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Tissue protozoa play a significant role as a cause of economic losses in reproductive and productive aspects in camels beside the zoonotic importance. A Cross-sectional study was designed to estimate the seroprevalence and risk factors of Toxoplasma gondii and Neospora caninum in dromedary camels in Matrouh and Aswan border governorates of Egypt. One hundred and eighty-two serum samples from apparently healthy dromedary camels and tested serologically for neosporosis and toxoplasmosis. The prevalence of possible related risk factors was investigated from December 2020 to November 2021. The serological testing of 182 camel serum samples revealed a prevalence (15.93%, 29/182) for Neospora caninum, (58.24%, 106/182) for Toxoplasma gondii and (9.34%, 17/182) for both infections together. The total seroprevalence rate was (64.84%, 118/182). Camels in Matrouh governorate were at high risk (p < 0.05) of infection with tissue protozoa 3.74 times more than camels in Aswan governorate. Maghrabi camels were found more significant (p < 0.05) prevalent for T. gondii infection (62/78, 79.49%) by 5.28 times than Sudani camels, Otherwise, in Sudi camels, N. caninum is almost 2.70 times Maghrabi camels. The results showed that age and sex were mostly significant for N. caninum and T. gondii. This study revealed that camels are a possible source of infection for the studied tissue protozoa, some of which are significant for public health. Further research is needed to describe their true situation and epidemiology in dromedary camels. [ABSTRACT FROM AUTHOR]

Neospora caninum, an intracellular protozoan parasite, causes neosporosis resulting in major losses in the livestock industry worldwide. However, no effective drugs or vaccines have been developed to control neosporosis. An in-depth study on the immune response against N. caninum could help to search for effective approaches to prevent and treat neosporosis. The host unfolded protein response (UPR) functions as a double-edged sword in several protozoan parasite infections, either to initiate immune responses or to help parasite survival. In this study, the roles of the UPR in N. caninum infection in vitro and in vivo were explored, and the mechanism of the UPR in resistance to N. caninum infection was analyzed. The results revealed that N. caninum triggered the UPR in mouse macrophages, such as the activation of the IRE1 and PERK branches, but not the ATF6 branch. Inhibition of the IRE1α-XBP1s branch increased the N. caninum number both in vitro and in vivo, while inhibition of the PERK branch did not affect the parasite number. Furthermore, inhibition of the IRE1α-XBP1s branch reduced the production of cytokines by inhibiting NOD2 signalling and its downstream NF-κB and MAPK pathways. Taken together, the results of this study suggest that the UPR is involved in the resistance of N. caninum infection via the IRE1α-XBP1s branch by regulating NOD2 and its downstream NF-κB and MAPK pathways to induce the production of inflammatory cytokines, which provides a new perspective for the research and development of anti-N. caninum drugs. [ABSTRACT FROM AUTHOR]

Neosporosis is internationally documented as one of the most popular diseases in cattle that cause economic losses due to high levels of abortion cases. Although Neospora caninum has been recently classified as a new species, it is still sharing many features with Toxoplasma gondii. This study aimed to detect and Imaging N. caninum in the blood of aborted cows, and prevalence study of N. caninum infection based on age, region and month. Blood samples from 106 aborted cows were collected using the appropriate method. First, these samples were examined microscopically via blood smears using Giemsa dye to diagnose the N. caninum within RBC. A qPCR technique was carried out to detect accurately 18S rRNA gene accurately. The results revealed that 65% of total aborted cases were positive for 18S rRNA detection of N. caninum, although this parasite was found microscopically in 15% of blood smear samples. According to PCR results, the prevalence study showed that the highest rate of infection was signed in the Al-Qassim district (75%) followed by the Al-Mahaweel district (74%) and decreased in Western Hamza district (48%). According to study months, November recorded the peak of infection (88%), then August (71%), whereas July recorded the lowest percentage (50%). The statistical analysis revealed there was no significant difference between the subjected regions and study months based on (P<0.05). On the other hand, it was found that cows less than 3 years old were more susceptible to infection than those over 3 years old. the results revealed that 71% of infected cows were less than 3 years old, while 29% were at age over 3 years old with a significant difference (P<0.005). In conclusion, N. caninum can be detected through blood within RBC. Age and regional factors in cows play an important role in resisting infection with this pathogen. [ABSTRACT FROM AUTHOR]

[This corrects the article DOI: 10.3389/fmicb.2016.01456.].

Neospora caninum is a coccidian parasite that occurs worldwide and is one of the most important causes of abortion, especially in cattle. However, no studies have been performed in Namibia to determine the N. caninum status in livestock. Therefore, this study aimed to determine the seroprevalence of N. caninum in cattle and the associated risk factors in the Khomas region of Namibia. A total of 736 sera were collected from cows in 32 farming establishments. These comprised 698 beef and 38 dairy cattle sera and were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Questionnaires were concurrently administered to determine possible risk factors associated with N. caninum seropositivity. A total of 42 sera were positive (all beef), giving an animal-level seroprevalence rate of 5.7%. Eight of the 32 establishments had at least one positive animal, giving a herd-level seroprevalence of 25%. There was no significant association between seropositivity and the presence of dogs, jackals, history of abortions, farm size, number of cattle or average annual rainfall. The establishments with moderate to high numbers of Feliformia were 9.8 times more likely to be seropositive to N. caninum than those with none to low levels of the former (p = 0.0245). The authors concluded that the seroprevalence level of N. caninum in the Khomas region was relatively low compared with other parts of the world and that the role of Feliformia in the epidemiology of bovine neosporosis needed to be further investigated. Contribution: Serological evidence of bovine neosporosis and the associated risk factors are reported in Namibia for the first time. This study contributes to the scientific body of knowledge on N. caninum in Africa, which is currently limited.

Neospora caninum is a coccidian parasite that occurs worldwide and is one of the most important causes of abortion, especially in cattle. However, no studies have been performed in Namibia to determine the N. caninum status in livestock. Therefore, this study aimed to determine the seroprevalence of N. caninum in cattle and the associated risk factors in the Khomas region of Namibia. A total of 736 sera were collected from cows in 32 farming establishments. These comprised 698 beef and 38 dairy cattle sera and were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Questionnaires were concurrently administered to determine possible risk factors associated with N. caninum seropositivity. A total of 42 sera were positive (all beef), giving an animal-level seroprevalence rate of 5.7%. Eight of the 32 establishments had at least one positive animal, giving a herd-level seroprevalence of 25%. There was no significant association between seropositivity and the presence of dogs, jackals, history of abortions, farm size, number of cattle or average annual rainfall. The establishments with moderate to high numbers of Feliformia were 9.8 times more likely to be seropositive to N. caninum than those with none to low levels of the former (p = 0.0245). The authors concluded that the seroprevalence level of N. caninum in the Khomas region was relatively low compared with other parts of the world and that the role of Feliformia in the epidemiology of bovine neosporosis needed to be further investigated. Contribution: Serological evidence of bovine neosporosis and the associated risk factors are reported in Namibia for the first time. This study contributes to the scientific body of knowledge on N. caninum in Africa, which is currently limited. [ABSTRACT FROM AUTHOR]

Toxoplasmosis and neosporosis are major protozoan diseases of global distribution. Toxoplasma gondii is the cause of toxoplasmosis, which affects almost all warm-blooded animals, including humans, while Neospora caninum induces neosporosis in many animal species, especially cattle. The current defective situation with control measures is hindering all efforts to overcome the health hazards and economic losses of toxoplasmosis and neosporosis. Adequate understanding of host-parasite interactions and host strategies to combat such infections can be exploited in establishing potent control measures, including vaccine development. Macrophages are the first defense line of innate immunity, which is responsible for the successful elimination of T.gondii or N. caninum. This action is exerted via the immunoregulatory interleukin-12 (IL-12), which orchestrates the production of interferon gamma (IFN-γ) from various immune cells. Cellular immune response and IFN-γ production is the hallmark for successful vaccine candidates against both T. gondii and N. caninum. However, the discovery of potential vaccine candidates is a highly laborious, time-consuming and expensive procedure. In this review, we will try to exploit previous knowledge and our research experience to establish an efficient immunological approach for exploring potential vaccine candidates against T. gondii and N. caninum. Our previous studies on vaccine development against both T. gondii and N. caninum revealed a strong association between the successful and potential vaccine antigens and their ability to promote the macrophage secretion of IL-12 using a murine model. This phenomenon was emphasized using different recombinant antigens, parasites, and experimental approaches. Upon these data and research trials, IL-12 production from murine macrophages can be used as an initial predictor for judgment of vaccine efficacy before further evaluation in time-consuming and laborious in vivo experiments. However, more studies and research are required to conceptualize this immunological approach. [ABSTRACT FROM AUTHOR]

Sarcocystis spp., Neospora caninum and Toxoplasma gondii are globally ubiquitous pathogens, and domestic sheep are considered to be one of the intermediate hosts. 83 myocardial samples of sheep were collected from 12 retail stores in Beijing, China. Sarcocystis spp., N. caninum and T. gondii were identified by PCR amplification of the 18S rRNA gene, Nc-5 gene and 529bp DNA fragment with a prevalence of 86.7% (95% CI: 77.5–93.2) and 43.4% (95% CI: 32.5–54.7) for Sarcocystis spp. and N. caninum infections, respectively, and no T. gondii was detected. The co-infection prevalence of Sarcocystis and N. caninum was 38.6% (95% CI: 28.1–49.9). Two Sarcocystis species were subtyped by analyzing 18SrRNA sequences and were identified as Sarcocystis tenella and Sarcocystis arieticanis. The prevalence of S. tenella and S. arieticanis infections was 84.3% (95% CI: 74.7–91.4) and 56.6% (95% CI: 45.3–67.5), respectively. This study shows that sheep have a high risk of infection with Sarcocystis and N. caninum, suggests that effective prevention measures are needed to avoid the spread of these parasites in sheep. Toxoplasmosis in sheep poses a threat to human and animal health and requires monitoring and preventing continuously.

Rock pigeons are synanthropic birds and live in close association within human settlements. Synanthropic birds are considered potential carriers of diseases and pose a subsequent risk of their transmission to humans. Neospora caninum and Toxoplasma gondii are intracellular protozoans infecting a wide range of hosts, as well as birds. Data related to the incidence of these protozoans in rock pigeons in Pakistan are scant. The aims of the study were to detect T. gondii and N. caninum DNA in tissue samples from rock pigeons (Columba livia) and to identify possible risk factors associated with infection. To accomplish this, pectoral muscle and brain samples were carefully collected from rock pigeons (n = 120) belonging to three zones located in ten districts of Punjab during a 4-month sampling span (July 2018 to October 2018). Data related to sex, age, sampling site, districts, seasonality, and ecological zones were recorded. DNA from brain and pectoral muscle samples was screened for both T. gondii and N. caninum by PCR assays. Chi-square analysis was used to check the association between positive samples and risk factors. The level of significance was p ≤ 0.05. T. gondii was detected in 46 (38.3%) brain samples, while 24 pectoral muscle samples (20%) were positive for N. caninum. Agroecological zones were statistically associated with the detection of N. caninum DNA. The outcomes of this study provide an understanding of the epidemiological pattern of N. caninum and T. gondii infection in rock pigeons in different regions of Punjab, Pakistan. [ABSTRACT FROM AUTHOR]

Neospora caninum is an intracellular protozoan that mainly infects cattle to cause abortion and significant economic losses worldwide. A better understanding of the immune evasion mechanisms of N. caninum could help to search for an effective approach to prevent and treat neosporosis. Mitophagy is used by some viruses to evade host immune surveillance. However, host cell mitophagy and its effect on N. caninum infection is unclear. In the present study, N. caninum -induced host cell mitophagy and its role in parasite infection were investigated in vitro and in vivo. Furthermore, the regulation of N. caninum -induced host cell mitophagy on the production of Reactive Oxygen Species (ROS), the secretions of proinflammatory cytokines, and the signals of p38, ERK, and Nlrp3 inflammasome were explored. Our results showed that autophagosomes and co-localization of LC3 with mitochondria were observed in N. caninum- infected macrophages. The mtDNA/nDNA ratio and the levels of mitochondrial marker proteins (Hsp60 and Tim23) were decreased with the increase of N. caninum numbers or infection time. N. caninum could induce mitophagy in brain and peritoneal lavage fluid cells of mice. Promoting mitophagy via mitophagy inducers (CCCP) could shorten survival time, decrease body weight, increase parasite load, and attenuate secretion of cytokines in N. caninum infected mice. CCCP treatment decreased the production of cytokines and Reactive Oxygen Species (ROS), and increased parasite burden in N. caninum -infected macrophages. Furthermore, CCCP or NAC (ROS inhibitor) treatment could inhibit ERK signal, Nlrp3 inflammasome, and cytokine production, while promote p38 signal in N. caninum -infected macrophages. The opposite results were obtained when using a mitophagy inhibitor (Mdivi1). Taken together, N. caninum -induced mitophagy could regulate the activations of p38, ERK, Nlrp3 inflammasome to inhibit the production of inflammatory cytokines in a ROS-dependent manner to escape host immune surveillance. [ABSTRACT FROM AUTHOR]

Neospora caninum is an intracellular protozoan that mainly infects cattle to cause abortion and significant economic losses worldwide. A better understanding of the immune evasion mechanisms of N. caninum could help to search for an effective approach to prevent and treat neosporosis. Mitophagy is used by some viruses to evade host immune surveillance. However, host cell mitophagy and its effect on N. caninum infection is unclear. In the present study, N. caninum-induced host cell mitophagy and its role in parasite infection were investigated in vitro and in vivo. Furthermore, the regulation of N. caninum-induced host cell mitophagy on the production of Reactive Oxygen Species (ROS), the secretions of proinflammatory cytokines, and the signals of p38, ERK, and Nlrp3 inflammasome were explored. Our results showed that autophagosomes and co-localization of LC3 with mitochondria were observed in N. caninum-infected macrophages. The mtDNA/nDNA ratio and the levels of mitochondrial marker proteins (Hsp60 and Tim23) were decreased with the increase of N. caninum numbers or infection time. N. caninum could induce mitophagy in brain and peritoneal lavage fluid cells of mice. Promoting mitophagy via mitophagy inducers (CCCP) could shorten survival time, decrease body weight, increase parasite load, and attenuate secretion of cytokines in N. caninum infected mice. CCCP treatment decreased the production of cytokines and Reactive Oxygen Species (ROS), and increased parasite burden in N. caninum-infected macrophages. Furthermore, CCCP or NAC (ROS inhibitor) treatment could inhibit ERK signal, Nlrp3 inflammasome, and cytokine production, while promote p38 signal in N. caninum-infected macrophages. The opposite results were obtained when using a mitophagy inhibitor (Mdivi1). Taken together, N. caninum-induced mitophagy could regulate the activations of p38, ERK, Nlrp3 inflammasome to inhibit the production of inflammatory cytokines in a ROS-dependent manner to escape host immune surveillance.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

Toxoplasmosis and neosporosis are major protozoan diseases of global distribution. Toxoplasma gondii is the cause of toxoplasmosis, which affects almost all warm-blooded animals, including humans, while Neospora caninum induces neosporosis in many animal species, especially cattle. The current defective situation with control measures is hindering all efforts to overcome the health hazards and economic losses of toxoplasmosis and neosporosis. Adequate understanding of host-parasite interactions and host strategies to combat such infections can be exploited in establishing potent control measures, including vaccine development. Macrophages are the first defense line of innate immunity, which is responsible for the successful elimination of T.gondii or N. caninum. This action is exerted via the immunoregulatory interleukin-12 (IL-12), which orchestrates the production of interferon gamma (IFN-γ) from various immune cells. Cellular immune response and IFN-γ production is the hallmark for successful vaccine candidates against both T. gondii and N. caninum. However, the discovery of potential vaccine candidates is a highly laborious, time-consuming and expensive procedure. In this review, we will try to exploit previous knowledge and our research experience to establish an efficient immunological approach for exploring potential vaccine candidates against T. gondii and N. caninum. Our previous studies on vaccine development against both T. gondii and N. caninum revealed a strong association between the successful and potential vaccine antigens and their ability to promote the macrophage secretion of IL-12 using a murine model. This phenomenon was emphasized using different recombinant antigens, parasites, and experimental approaches. Upon these data and research trials, IL-12 production from murine macrophages can be used as an initial predictor for judgment of vaccine efficacy before further evaluation in time-consuming and laborious in vivo experiments. However, more studies and research are required to conceptualize this immunological approach.

Fig. 9. The role of TLR2 and TLR3 in controlling N. caninum infection. When N. caninum invaded, TLR2 and TLR3 signals were activated to induce the phosphorylation of AKT signal, and in turn regulated the phosphorylation of ERK signal, which induced the secretion of IL-12p40 to achieve the purpose of resistance to N. caninum infection. Besides, IL-12p40 exerted an inhibitory effect on N. caninum by promoting IFN-γ secretion to induce NO and GBP2 production during N. caninum infection. We speculated that IL-12p40 could induce the activation of NK and CTL cells and the differentiation of Th1-type lymphocytes via IFN-γ to eliminate N. caninum. [Display omitted] • TLR2/TLR3-AKT-ERK signal axis regulated N. caninum induced IL-12p40 production in mouse macrophages. • IL-12p40 played a crucial role in anti- N. caninum via inducing IFN-γ, NO, and GBP2 production in mouse macrophages. • LTLR2 was more predominant than TLR3 in regulating IL-12p40 production in mice during N. caninum infection. TLR2/3-IL-12p40 axis controlled the N. caninum proliferation in bovine macrophages. Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum -induced IL-12p40 production and the role of TLR2/TLR3 in anti- N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2−/− mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3−/− mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2−/− and TLR3−/− mouse macrophages. In N. caninum- infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2−/− and TLR3−/− mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2−/−, and TLR3−/− mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum , and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production. [ABSTRACT FROM AUTHOR]

Abstract Between December 2016 and April 2017, a spate of abortions occurred in a closed dairy herd from the central eastern region of Paraná, Brazil, in which 75 cows aborted. To identify its cause, organ fragments were collected from an aborted fetus for histopathology, and the blood samples from a stillborn, 4 aborted fetuses, and 9 farm dogs for indirect fluorescent antibody technique (IFAT). These tests found multifocal non-suppurative encephalitis, periportal hepatitis, and multifocal lymphoplasmacytic myocarditis, and detected anti-Neospora antibodies in all aborted fetuses, and in 5 of the 9 dogs. DNA of Neospora caninum was detected in the brain tissue of an aborted fetus. Blood samples of 340 cows and 146 heifers showed 33.5% and 30.8% seropositivity, respectively. In this closed herd, the parasite was probably introduced by infected domesticated or wild carnivores inhabiting the farm, through the infective oocysts present in their stool.

Vaccination is the only feasible way for appropriate prevention of Neospora caninum infection. The present in silico study was done to evaluate the physico-chemical properties and determine immunogenic epitopes of N. caninum SAG1 protein as a possible vaccine candidate. Web-based tools were used to predict physico-chemical properties, antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, transmembrane domains and signal peptide, secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind mouse major histocompatibility complex (MHC) and cytotoxic T lymphocyte (CTL). The protein was stable in a test tube, had 319 residues with a molecular weight of 33.07 kDa, representing aliphatic index of 76.68 (thermotolerant) and GRAVY score of 0.031 (hydrophobic). There were 42 PTM sites and an N-terminally-located signal peptide in the sequence. Secondary structure comprised mostly by random coils, followed by strands and helices. Ramachandran plot of the refined model showed 71.7%, 24.9%, 3.0% and 0.4% residues in the favored, additional allowed, generously allowed and disallowed regions, correspondingly. Additionally, various potential B-cell (linear and conformational), CTL and MHC-binding epitopes were predicted for N. caninum SAG1. The findings of the present in silico study are a premise for vaccination strategies against neosporosis.

Neospora caninum poses as a considerable threat to animal health and generates significant economic impact in livestock production worldwide. Here, we have investigated the mechanism that underlies the participation of the inflammasome complex and Reactive Oxygen Species (ROS) in the regulation of immune responses during N. caninum infection. For that purpose, we used in vitro (bone marrow derived macrophages) and in vivo mouse models of infection. Our results show that NLRP3 and NLRC4 receptors, alongside with ASC and Caspase-1, are required for proper activation of the inflammasome during N. caninum infection. As expected, the engagement of these pathways is crucial for IL-1α, IL-1β, and IL-18 production, as well as the induction of pyroptosis. Our results also show that N. caninum induces ROS production dependent of the inflammasome assembly, which in its turn also depends on MyD88/NF-κB-induced ROS to maintain its activation and, ultimately, lead to restriction of parasite replication.

Abstract Background Dairy production in Bosnia and Herzegovina exhibits limited productivity, which may partly, be explained by extensive reproductive problems of non-infectious and infectious origin. Brucella spp., Chlamydia abortus, Coxiella burnetii and Neospora caninum are common infectious causes of decreased reproductive outcomes in cattle worldwide. Little is, however, known about the disease status of herds with reduced reproductive performances. A cross-sectional study was designed to document the status of these pathogens in dairy cattle in Bosnia and Herzegovina. A total of 1970 serum samples were collected from cattle in farms located in three cantons (regions). Enzyme linked immunosorbent assays were used to screen for seropositivity against four selected pathogens. Results The overall seroprevalence was estimated at both the herd level and at individual level for each pathogen. At the individual animal level, the prevalence for C. abortus, C. burnetii, N. caninum and Brucella spp. was 52.1% (95% CI: 41.2–62.7), 8.8% (95% CI: 5.3–14.2), 9.2% (95% CI: 6.0–12.3 and 0.2% (95% CI: 0.1–0.5), respectively. The corresponding estimates for herd level were 87.9% (95% CI: 82.6–91.8), 19.6% (95% CI: 14.6–25.8), 35.2% (95% CI: 28.8–42.1), and 1.5% (95% CI: 0.5–4.6). A substantial overlap was observed in the presence of N. caninum, C. abortus and C. burnetii at individual and herd level. Conclusion Our study demonstrated a high level of antibodies to Chlamydia abortus. Considering the association of this agent with reproductive disorders in cattle, future studies should be directed to the epidemiological traits of this infection. Additionally, the relatively high levels of exposure to C. burnetii and N. caninum found in this study highlights the need for targeted control of infectious causes of reproductive disorders in dairy cattle of the studied areas. Given the low seroprevalence, Brucella spp. does not seem to represent a problem in the reproductive health of cattle in the studied areas.

Neospora caninum poses as a considerable threat to animal health and generates significant economic impact in livestock production worldwide. Here, we have investigated the mechanism that underlies the participation of the inflammasome complex and Reactive Oxygen Species (ROS) in the regulation of immune responses during N. caninum infection. For that purpose, we used in vitro (bone marrow derived macrophages) and in vivo mouse models of infection. Our results show that NLRP3 and NLRC4 receptors, alongside with ASC and Caspase-1, are required for proper activation of the inflammasome during N. caninum infection. As expected, the engagement of these pathways is crucial for IL-1α, IL-1β, and IL-18 production, as well as the induction of pyroptosis. Our results also show that N. caninum induces ROS production dependent of the inflammasome assembly, which in its turn also depends on MyD88/NF-κB-induced ROS to maintain its activation and, ultimately, lead to restriction of parasite replication. [ABSTRACT FROM AUTHOR]

Neosporosis is a major cause of abortions in cattle worldwide. Recently a live attenuated vaccine showing promising results in preventing abortions, when administered at mid-pregnancy to seropositive cows, was developed. In this study, vaccination with 2 × 108 live frozen N. caninum tachyzoites (NcIs491) was used to immunize naturally infected seropositive pregnant dairy dams. The study was performed under field conditions in four herds, and a follow-up of three subsequent pregnancies was analyzed. A total of 1136 cows were serologically examined. Total seroprevalence was 41.4%, with 25.1% of the cows having titers of 1:800 or higher. Abortion rates were significantly higher in cows with high antibody titers (≥1:800) for two consecutive pregnancies. Vaccination was administered to 114 out of 285 cows with antibody titers higher than 1:800. Immunization resulted in lower abortion rates at three of the farms. Vaccine efficacy ranged from −19.8% to 75% at different farms, with overall efficacy of 28.4% in all four farms and overall efficacy of 58.2% in the three farms with positive results. Our results showed different vaccine efficacy in studied farms, suggesting that frozen live vaccination may generally be an effective method to control neosporosis in cattle.

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.

Coxiella burnetii and Neospora caninum are frequent infectious causes of decreased reproductive outcomes in cattle around the world. So our study aimed to assess the performance of IgG-ELISA for diagnosis of N. caninum and C. burnetii and compare the sensitivity of ELISA and IFA in detection IgG to C.burnetii phase II, to achieve these, a total of 200 samples including milk and serum samples (100 for each) were collected from 100 dairy cattle suffering from infertility and abortion during the period from January to December 2017 in four governorates of upper Egypt, these samples were transferred to laboratory for serological examination. The results revealed that N.caninum antibodies were detected in 29% of serum samples and 10% of milk samples while C.burnetii antibodies were detected in 34% and 17% of serum and milk samples respectively. The IgGELISA of both N.caninum and C.burnetii were detected in the same serum and milk samples with percentage 14% and 4% respectively, amid dairy cattle in 4 governorates of upper Egypt. IgM-IFA indicated the acute infection of C.burnetii in 11% and 4% of serum and milk samples while chronic infection (IgG-IFA) was detected in 19% of serum samples and 8% of milk samples. The comparison between sensitivity of ELISA and IFA in detection of IGg for C.burnetii phase II showed that ELISA was more sensitive and excellent diagnostic method for C.burnetii infection. [ABSTRACT FROM AUTHOR]

The Apicomplexan parasite Neospora caninum, an obligate intracellular protozoan, causes serious diseases in a number of mammalian species, especially in cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle worldwide which have a major economic impact on the cattle industry. However, the mechanism by which N. caninum proliferates within host cells is poorly understood. Epidermal growth factor receptor (EGFR) is a protein kinase ubiquitously expressed, present on cell surfaces in numerous species, which has been confirmed to be essential in signal transduction involved in cell growth, proliferation, survival, and many other intracellular processes. However, the presence of EGFR in N. caninum and its role in N. caninum proliferation remain unclear. In the present study, we identified a putative EGFR-like kinase in N. caninum, which could be activated in tachyzoites by infection or treatment with rNcMIC3 [containing four epidermal growth factor (EGF) domains] or human EGF. Blockade of EGFR-like in tachyzoites by AG1478 significantly reduced parasite proliferation in host cells. Our data suggested that the activation of tachyzoite EGFR-like might facilitate the intracellular proliferation of N. caninum.

Neospora caninum, an obligate intracellular protozoan parasite, can infect a large variety of vertebrate hosts including the most economically important cattle. Infection with N. caninum is a main cause of abortion in both dairy and beef cattle, which causes great economic losses worldwide. However, the mechanism of host cell infection by N. caninum has not been fully elucidated, especially in terms of inflammatory responses. In this study, the effect of TLR-ERK signaling pathway on the synthesis of pro-inflammatory interleukin-12p40 in mouse peritoneal macrophages (PMϕ) challenged by N. caninum was investigated. Our results suggested that N. caninum infection quickly activated MEK-ERK signaling via TLR11 in PMϕ. In addition, N. caninum infection also caused upregulated production of IL-12p40 by PMϕ, which was significantly reduced with the blockade of TLR11/MEK/ERK pathway, suggesting that this upregulation of IL-12 p40 was TLR11 and MEK-ERK-activation dependent.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

In order to estimate the seroprevalence and to assess risk factors of Toxoplasma gondii and Neospora caninum in the province of Nyanga, in southern Gabon, a cross-sectional study was conducted in sheep and goats in the county of Mongo. Serological screening was performed using an indirect multi-species enzyme-linked immunosorbent assay and a commercial direct agglutination test to test serum samples for the presence of anti- N . caninum and anti- T. gondii Immunoglobulines (Ig) G antibodies, respectively. From a total of 201 small ruminants, including 95 sheep and 106 goats, the overall anti- N. caninum and anti- T. gondii IgG seroprevalences were 31.3% (n = 63) and 45.8% (n = 95), respectively. Statistical analyses showed that adult small ruminants were 4 times more likely to be infected with T. gondii than young animals (p < 0.001; OR = 4.27; 95% CI: 2.07–8.81). The locality was significantly associated with T. gondii seropositivity (p = 0.001). The Dilemba (p < 0.001; OR = 0.07), Moulengui-Binza (p = 0.023; OR = 0.05) and Rina-Nzala localities (p = 0.005; OR = 0.1) were not identified as risk localities associated with T. gondii infection. The seroprevalence of N. caninum was significantly higher in sheep (42.1%) than goats (21.7%). The species was not associated with N. caninum infection (p = 0.005; OR = 0.27; 95% CI: 0.11–0.68). In Bibora, small ruminants were almost 4 times more likely to get Neospora infection than the animals in the other localities (p = 0.04; OR = 3.98; 95% CI: 1.06–14.93). The serological prevalence of N. caninum and T. gondii could suggest a considerable impact on the reproductive process from these pathogens in sheep and goats within the Mongo County and a high exposure of humans to T. gondii . [ABSTRACT FROM AUTHOR]

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis. [ABSTRACT FROM AUTHOR]

Neosporosis, caused by the intracellular protozoan Neospora caninum , is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum -seropositive pregnant dams were inoculated with 10 8 live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively ( P = 0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows. [ABSTRACT FROM AUTHOR]

To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 10 7 N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20 mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninum -related brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril. [ABSTRACT FROM AUTHOR]

Recent reports indicate N. caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the mode of transmission of neosporosis in sheep in New Zealand is limited. This study aimed to determine the rate of vertical transmission that would occur in lambs born from experimentally inoculated ewes and to determine if previous inoculation would protect the lambs from N. caninum infection. A group of 50 ewes was divided into 2 groups with one group being inoculated with 5 × 10 6 N. caninum tachyzoites prior to pregnancy in Year 1. In Year 2, each of these groups was subdivided into 2 groups with one from each original group being inoculated with 1 × 10 7 N. caninum tachyzoites on Day 120 of gestation. Inoculation of N. caninum tachyzoites into ewes prior to mating resulted in no congenital transmission in lambs born in Year 1 but without further inoculation, 7 out of 11 lambs in Year 2 were positive for N. caninum infection. Ewes that were inoculated in both years resulted in all 12 lambs born in Year 2 being positive for N. caninum infection. This indicates that previous inoculation in Year 1 did not result in any vertical transmission in that year but did not provide any protection against vertical transmission in Year 2. These results suggest that vertical transmission occurs readily once the ewe is infected. [ABSTRACT FROM AUTHOR]

Sarcocystis spp., Neospora caninum and Toxoplasma gondii are globally ubiquitous pathogens, and domestic sheep are considered to be one of the intermediate hosts. 83 myocardial samples of sheep were collected from 12 retail stores in Beijing, China. Sarcocystis spp., N. caninum and T. gondii were identified by PCR amplification of the 18S rRNA gene, Nc-5 gene and 529bp DNA fragment with a prevalence of 86.7% (95% CI: 77.5-93.2) and 43.4% (95% CI: 32.5-54.7) for Sarcocystis spp. and N. caninum infections, respectively, and no T. gondii was detected. The co-infection prevalence of Sarcocystis and N. caninum was 38.6% (95% CI: 28.1-49.9). Two Sarcocystis species were subtyped by analyzing 18SrRNA sequences and were identified as Sarcocystis tenella and Sarcocystis arieticanis . The prevalence of S. tenella and S. arieticanis infections was 84.3% (95% CI: 74.7-91.4) and 56.6% (95% CI: 45.3-67.5), respectively. This study shows that sheep have a high risk of infection with Sarcocystis and N. caninum , suggests that effective prevention measures are needed to avoid the spread of these parasites in sheep. Toxoplasmosis in sheep poses a threat to human and animal health and requires monitoring and preventing continuously., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Chen, Yang, Wang, Liu and Liu.)

N. caninum is a protozoan parasite of the phylum Apicomplexa, and has been recognized as a major cause of abortion and reproductive failure in cattle in the world, Although in Colombia there are some studies on the seroprevalence of this disease, even so the information remains limited. Objective. The aim of this study was to establish the seroprevalence of N. caninum in dairy cows of Tuta, (Boyacá, Colombia). Materials and methods. 375 animals were sampled. The samples were processed under indirect ELISA technique, a statistical analysis was performed with the Chi-square association-independence test to determine if there was a relationship between the presence of antibodies against N. caninum and the different reproductive variables. Results. It was observed that 52% of the individuals were positive for antibodies against N. caninum. the only reproductive variable that presented a statistical relationship with the presence of the protozoan was repetition of estrus, there was no relationship between age and breed of the cattle and the presentation of N.caninum. Conclusions. The seroprevalence is high if they if you keep in mind the seroprevalence data previously reported in other regions of the country, however, these results do not demonstrate the presence of disease in the animals analyzed, but the antigenic evidence, which implies that at some point life they became infected with the agent and promoted the formation of specific antibodies. [ABSTRACT FROM AUTHOR]

Neosporosis is a major cause of abortions in cattle worldwide. Recently a live attenuated vaccine showing promising results in preventing abortions, when administered at mid-pregnancy to seropositive cows, was developed. In this study, vaccination with 2 × 108 live frozen N. caninum tachyzoites (NcIs491) was used to immunize naturally infected seropositive pregnant dairy dams. The study was performed under field conditions in four herds, and a follow-up of three subsequent pregnancies was analyzed. A total of 1136 cows were serologically examined. Total seroprevalence was 41.4%, with 25.1% of the cows having titers of 1:800 or higher. Abortion rates were significantly higher in cows with high antibody titers (≥1:800) for two consecutive pregnancies. Vaccination was administered to 114 out of 285 cows with antibody titers higher than 1:800. Immunization resulted in lower abortion rates at three of the farms. Vaccine efficacy ranged from −19.8% to 75% at different farms, with overall efficacy of 28.4% in all four farms and overall efficacy of 58.2% in the three farms with positive results. Our results showed different vaccine efficacy in studied farms, suggesting that frozen live vaccination may generally be an effective method to control neosporosis in cattle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• ciELISA tSAG1 can differentiate N. caninum -infected and uninfected sheep and goats. • ciELISA tSAG1 was highly sensitive and specific to detect anti- N. caninum antibodies. • Cross-reactions were not observed between tSAG1 and anti- Toxoplasma gondii antibodies. • At field evaluation the concordance between ciELISA tSAG1 and IFAT was very good. • The production of tSAG1 recombinant protein is simple and standardized. Neospora caninum is a protozoan parasite that causes abortion and reproductive failure in small ruminants. We validated and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1) from N. caninum for the detection of anti- N. caninum antibodies in sheep and goat flocks. The assay was validated using 80 positive and 142 negative serum samples from sheep and goats analyzed by IFAT and immunoblot (IB). ciELISA tSAG1 was then used to evaluate the prevalence of anti- N. caninum antibodies in 1449 goats from 143 flocks and 385 sheep from 40 flocks and compared to IFAT. The prevalence of anti- Toxoplasma gondii antibodies was evaluated by IFAT. The ciELISA tSAG1 cut-off was ≥ 36 percent inhibition, with a diagnostic sensitivity of 100.0 % (95 % CI = 95.4–100.0 %) and a diagnostic specificity of 98.6 % (95 % CI = 95.0–99.8 %) relative to the agreement between IFAT and IB. The field evaluation revealed a concordance between ciELISA tSAG1 and IFAT of 97.4 %, with an agreement (κ) of 0.90 for sheep sera, and a concordance of 96.5 % with κ = 0.85 for goat sera. The overall prevalence of anti- N. caninum antibodies in sheep was 14.3 % by IFAT and 15.8 % by ciELISA tSAG1. In goats, prevalence was 12.9 % by IFAT and 14.6 % by ciELISA tSAG1. The overall prevalence of anti- T. gondii antibodies was 28.8 % in goats and 43.8 % in sheep. The ciELISA tSAG1 could be useful for large-scale detection of anti- N. caninum antibodies in sheep and goats, and for seroepidemiological investigations due to its appropriate sensitivity and specificity, and the simplicity of production. [ABSTRACT FROM AUTHOR]