TNF-TNFR1 Signaling Enhances the Protection Against Neospora caninum Infection

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 (Tnfr1-/-). We observed that Tnfr1-/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1-/- mice showed a reduction in nitric oxide (NO) levels in vivo. We also observed that Tnfr1-/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum.

Neospora caninum Infection in Cattle in the Province of Kohgiluyeh and Boyer Ahmad, Southwest of Iran: Seroprevalence and Molecular Assessment

Introduction. Among the protozoa of veterinary importance, Neospora caninum is responsible for large economic and productive losses in cattle herds. Dogs are being considered the definitive hosts of the parasite. Neospora caninum has gained considerable attention through its role in the etiology of bovine abortion. The current study aimed at assessing the status of Neospora infection in cattle in Boyer-Ahmad County in Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. Methods. In this cross-sectional study, 150 cattle blood samples were collected and samples were screened for N. caninum antibodies using a modified direct agglutination test (MAT). For the same samples, 130 buffy coats were collected and tested, by PCR, for the presence of N. caninum DNA, targeting the Nc-5 gene. Results. Anti-N. caninum antibodies were detected in the sera of 49 out of 150 cattle which is corresponding to a seroprevalence rate of 32.7%. Neospora DNA was detected in buffy coats of 26 out of 127 (20.47%) cattle. Even though Neospora infection was more common in females and in five-year-old cattle by serology and molecular methods, yet there was no statistically significant difference between age, sex, and Neospora infection in both molecular and serological methods ( p > 0.05 ). Conclusion. Findings of the current study indicate a high rate of N. caninum infection in cattle of Boyer-Ahmad region in the southwest of Iran. This issue should be further investigated and the prevention and control of this parasite in livestock, due to the high financial burden of this parasite in the livestock industry, should be considered.

The Protective Role of TLR2 Mediates Impaired Autophagic Flux by Activating the mTOR Pathway During Neospora caninum Infection in Mice

Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.

Clinical presentation, diagnosis and therapeutic management of Dipylidium caninum (Cestoda: Dilepididae) infection in a domestic cat (Felis catus): a case report

Background: Dipylidium caninum, a zoonotic cyclophyllidean tapeworm, mainly infects dogs, cats, and occasionally humans as well. Here, we present D. caninum infection in a domestic cat. A cat of about one year of age with a history of intermittent diarrhea and shedding stool containing whitish cooked rice like soft particles. Methods: The case was identified by thorough clinical, coprological, and parasitological examinations, and treated accordingly. Results: During the physical examination, the cat was found to be infested with flea, and coprological investigation revealed the presence of gravid segments of cestodes. By preparing a permanent slide, we conducted a microscopic examination, and the cestode was confirmed as D. caninum. The cat was treated with albendazole and levamisole, which were ineffective; additionally, levamisole showed toxicity. Then, we administered niclosamide which completely cured the animal. On re-examination after a week, feces were found negative for eggs/gravid segments of any cestode. Conclusion: Niclosamide was found effective against dipylidiasis and can be treated similar infections in pets.

Neosporosis: a neglected abortifacient disease in Egypt, seroprevalence and farmers’ knowledge, attitudes and practices

Neosporosis has emerged as a serious disease of cattle and dogs worldwide, causing abortions, neonatal mortalities and massive economic losses in dairy herds. The current study aimed to investigate the burden of Neospora (N.) caninum infection among cows that aborted and to assess knowledge, attitudes, and practices (KAPs) of farmers toward neosporosis in Kafrelsheikh governorate, Egypt. A total of 92 cows that aborted and 25 heifers born from cows that aborted and seropositive for N. caninum infection from 15 dairy herds in different districts of Kafrelsheikh governorate were examined serologically against N. caninum infection using ELISA. A structured questionnaire was built and distributed to 41 farmers in the study area. The overall seroprevalence of N. caninum infection among the examined cows that aborted was 38.04% (35/92). On the other hand, the prevalence of N. caninum infection among the 25 examined heifers born from seropositive cows that aborted was 28% (7/25). The KAPs analysis showed that farmers lack the required information on N. caninum infection and its consequences on dairy farms. The farmers performed risky practices which are responsible for disease entrance and spread on the farm; buy and keeping animals after abortion as well as their heifers. In addition, risk management practices were widespread in dairy farms such as: free movement of dogs, especially stray ones, in 100% of the farms, dogs were able to access and defecate in cattle feed and drinking water sources and had the chance to eat placentas and abortion materials. The current study points out neosporosis as a neglected cause of abortion among cattle in Kafrelsheikh governorate, as well as there is a lack of knowledge and risky practices by the local farmers. These findings may be extrapolated to nearby areas and countries of the same cattle husbandry practices, and veterinary services in such countries should consider neosporosis in their surveillance and control programs.

Comparative Evaluation of Four Potent Neospora caninum Diagnostic Antigens Using Immunochromatographic Assay for Detection of Specific Antibody in Cattle

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.

A Literature Review of Neospora caninum Infection in Humans

Background: Neosporosis is a parasitic disease caused by Neospora caninum. This parasite is an obligate intracellular coccidia similar to Toxoplasma gondii with a global distribution. With regard to the experimental studies, vertical transmission of the parasite in the monkey (non-human primates) has increased the concern about the zoonotic potential of this disease. The principal aim of the current research was to perform a mini-review on investigations regarding the Neospora infection in humans on a global scale for the first time. Methods: All peer-reviewed articles (published until April 2021) on the Neospora infection in humans were searched in English databases such as Google Scholar, ScienceDirect, Scopus, PubMed, and ProQuest. Results: Based on data in the available articles, the presence of antibodies against the Neospora infection was between 0 and 37.7% in people from different countries. The seroprevalence rate of this infection in HIV-positive individuals was higher (26.6% and 37.7%) compared to other cases. Finally, the genomic DNA of Neospora was detected up to 1% using molecular biology techniques. Conclusions: Overall, the detection of anti-Neospora antibodies in humans indicated that people have been exposed to the parasite. Comprehensive research studies are essential for clarifying the risk factors associated with the Neospora infection in humans. This report provides the baseline information for future researchers. Molecular investigations and genotypic works on N. caninum isolates are highly recommended as well.

Molecular Identification of Neospora caninum Infection in Abort-ed Fetuses of Sheep, Cattle, and Goats in Mazandaran Province, Northern Iran

Background: We aimed to identify Neospora caninum DNA in the brain samples of aborted fetuses of cattle, goats, and sheep in Mazandaran, northern Iran, using PCR. Methods: In total, 133 aborted fetuses (51 sheep, 78 cattle, and 4 goats) were randomly collected from different stages of gestation in various regions of Mazandaran, Iran, from Mar 2016 to May 2017. The DNA was extracted from all the brain samples using phenol chloroform isoamyl alcohol instructions. The Nc-5 gene was used for the detection of N. caninum DNA by nested-PCR assay. Results: The detection of N. caninum DNA was confirmed by the observation of a 227 bp band in 24 samples of 133 aborted fetuses (18.1%). The highest prevalence rate of N. caninum was detected in the cattle (20.5%) followed by the sheep (15.6%); however, no positive cases were reported in the goats. The highest and lowest prevalence rates of the infection were reported as 23.8% and 8.6% in Qaemshahr, and Behshahr, respectively. The prevalence rate of infection (32%) in the early gestational period was higher than those in the middle (15%) and late (3.8%) gestational periods. Conclusion: The obtained data of the present study indicated that N. caninum infection may partly be responsible for abortion and economic loss in livestock farming in Mazandaran Province.