaccine based on dead N. caninum tachyzoites is currently available for prophylaxy; this vaccine is thought to confer about 46% protection against N. caninum-induced abortion in cattle (36). However, in a number of countries, this vaccine has not been licensed, since more complete scientific documentation is required to authorize the use of a vaccine against N. caninum (8). The need for a more effective vaccine against transplacental infection in cattle is therefore of the utmost importance. Live vaccines are thought to induce complete protective immunity against N. caninum infection. In vaccination trials with the mouse model, the use of N. caninum tachyzoite crude extract as the immunogen resulted in an absence of protection against parasite-related neurological illness and death (5, 27). Such vaccinations have also proved ineffective for the prevention of abortion in cattle, even in the presence of adjuvants (42).Given that protective immunity against intracellular pathogens such as T. gondii and N. caninum involves T-cell-mediated immunity (12, 21) and that experimental evidence of protection against N. caninum transplacental transmission has been shown to involve high levels of gamma interferon (IFN-γ) production (17, 42), we propose an innovative approach based on heterologous vaccination.Taking into consideration the antigenic similarities between N. caninum and T. gondii, we used an attenuated strain of T. gondii as a heterologous vaccine against N. caninum. A mutant RH strain of T. gondii tachyzoites lacking the mic1 and mic3 genes was constructed in our laboratory, the “mic1-3KO strain.” The disruption of these two genes, both of which code for proteins involved in tachyzoite adhesion and invasion, greatly decreases virulence in mice (7). Vaccination with the mic1-3KO strain provides strong protection against chronic and congenital toxoplasmosis in mice through the induction of strong humoral and Th1 cellular immune responses (18). In this study, we used this attenuated strain as a heterologous vaccine. Our results provide evidence for protection against lethal N. caninum infection. This protection was associated with strong cross-reactive humoral and Th1 cellular immune responses, overcoming the biological and antigenic differences between the two species.     

Seroepidemiology of Toxoplasma gondii, Neospora caninum, and Leishmania spp. infections and risk factors for cats from Brazil

The seroprevalence of infection by Toxoplasma gondii, Neospora caninum, and Leishmania spp. was detected through an indirect immunofluorescence in 70 cats from the Andradina Municipality, S?o Paulo State, Brazil. Anti-T. gondii antibodies (titer >64) were detected in 15.7% (11/70) of animals, whereas positivity for N. caninum (titer 16) was not observed in any animal. Of the cats from urban and rural areas, 10.4% (5/48) and 27.2% (6/22) were positive for T. gondii, respectively. Breed, age, food, and contact with animals of other species were significant for considering the positivity for T. gondii (P ≤ 0.0001). Cats having access to streets (17.1%, 11/64), cats cohabiting with rats (19.6%, 10/51), and cats feeding on homemade food and raw milk (27.2%, 6/22) were positive for T. gondii. In addition, 4.2% (3/70) of the cats were positive for Leishmania spp. by ELISA technique and negative by IFAT without coinfection with T. gondii and Leishmania spp. There was no serological positivity against feline immunodeficiency virus or feline leukemia virus. In conclusion, T. gondii infection in part of the feline population from Andradina is not linked to immunosuppressions or coinfections but probably to postnatal infection in association with the type of diet and presence of rats.     

Differentiation between mouse-virulent and -avirulent strains of Toxoplasma gondii by a monoclonal antibody recognizing a 27-kilodalton antigen.

Using a murine monoclonal antibody, we were able to differentiate between mouse-virulent and -avirulent strains of Toxoplasma gondii. Monoclonal antibody TB6G5 was reactive with eight clinical mouse-avirulent isolates but not with mouse-virulent laboratory strains RH and BK. The antibody-reactive antigen was identified by indirect immunofluorescence and immunoblot as a 27-kDa cytoplasmic protein expressed by tachyzoites as well as by bradyzoites.     

Identification of a virulence-associated antigen of Toxoplasma gondii by use of a mouse monoclonal antibody.

A monoclonal antibody generated against the mouse-lethal RH strain of Toxoplasma gondii was developed. Tachyzoites of virulent and avirulent T. gondii isolates grown in permanent macrophage cell cultures were examined for differences in reactivity with this antibody. Virulence of these Toxoplasma isolates was quantified by injecting different numbers of tachyzoites into NMRI mice and observing the animals for signs of infection or death. The monoclonal antibody identified a 23-kDa antigen expressed by the mouse-lethal strains BK and RH, whereas this antigen was not detected in low-mouse-virulent strains, which were all clinical isolates from Europe. Using Western blot (immunoblot), immunofluorescence, and immunoelectron microscopy, we localized the 23-kDa antigen to the membrane compartment. From these results, we suggest that this 23-kDa antigen is a marker of strain virulence upon which a virulence classification of T. gondii may be based.     

Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.