Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108?±?2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.     

Seroprevalence of Neospora caninum in cats from the Czech Republic

Sera of 414 cats coming from different parts of the Czech Republic were tested for N. caninum antibodies. Sera samples were collected during years 2002–2011. N. caninum antibodies were detected by a commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA) with cut off ≥30% inhibition. Samples positive in cELISA were confirmed by an indirect fluorescence antibody test (IFAT); titre ≥50 was considered positive. In total, 137 (33%) cats reacted positively in cELISA; N. caninum antibodies in IFAT were detected in 16 (3.86%) cats with titres 50 and 100. In 6 cats, positive for N. caninum antibodies, T. gondii antibodies were also detected by IFAT. It is the first report of N. caninum antibodies in domestic cats from the Czech Republic and third report in Europe.     

Serological Evidence of Human Infection with the Protozoan Neospora caninum

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.     

PCR detection of Neospora caninum in water buffalo foetal tissues

The seroprevalence of Neospora caninum was surveyed by an ELISA kit on two water buffalo herds of Southern Italy. Seropositive samples were detected in 47% and 59% of individuals, respectively, thus indicating high level of exposure to the parasite even if the possibility of vertical transmission cannot be excluded. Tissue samples collected from three aborted fetuses from the same herds were investigated for N. caninum presence by PCR assays targeting the 18S and the Nc5 DNA sequences, respectively. Both methods have shown the presence of N. caninum DNA in heart and brain. Sequencing of the Nc5 genomic DNA confirmed the presence of N. caninum in the samples; phylogenetic analysis of the obtained sequences showed high homology among the Neospora recovered from different samples. The present study suggests an important role of N. caninum as a possible abortive agent for water buffaloes.     

Neospora caninum surface antigen (p40) is a potential diagnostic marker for cattle neosporosis

Neospora caninum is an intracellular protozoan that infects domestic and wild canids as well as many warm-blooded animals as shown by the isolation of viable parasites. The effectiveness of diagnostic tests for detecting specific antibodies against N. caninum is hampered by potential cross-reaction with other Coccidia. So, there is currently an urgent need for a sensitive and specific diagnostic assay for detecting N. caninum in animals. The N. caninum 40-kD surface antigen (p40), similar to NcSAG1 and NcSRS2, was shown to belong to surface antigen super family and thus represents an excellent marker for the diagnosis of neosporosis. In order to test the hypothesis, recombinant Ncp40 (rNcp40) was expressed in Escherichia coli, and an indirect ELISA test was developed using recombinant NCp40 antigen for N. caninum serodiagnosis. The antigen used in this study did not have cross-reactivity with anti-Toxoplasma gondii serum. Anti-p40 antibodies were detected by ELISA in the sera of Yellow cattle and were compared with (IFAT). Optimal sensitivity and specificity (98.2 and 98.6 %) were identified by IFAT. Additionally, 37 positive sera of T. gondii were detected and there was no significant difference with the negative serum of N. caninum. The rNcp40 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene

Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarites and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis.     

Characterization of a Swedish bovine isolate of Neospora caninum

The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.     

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (≤200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%–29% and 22–25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.     

Antigen-Specific CD8+ T Cells Protect against Lethal Toxoplasmosis in Mice Infected with Neospora caninum

Neospora caninum is a coccidial protozoan parasite that appears morphologically indistinguishable from Toxoplasma gondii and that infects a large range of mammals. Both inbred and outbred strains of mice exhibit a high degree of resistance to infection with N. caninum. Three inbred strains of mice (A/J, BALB/c, and C57BL/6) that were infected intraperitoneally with N. caninum were protected against a lethal challenge from T. gondii. Vaccine-induced protection was Neospora dose dependent. A rise in the CD8⁺ T-cell population in mice that had been vaccinated with N. caninum and challenged with T. gondii was observed. Adoptive transfer of CD8⁺ T-cell splenocytes from N. caninum-infected mice was protective against challenge with Toxoplasma. The CD8⁺ T cells from Neospora-infected mice proliferate to both Neospora and Toxoplasma antigens in vitro and secrete substantial quantities of gamma interferon when pulsed with the parasite antigen. These observations demonstrate that N. caninum protects against lethal T. gondii infection by the induction of CD8⁺ T cells that are immunoreactive to both parasites.     

Evaluation of parasitological and immunological aspects of acute infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs

This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L₃ larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.     

Sensitive and Specific Identification of Neospora caninum Infection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Macrophage Depletion Prior to Neospora caninum Infection Results in Severe Neosporosis in Mice

We observed that murine macrophages showed greater activation and increased interleukin 6 (IL-6), IL-12p40, and interferon gamma (IFN-γ) production during Neospora caninum infection. Many macrophages migrated to the site of infection. Furthermore, macrophage-depleted mice exhibited increased sensitivity to N. caninum infection. This study indicates that macrophages are required for achieving protective immunity against N. caninum.     

Application of chicken embryonated eggs as a new model for evaluating the virulence of Neospora caninum tachyzoites

Neospora caninum is a cyst-forming coccidian parasite, which is highly pathogenic for cows and dogs. Determination of parasite virulence has been applied in the development of live vaccines against protozoan parasites. In this sense, we wanted to determine whether chicken embryonated egg could be an animal model to show different virulence of N. caninum tachyzoites. In this study, the first N. caninum isolate was considered low passage (no passage) and then passaged for 80 times in vitro as high passage. Groups of 8-day-old embryonated eggs with 10 eggs in each group were inoculated with 10 and 10² of low or high-passage N. caninum tachyzoites, and any mortality was recorded. Suitable samples from different tissues (liver, heart, and brain) of the dead embryos were collected for histopathological and immunohistochemistry (IHC) study. In this study, the mortality rate decreased in the high passage infection in each group and IHC method showed the presence of the parasite in chicken tissues receiving high and low passage parasites. The present investigation showed that the chicken embryonated eggs can be a valuable alternative approach for in vivo testing of live attenuated potential vaccine, and also, these results confirm that an increase in passage could decrease the N. caninum virulence in chicken embryonated egg.     

Comparative study for the detection of antibodies to Neospora caninum in milk and sera in dairy cattle in southern Romania

The study aimed to assess the within-herd Neospora caninum exposure in dairy cattle in southern Romania, based on the detection of specific antibodies in milk and serum. A total of 104 paired samples of milk and serum were collected from four dairy farms. Individual samples were analyzed for N. caninum antibodies by ELISA: IDEXX Neospora Ab (Idx) (three farms: A, B, C; n = 60) and ID-VET Lab (Idv) (farm D; n = 44). Additionally, four pooled milk samples, one per each farm (A, B, C) and a composed one (A+B+C), were analyzed with Idx ELISA. Optimized cut-off values for milk samples were determined by receiver operating characteristic (ROC) analysis, with serum results considered as true status. The agreement was expressed by K values. The overall seroprevalence of N. caninum infection was 45% in the farms tested by Idx ELISA and 56.8% in the farm tested by Idv ELISA. A good agreement between serum and milk was obtained for both ELISA kits (K = 0.72 and 0.77, respectively). The specificity and sensitivity at optimized cut-off of S/P>0.704 for Idx and S/P%>7.966% for Idv were 100% and 70.37% for Idx and 89.47% and 88% for Idv. Testing pooled milk samples, there were identified as N. caninum positive the dairy farms with a 15% or higher within-herd seroprevalence at the cut-off value of S/P>0.51. This is the first study in Romania in which milk samples were tested to determine the N. caninum infection status in dairy farms, providing a base for further researches.     

Molecular analyses on Neospora caninum-triggered NETosis in the caprine system

Neospora caninum is an obligate intracellular protozoan parasite causing serious reproductive disorders in large and small ruminants worldwide. Polymorphonuclear neutrophils (PMN) react against multiple invading pathogens through different mechanisms including the release of neutrophil extracellular traps (NETs). Here, in vitro interactions of caprine PMN and N. caninum tachyzoites were studied. Scanning electron microscopic- and immunofluorescence-analyses demonstrated that caprine PMN undergo NETosis upon contact with tachyzoites of N. caninum, extruding filaments that entrap parasites. Detailed co-localization studies of N. caninum tachyzoite-induced NETs revealed the presence of PMN-derived DNA being decorated with histones (H1, H2A/H2B, H3,H4) and neutrophil elastase (NE) corroborating the molecular characteristics of classical mammalian NETs. As a new result for parasite-induced NETosis, we identified pentraxin and cathepsin B in N. caninum-triggered NETs. Nonetheless, functional inhibition assays revealed that during caprine NET formation triggered by N. caninum different molecular signaling pathways are induced, when compared to other apicomplexan parasites or host species. As such, N. caninum-induced NETosis appears to be influenced by MPO but independent of NADPH oxidase, SOCE, ERK1/2 and p38 MAPK activities. Furthermore, the inhibition of PMN autophagy via blockage of the PI3K-mediated signaling pathway failed to influence tachyzoite-induced NETosis. Since N. caninum-tachyzoites induced caprine NETosis, this effector mechanism should be considered as an early host immune response during acute caprine neosporosis.     

Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces,Thailand

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.     

Detection of Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi in the brains of common voles (Microtus arvalis) and water voles (Arvicola terrestris) by gene amplification techniques in western Austria (Vorarlberg)

Knowledge about the protozoan parasite fauna in voles (Arvicolinae) in Austria is rather limited, although some of these pathogens play an important role in human medicine and cause zoonoses (e.g., Toxoplasma gondii and Encephalitozoon cuniculi). Others are of relevance in veterinary medicine and have a negative economic impact (e.g., Neospora caninum). Two hundred sixty-eight common voles (Microtus arvalis) and 86 water voles (Arvicola terrestris) from the most western Austrian province, Vorarlberg, were analyzed with PCR techniques for infections with T. gondii, N. caninum, and E. cuniculi. Brain tissues of two common voles (0.7%) and of four water voles (4.7%) tested positive for T. gondii. Furthermore, analysis of four common voles (1.5%) and two water voles (2.3%) generated positive findings for N. caninum, and brain tissues of 16 common voles (6%) and six water voles (7%) tested positive for E. cuniculi. Accordingly, this study not only demonstrates the autochthonous existence of the zoonotic parasites T. gondii and E. cuniculi in voles in Vorarlberg, it also provides the first evidence of an occurrence of N. caninum in animals of the subfamily Arvicolinae, and it is an additional contribution to investigations of the sylvatic cycle of N. caninum.     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Canine neosporosis: clinical and pathological findings and first isolation of Neospora caninum in Germany

Neosporosis was diagnosed in an 11-week-old puppy of the breed Kleiner Münsterländer with progressive hindlimb paresis. Pathohistological and immunohistological examinations revealed a disseminated infection with Neospora caninum. Parasitic stages were demonstrated in the brain, spinal cord, retina, muscles, thymus, heart, liver, kidney, stomach, adrenal gland, and skin. Immunohistochemistry investigations were carried out using polyclonal rabbit antisera developed against N. caninum tachyzoites and the recombinant bradyzoite-specific antigen BAG-5 of Toxoplasma gondii, which is known to cross-react with N. caninum bradyzoites. BAG-5 antibodies recognized tissue cysts within the CNS and some protozoan stages that were not surrounded by a visible cyst wall. All parasite clusters in the retina and some in muscle tissue stained positively with the BAG-5 antiserum. N. caninum was isolated in cell culture and mice inoculated with brain and spinal cord of the puppy. The new isolate is the first reported in Germany and is designated NC-GER1.     

The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5′ coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3′ UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5′ coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.