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.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in dogs from Korea

Toxoplasma gondii and Neospora caninum are closely related protozoan parasites, they share many common hosts, and can cause neurological diseases in dogs. Dogs can have close contacts with humans and livestock and therefore they can act as reservoirs of these parasites. The aim of this study was to survey the seroprevalence of antibodies against T. gondii and N. caninum and their co-infection rate in dogs in Korea. In total, sera from 553 domestic dogs were collected from different breeds, sexes, and ages of dogs from nine provinces across the country of Korea during 2006 and 2007. The presence of antibodies against T. gondii and N. caninum was analyzed using the latex agglutination test (LAT) with a cut-off value of 1:32, and the indirect fluorescent antibody test (IFAT) using a serum titer of 1:100. In the total dog population, 71 (12.8%) dogs were positive for anti-T. gondii antibodies and only 20 (3.6%) were positive for anti-N. caninum antibodies. Relatively higher seropositive frequencies of antibodies against T. gondii (20.1%) and N. caninum (4.9%) were detected in the dog population from the Gyeonggi. A higher proportion of animals seropositive for anti-T. gondii antibodies was found in stray dog populations as compared to household dog populations: 18.5% (59/319) vs 5.1% (12/234), respectively. The Chi-square tests revealed significant differences in the seropositive frequencies of antibodies against T. gondii between stray and household dogs in the total population (p<0.0001), and in dogs from the Gyeonggi (p<0.01). No significant differences were observed for the presence of antibodies against T. gondii or N. caninum when compared across the sex or age (p>0.05). The first serological survey on antibodies against both T. gondii and N. caninum parasites across the entire country showed that co-infection was not common in these canine populations with a seropositive level of 0.72%. The significantly higher positive frequency of T. gondii antibodies in stray dogs in both, Gyeonggi and in the total dog populations suggests that further investigation on the seroprevalence of parasites should focus on stray dogs.     

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.     

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).     

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.     

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.     

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.     

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.     

Investigation of Neospora caninum, Hammondia sp., and Toxoplasma gondii in tissues from slaughtered beef cattle in Bahia, Brazil

Neospora caninum, Hammondia sp., and Toxoplasma gondii are parasites with morphological and genetic similarities. N. caninum and T. gondii are important abortive agents of cattle and sheep, respectively, and may infect numerous animal species. Hammondia sp. is not known to induce disease in animals, but may cause confusion in the identification of closely related coccidia. The aim of this study was to investigate infection rates caused by N. caninum, Hammondia sp., and T. gondii in beef cattle using a nested PCR for Toxoplasmatinae rDNA, followed by sequencing of the PCR products. Antibodies to N. caninum and T. gondii were also investigated in the tested animals. Brains and hearts were obtained from 100 beef cattle in a slaughterhouse in Bahia. Seven samples from brain tested positive for Toxoplasmatinae DNA. No positive reactions were found in heart tissues. After sequencing of the PCR products from all positive tissues, five sequences matched with N. caninum and two matched with T. gondii. Antibodies to N. caninum and T. gondii were found in 20% and 26% of the animals, respectively. The confirmation of N. caninum and the absence of Hammondia heydorni in the tested animals is suggestive that cattle are not efficient intermediate hosts of H. heydorni; however further studies need to be performed using a greater variety of tissues and a higher sample size. The detection of T. gondii DNA in bovine tissues reinforces the potential risk of transmission of this parasite to humans and other animals through the consumption of bovine meat.     

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.     

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.     

Neospora spp. and Toxoplasma gondii antibodies in horses in the Czech Republic

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had ≥50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.     

The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii and has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) from N. caninum tachyzoites that are the predominant antigens recognized by antisera from Neospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverse N. caninum isolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to the T. gondii surface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins of T. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.     

Apical membrane antigen 1 is a cross-reactive antigen between Neospora caninum and Toxoplasma gondii, and the anti-NcAMA1 antibody inhibits host cell invasion by both parasites

The cross-reactive antigens of Neospora caninum and Toxoplasma gondii are important in the exploration to determine the common mechanisms of parasite-host interaction. In this study, a gene encoding N. caninum apical membrane antigen 1 (NcAMA1) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with recombinant T. gondii apical membrane antigen 1 (TgAMA1). NcAMA1 was encoded by an open reading frame of 1695 bp, which encoded a protein of 564 amino acids. The single-copy NcAMA1 gene was interrupted by seven introns. NcAMA1 showed 73.6% amino acid identity to TgAMA1. Mouse polyclonal antibodies raised against the recombinant NcAMA1 (rNcAMA1) recognized a 69-kDa native parasite protein by Western blotting. Immunofluorescence analysis showed that NcAMA1 was localized to the apical end of tachyzoites. Two-dimensional electrophoresis and Western blotting indicated that an approximately 57-kDa cleavage product was released into the excretory/secretory products of N. caninum. Preincubation of free tachyzoites with anti-rNcAMA1 IgG antibodies inhibited the invasion into host cells by N. caninum and T. gondii. These results indicated that AMA1 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control two parasites.     

Role of TNF and IL-1 in infections with Toxoplasma gondii.

Mice lethally infected with the C56 strain of Toxoplasma gondii and treated with purified recombinant murine tumour necrosis factor (TNF, 1 microgram/day/mouse for 8 days), recombinant human interleukin-1 (IL-1 alpha or IL-1 beta, 100 ng/day/mouse for 5 days) or a single dose of a combination of TNF (1 microgram/mouse) and IL-1 alpha or IL-1 beta (100 ng/mouse) were significantly protected against death (P less than 0.05-0.001, as compared with untreated infected controls). Mice infected with 100,000 tachyzoites of the highly virulent RH strain of T. gondii released serum TNF in relation to the time after infection and were primed to secrete an enhanced level of serum TNF upon stimulation with bacterial lipopolysaccharide (LPS). In vitro studies showed that interferon-gamma (IFN-gamma) increased the antimicrobial activity of murine peritoneal macrophages whereas TNF, IL-1 alpha and IL-1 beta did not. TNF, however, synergized with the anti-toxoplasmic effect provided by IFN-gamma and this activity was blocked by anti-TNF antibodies. IFN-gamma induced the production of TNF and the anti-toxoplasmic effect provided by IFN-gamma seemed to be dependent partly on the production of TNF. We conclude that TNF and IL-1 may play a significant role in modulating the host's immune defence against T. gondii infection.     

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs)

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.     

Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium

Anaplasmosis and babesiosis are major tick-borne diseases with a high economic impact but are also a public health concern. Blood samples collected in the spring, summer, and autumn of 2010 from 65 cows in seven different farms in Belgium were monitored with an indirect immunofluorescence antibody test to assess seroprevalence against these pathogens. Seroprevalences to Babesia spp. were measured as 10.7%, 20%, and 12.3% in spring, summer, and autumn, respectively, whereas seroprevalences to Anaplasma phagocytophilum were 30.8%, 77%, and 56.9%, respectively. A total of 805 Ixodes ricinus ticks were collected at the same time from both cattle (feeding ticks) and grazed pastures (questing ticks). The infection level of ticks, assessed by PCR assay, for Babesia spp. DNA was 14.6% and 7.9% in feeding and questing ticks, respectively, whereas 21.7% and 3% of feeding and questing ticks were found be positive for A. phagocytophilum cDNA. Fifty-five PCR-positive samples were identified by sequencing as Babesia sp. EU1, of which five from feeding ticks were positive for both A. phagocytophilum and Babesia sp. EU1. The high density of wild cervids in the study area could explain these observations, as deer are considered to be the main hosts for adults of I. ricinus. However, the absence of Babesia divergens both in feeding and questing ticks is surprising, as the study area is known to be endemic for cattle babesiosis. Increasing cervid populations and comorbidity could play an import role in the epidemiology of these tick-borne diseases.