Search Results (5)

Neospora caninum is widely recognised as one of the most significant causes of abortion in cattle, with infections also occurring in sheep and goats. To prevent and control animal neosporosis, it is crucial to develop sensitive and specific methods for detecting N. caninum infection. Recently, several recombinant proteins have been utilised in serological assays for the diagnosis of neosporosis. In this study, we used commercial gene synthesis to produce dense granular antigen 4 (NcGRA4) recombinant protein. NcGRA4 plasmids were expressed in the Escherichia coli system and then purified. The purified recombinant protein was analysed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis. To evaluate the diagnostic potential of recombinant NcGRA4 protein, we tested 214 serum samples from goat farms via indirect enzyme-linked immunosorbent assay (iELISA) and compared the results to those from the indirect fluorescent antibody test (IFAT). Western blotting analysis revealed a single NcGRA4 band with an expected molecular weight of 32 kDa. The specific IgG against N. caninum was detected in 34.1% and 35% of samples evaluated by NcGRA4 iELISA and IFAT, respectively. The sensitivity and specificity of the NcGRA4 iELISA were 71.6% and 86.3%, respectively, when compared with the results from IFAT. Our results demonstrate that a recombinant protein that can be used to detect animal neosporosis can be produced using a synthetic NcGRA4 gene. Overall, recombinant NcGRA4 shows promise as a sensitive and specific serological marker for identifying target IgG in goat samples. Full article

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection. Full article

Toxoplasma gondii and Neospora caninum belong to the Apicomplexan protozoa which is an obligate intracellular parasite, causing toxoplasmosis and neosporosis throughout the world. Cats and dogs are the definitive hosts of these two parasites. However, information on the epidemiology of toxoplasmosis and neosporosis in stray cats and dogs in the Qinghai–Tibetan Plateau Area (QTPA) is limited, and little is known about the diversity of the diseases. Therefore, the aim of this study was to perform indirect ELISA tests based on recombinant TgSAG1, TgGRA1, NcSAG1 and NcGRA7 proteins to establish a detailed record of the seroprevalence of T. gondii and N. caninum-specific IgG and IgM antibodies in serum samples and to develop qPCR amplification based on TgB1 and NcNc5 genes to conduct molecular epidemiology in feces from stray cats and dogs in the QTPA. In the current study, a total of 128 cat serum samples were analyzed through serological tests in which 53 (41.4%) and 57 (44.5%) samples were found positive for T. gondii specific-IgG and IgM antibodies, and 2 (1.6%) and 74 (57.8%) samples were confirmed positive for N. caninum specific-IgG and IgM antibodies, respectively. Out of 224 stray dog sera, 59.8% and 58.9% were recorded as positive against anti-Toxoplasma IgG and IgM antibodies, 17.9% and 64.7% were detected positive against Neospora IgG and IgM. On the other hand, 1 of 18 cat fecal samples was successfully amplified within the Ct value of 10 to 30 while no cat was positive for neosporosis. Moreover, a higher prevalence of toxoplasmosis in stray dogs (14.5%, 16/110) than of neosporosis (5.5%, 6/110) with different parasite numbers were found. Further analysis showed that no significant sex differences were found nor between the overall infection rates of T. gondii and N. caninum in this study. This study suggests that stray cats and dogs play key roles in the transmission and prevalence of T. gondii and N. caninum in the plateau area. Full article

Neospora caninum is an intracellular protozoan parasite responsible for numerous abortion outbreaks and neonatal abnormalities in cattle. Rapid and accurate diagnosis is critical for N. caninum control owing to the lack of vaccine or drug-based control strategies. Herein, we evaluated the performance of four frequently used antigens in the diagnosis of N. caninum infection using immunochromatographic tests (ICTs) as a rapid, affordable, and field applicable tool. These antigens included recombinant proteins of N. caninum surface antigen 1 (NcSAG1), dense granule proteins 7 (NcGRA7) and 6 (NcGRA6), in addition to native Neospora lysate antigen (NLA). Our study revealed the utility of all antigen-based ICTs for detection of specific antibodies to N. caninum. However, the NcSAG1-based ICT was the best for detection of all control N. caninum-infected mouse or cattle sera, while NcGRA7 and NcGRA6-based ICTs exhibited specific ability to detect samples from acute and sub-acute infection in mice and cattle, respectively. Analyses of the NcSAG1-based ICT against enzyme-linked immunosorbent assays (ELISAs) of the same antigen revealed its efficiency in detection of field cattle samples as observed in high sensitivity (84.2%), specificity (93.5%), agreement (90%), and kappa value (0.78). The current knowledge provides an efficient platform for N. caninum control through on-site diagnosis of infected cattle. Full article

Vaccination is the mainstay of preventative measures for numerous infectious diseases. Neospora caninum infection induces storms of abortion in pregnant cows and ewes, resulting in drastic economic losses because of fetal losses and culling of the dams. Herein, we evaluated the potential of recombinant protein of N. caninum dense granule protein 6 fused with glutathione-S-transferase (NcGRA6+GST) as a vaccine candidate against neosporosis in a pregnant mouse model. The protective efficacy was investigated by subcutaneous inoculation of BALB/c mice with recombinant NcGRA6+GST (25 pmol), and GST alone (25 pmol) or phosphate-buffered saline (PBS) as the controls. This study revealed the partial ability of NcGRA6+GST to protect the dams and offspring from N. caninum infection during the critical period of pregnancy. This ability was revealed by higher survival rate and lower parasite burden in brains of offspring of the NcGRA6+GST-immunized group in comparison with the control groups. In addition, mouse dams from NcGRA6+GST-immunized groups exhibited lower clinical score and minimum alteration in body weight in comparison with PBS or GST groups after challenge with N. caninum tachyzoites. Taken together, our results suggest the efficacy of recombinant NcGRA6 for interrupting the vertical transmission of N. caninum in mice by reducing the severity of infections in dams and offspring. Full article