Working towards the development of vaccines and chemotherapeutics against neosporosis-With all of its ups and downs-Looking ahead.

Neospora caninum is an apicomplexan and obligatory intracellular parasite, which is the leading cause of reproductive failure in cattle and affects other farm and domestic animals, but also induces neuromuscular disease in dogs of all ages. In cattle, neosporosis is an important health problem, and has a considerable economic impact. To date there is no protective vaccine or chemotherapeutic treatment on the market. Immuno-prophylaxis has long been considered as the best control measure. Proteins involved in host cell interaction and invasion, as well as antigens mediating inflammatory responses have been the most frequently assessed vaccine targets. However, despite considerable efforts no effective vaccine has been introduced to the market to date. The development of effective compounds to limit the effects of vertical transmission of N. caninum tachyzoites has emerged as an alternative or addition to vaccination, provided suitable targets and safe and efficacious drugs can be identified. Additionally, the combination of both treatment strategies might be interesting to further increase protectivity against N. caninum infections and to decrease the duration of treatment and the risk of potential drug resistance. Well-established and standardized animal infection models are key factors for the evaluation of promising vaccine and compound candidates. The vast majority of experimental animal experiments concerning neosporosis have been performed in mice, although in recent years the numbers of experimental studies in cattle and sheep have increased. In this review, we discuss the recent findings concerning the progress in drug and vaccine development against N. caninum infections in mice and ruminants.

The seasonality as a relevant aspect to be considered for differential diagnosis of Trypanosoma vivax infection and co-infections in female cattle.

Bovine Trypanosomiasis and other infectious diseases cause relevant loss for the livestock industry impacting productive/reproductive indices. This study intended to better understand the frequency, seasonality, and profile of infections associated with Bovine Trypanosomiasis. A total of 1443 serum samples were screened for T. vivax infection and other infectious diseases: Neosporosis, Leptospirosis, Bovine Leukosis Virus infection/(BLV), Infectious Bovine Rhinotracheitis/(IBR) or Bovine Viral Diarrhea/(BVD). Distinct methods were used for screening and diagnosis: immunofluorescence assay (Trypanosomiasis), ELISA (Neosporosis,BLV,IBR,BVD) and microscopic agglutination test (Leptospirosis). Our findings demonstrated that the seropositivity for Trypanosomiasis=57% was similar to Neosporosis=55%, higher than Leptospirosis=39% and BVL=34%, but lower than IBR=88% and BVD=71%. The seropositivity for Trypanosomiasis was higher in the autumn and lower in the winter. Regardless the season, the IBR seropositivity (min=73%;max=95%) was higher than Trypanosomiasis (min=48%;max=68%). Moreover, Neosporosis (min=71%;max=100%) and BVD (min=65%;max=76%) were more frequent than Trypanosomiasis in the summer, winter and spring. The diagnosis outcome revealed that Trypanosomiasis&IBR=43% and Trypanosomiasis&Neosporosis=35% were the most frequent co-infections with higher seropositivity in the autumn (58%) and summer (80%), respectively. Noteworthy, high seropositivity to Trypanosomiasis&BVD was registered in the autumn (46%). Together, our data re-enforce the relevance of differential diagnosis between Trypanosomiasis with other bovine infectious diseases and that differences in the seasonality profile is a relevant aspect to be considered while selecting the differential diagnosis to be applied.

Seroprevalence and molecular detection of Toxoplasma gondii and Neospora caninum in beef cattle and goats in Hunan province, China.

BACKGROUND: Toxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China. METHODS: Sera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti-N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP). RESULTS: Specific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats (P < 0.05), and age > 3 years was a significant risk factor for N. caninum infection in beef cattle (P < 0.05). PCR positivity for T. gondii was observed in three beef samples (1.9%; 3/160) and seven chevon samples (4.4%; 7/160). Genotyping of PCR positive samples identified one to be ToxoDB#10. The N. caninum DNA was observed in one beef sample (0.6%; 1/160) but was negative in all chevon samples. CONCLUSIONS: To our knowledge, this is the first large-scale serological and molecular investigation of T. gondii and N. caninum and assessment of related risk factors in beef cattle and goats in Hunan Province, China. The findings provide baseline data for executing prevention and control of these two important parasites in beef cattle and goats in China.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

Neospora caninum antibodies in bulk tank milk from dairy cattle herds in Italy in relation to reproductive and productive parameters and spatial analysis.

Among the available diagnostic techniques, antibody detection in bulk tank milk (BTM) represents a useful tool to estimate and monitor Neospora caninum herd prevalence. To evaluate the prevalence of N. caninum and the effect of parasite infection on herd performances, BTM samples collected from 586 dairy herds located in one of the largest dairy production areas in Italy (Lombardy) were analyzed by an indirect ELISA to detect anti-N. caninum specific antibodies. Generalized linear models (GLMs) were developed. A purely spatial analysis scanning for clusters with high or low rates for N. caninum using the Bernoulli model was performed. A maximum entropy approach was used to estimate the probability of distribution of the parasite based on occurrence records together with environmental variables. Overall, 180 herds resulted positive for N. caninum antibodies on bulk tank milk (P = 30.7 %). A higher risk of seropositivity was evidenced in the provinces of Milano, Cremona, Brescia, and Bergamo (P = 32-40 %); a lower risk was evidenced in Lodi, Pavia, and Mantova (P = 13-24 %). A higher risk of seropositivity was revealed for small-medium farms (101-300 animals) (O.R.=2.8) and for older animals with more than 4 years (O.R.=4.4). Regarding the effect of N. caninum infection on herd performances, the number of inseminations for conception was higher (> 3 inseminations), and the period from calving to conception was longer (> 150 days) for positive farms (O.R.=2.0 and O.R.=2.3, respectively); besides, lower head daily milk production (<20 kg and 21-25 kg) and mature equivalent milk yield (<11,000), and somatic cell counts higher than 300,000 cells/ml were observed for N. caninum positive herds (O.R.=0.4, O.R.=0.4 and O.R.=1.9 respectively). The geographical distribution of N. caninum positive farms with the highest level of probability covers the central sector of the Po Plain where a significant cluster for high risk of parasite infection was shown by spatial scan statistic and Maximum entropy ecological niche modelling. A further significant cluster of low risk occurred in the southern. The climatic and environmental variables with the highest training gain when used in isolation resulted altitude, land use/land cover, and other variables related to temperature and precipitation. Neosporosis is widely distributed in Italian dairy herds and an impact of the parasite on herd performances could be hypothesized. Even if the role of N. caninum in alterations of reproductive and productive parameters should be further explored, veterinarians and farmers should be aware of neosporosis, and control plans should be adopted.

Evaluation of species-specific polyclonal antibodies to detect and differentiate between Neospora caninum and Toxoplasma gondii.

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti-Neospora-rNcSRS2 and anti-Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti-Neospora-rNcSRS2 serum labeled specifically only N. caninum-infected tissue blocks, and the anti-Toxoplasma-rTgSRS2 serum was specific to only T. gondii-infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.

Serological survey of Neospora caninum and Toxoplasma gondii in shelter-housed cats infected with feline immunodeficiency virus, Brazil / Inquérito sorológico para Neospora caninum e Toxoplasma gondii em um abrigo de gatos infectados com o vírus da imunodeficiência felina, Brasil

Felines play a leading role in the epidemiology of Toxoplasma gondii infection, but there is scarce information about the epidemiology of Neospora caninum, particularly in feline immunodeficiency virus (FIV)-infected cats. Cats seropositive to T. gondii do not usually show symptoms unless they are immunosuppressed, such as FIV-infected cats. The same relationship remains poorly known for N. caninum, although it has been associated with neurological disorders in HIV-infected people. Since FIV-infected cats are prone to develop encephalitis of unknown etiology, this study aimed to evaluate the presence of specific antibodies to T. gondii and N. caninum in a shelter for stray cats naturally infected with FIV. A total of 104 serum samples from cats living in a shelter, located in São Paulo city (Brazil), was assessed for T. gondii and N. caninum specific antibody by indirect fluorescent-antibody test (IFAT). Of the 104 cats, 25 (24%) were infected with FIV and, aside from these, 8 (32%) had antibodies against T. gondii (titers from 16 to 128). Only 1 (4%) of the FIV-infected cats had antibodies against N. caninum, which was the first record of coinfection. Among the FIV-naïve cats, 11 (14%) were positive for T. gondii(titers from 16 to 256) and only 1 (1.2%) had antibodies against N. caninum. Serologically positive reactions to T. gondii and N. caninum were not correlated with age or sex (p>0.05), and there was no correlation between FIV and the occurrence of anti-T. gondii or anti-N. caninum antibodies (p>0.05). Further studies encompassing larger cat populations from different origins and locations are essential to clarify the prevalence of T. gondii and N. caninum antibodies in FIV-positive cats.(AU)

Os felinos têm um papel importante na epidemiologia da infecção por Toxoplasma gondii, mas pouco se sabe sobre a epidemiologia da infecção por Neospora caninum em gatos, particularmente em gatos infectados com o vírus da imunodeficiência felina (FIV). Gatos soropositivos para Toxoplasma gondii geralmente não apresentam sintomas a não ser que estejam imunossuprimidos, como gatos infectados com FIV. A mesma relação ainda é pouco conhecida para N. caninum, embora tenha sido associada a distúrbios neurológicos em pessoas infectadas pelo HIV. Considerando que gatos infectados com FIV são propensos a desenvolver encefalite de etiologia desconhecida, o presente estudo teve como objetivo avaliar a presença de anticorpos específicos para T. gondii e N. caninum em gatos infectados com FIV. Um total de 104 amostras de soro de gatos residentes em um abrigo na cidade de São Paulo, Brasil, foram avaliadas para a presença de anticorpos contra T. gondii e N. caninum pelo teste de imunofluorescência indireta (RIFI). Dos 104 gatos, 25 (24%) estavam infectados com FIV e destes 8, (32%) tinham anticorpos contra T. gondii (titulação entre 16 e 128). Apenas 1 (4%) dos gatos infectados com FIV apresentava anticorpos contra N. caninum, sendo este o primeiro registro dessa coinfecção. Entre os gatos não infectados com FIV, 11 (14%) foram positivos para T. gondii (titulação entre 16 e 256) e apenas 1 (1,2%) tinha anticorpos contra N. caninum. A reação sorologicamente positiva para T. gondii e N. caninum não foi correlacionada com a idade ou sexo (p> 0,05), nem houve correlação entre FIV e ocorrência de anticorpos para T. gondii ou N. caninum(p> 0,05). Estudos subsequentes abrangendo populações maiores de gatos de diferentes origens e locais são essenciais para esclarecer a prevalência de anticorpos contra T. gondii e N. caninum em animais acometidos por FIV.(AU)

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

Macrophage Stimulation as a Useful Approach for Immunoscreening of Potential Vaccine Candidates Against Toxoplasma gondii and Neospora caninum Infections.

Toxoplasmosis and neosporosis are protozoan diseases that adversely affect the medical and additionally veterinary sectors, respectively. Toxoplasmosis is caused by Toxoplasma gondii which infects almost all warm-blooded animals including humans. While, neosporosis is caused by Neospora caninum, which induces infection in many animal species particularly in cattle. Currently, control measures for both infections are defective because of no effective vaccine or treatment. Macrophages constitute the first line of innate immunity, which contributes to the effective elimination of T. gondii or N. caninum. This action is mediated by IL-12, which is critical for the secretion of interferon gamma (IFN-γ). Successful vaccine candidates against both protozoan parasites should possess the ability to induce the cellular immune response and IFN-γ production. In this chapter, we will focus on an efficient immunological approach for discovery of potential vaccine candidates against above-mentioned parasites. Our previous studies revealed a strong correlation between vaccine antigens that enhanced the macrophage secretion of IL-12 and their efficacy as potential vaccine candidates in murine model. In case of T. gondii, peroxiredoxin 1 (TgPrx1) and peroxiredoxin 3 stimulated the production of IL-12 from murine peritoneal macrophages and conferred strong to moderate protection in C57BL/6 mice, respectively. At the same context, Neospora antigens of dense granule protein 6 (NcGRA6) and cyclophilin entrapped with oligo-mannose coated-liposomes stimulated macrophage IL-12 secretion and substantially protected immunized BALB/c mice. Therefore, we can deduce that macrophage stimulation evidenced in IL-12 production can be used as a useful approach for judgment of vaccine efficacy before further evaluation using in vivo experiments. Methods of vaccine preparation and macrophage stimulation will be fully described for TgPrx1 and NcGRA6 as potential vaccine candidates against toxoplasmosis and neosporosis, respectively.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice.

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.

Morphometric study of encephalic lesions in aborted bovine fetuses naturally infected by two subpopulations of Neospora caninum.

Neospora caninum is a major reproductive disease in cattle worldwide. In the Argentinian Humid Pampa, the seroprevalence, incidence of abortions, and economic losses due to neosporosis are considerably higher in dairy than in beef cattle. Despite this, we recently demonstrated that N. caninum subpopulations are indistinctly distributed in both dairy and beef production systems. The association between genotypic characteristics defined by microsatellite analysis and the virulence of the different strains-particularly with regard to the severity and extension of histological lesions-is largely unknown. Herein, we used a morphometric approach to analyze encephalic lesions in 62 bovine fetuses spontaneously infected by N. caninum. Morphometric parameters (average size of focal lesions, number of foci/cm2 and the percentage of the section affected by lesions) were compared according to the N. caninum subpopulations found in our previous microsatellite genotyping analysis, animal biotype (beef versus dairy), and fetal age (second stage of gestation versus third stage). The average size of the lesions differed significantly among fetuses with different gestational ages; however, no significant differences among animal biotypes or genotypic patterns were found. Further research into the genetic, molecular, and husbandry factors that could account for this greater impact in Argentinian dairy herds is needed.

A recent update about seroprevalence of ovine neosporosis in Northern Egypt and its associated risk factors.

Neospora caninum (Family: Sarcocystidae) is an obligate intracellular protozoan. It is one of the most critical abortifacients in ruminants. The seroprevalence of antibodies against N. caninum and its risk factors was investigated among 430 sheep from four North Egyptian governorates, Alexandria, Gharbia, Menofia, and Qalyubia, during the period from 2017 to 2018. Generally, the overall prevalence rate of N. caninum among sheep was 8.6%. The logistic regression analysis for the obtained data revealed that N. caninum increased significantly with age (OR = 2.4, 95% CI: 8.4-18.7) of the ewe (OR = 3.3, 95% CI: 7.6-14.9), particularly among sheep in contact with dogs (OR = 4.9, 95% CI: 7.5-14.3). Besides, locality, season, and pregnancy status of examined sheep had no significant effect on the appearance of N. caninum infection. the present findings confirm the presence of N. caninum among sheep in Egypt which probably play a role in reproductive failure in sheep. Therefore, sanitary measures and monitoring of the infection should be implemented to reduce the spreading of the infection.

Seroprevalence of Neospora caninum and Besnoitia besnoiti in Cattle in Oguzlar Region

Objective: This study aimed to investigate the seroprevalence of Neospora caninum and Besnoitia besnoiti in cattle in the Oguzlar district of Çorum province. Methods: Venous blood samples were collected from the vena jugularis of 100 cattle in the Oguzlar region and stored into anticoagulant-free tubes. Serum samples were examined with commercial c-ELISA kits for N. caninum (IDEXX, Switzerland) and B. besnoiti (ID.vet, France). Results: Two of serum samples were found to be N. caninum (2%) and five were B. besnoiti (5%) seropositive. No mixed infection was detected in any of serum samples. Conclusion: In this study, the presence of N. caninum and B. besnoiti was serologically determined in animals that are not imported in the Oguzlar region. This is the first study in the region to identify B. besnoiti in the seropositive cattle and is the third study in Turkey.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine.

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1ß, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis.

Development and validation of a time-resolved fluorescence immunoassay for the detection of anti-Toxoplasma gondii antibodies in goats.

Toxoplasma gondii is a worldwide distributed parasite causing abortions and fetal malformations in small ruminants. The aim of this study was to design and validate a new immunoassay based on the use of TgSAG1-GRA8 chimeric antigen for the detection of anti-T. gondii antibodies in serum of goats. First, a time-resolved fluorescence immunoassay (TgSAG1-GRA8-TRFIA) was developed. In addition, the diagnostic performance of TgSAG1-GRA8-TRFIA was compared with an optimized enzyme-linked immunosorbent assay (TgSALUVET-ELISA) and a Western Blot (WB), both based on whole T. gondii tachyzoite antigenic extract. The TgSAG1-GRA8-TRFIA has shown a high intra- and inter-assay precision, analytical sensitivity and accuracy. The ROC analysis of this assay showed an optimal cut-off of 217.4 Units of Fluorometry for T. gondii (UFT), with 92 % of sensitivity and 90.48 % of specificity. A positive and statistically significant Spearman's correlation with TgSALUVET-ELISA was detected, and kappa value was 0.83, presenting high agreement with both methods. However, TgSAG1-GRA8 protein showed cross-reactivity with specific anti-Neospora caninum antibodies. Thus, TgSAG-1-GRA8 chimeric antigen seems not to be an ideal option for the serodiagnosis of T. gondii infection in goats unless combined with the serodiagnosis of N. caninum infection in parallel. In the light of the results obtained, a comprehensive study on the existence of cross-reactivities between T. gondii antigens used in serological tests employed in animal health and specific antibodies directed against Toxoplasmatinae parasites should be performed.

Neospora caninum and Toxoplasma gondii infections and their relationship with reproductive losses in farmed red deer (Cervus elaphus).

The aims of the present study were to determine the Neospora caninum and Toxoplasma gondii seropositivity rates in farmed red deer hinds from Argentina and their relationship with reproductive losses. Over a 2-year period, 449 hinds from 4 commercial farms were serologically tested at late gestation for N. caninum and T. gondii by IFAT. During the first year, a sequential serological analysis was carried out at 3 different time points to analyze antibody dynamics from mating until the end of the gestation period. Fetal and postnatal mortality rates were estimated by 3 successive ultrasound scannings (us) annually and a breeding control carried out after the calving period. Ultrasound fetal measurements were used to estimate conception date and gestational age of abortions. The seropositivity rate for N. caninum was 25.5% (37/145) for the yearlings and 34.2% (104/304) for the adults, while for T. gondii was 64.3% (93/145) and 78.3% (238/304), respectively. Abortions detected at us1 and us2 were 13/21 (61.9%) with a range of gestational age of 30-87 days, while abortions detected at us3 were 8/21 (38.1%) with a range of gestational age of 49-209 days. The fetal mortality rate was 4% and 5.8%, while the postnatal mortality rate was 18.8% and 4.1% of 101 yearlings and 294 adult pregnant hinds, respectively. Most seropositive hinds to both protozoans showed a stable antibody titer pattern from mating to the end of gestation, and a lower proportion developed an increase in titers suggesting infection recrudescence. Seroconversion during the gestational period was demonstrated in 6 and 50 hinds for N. caninum and T. gondii, respectively. Hinds with fetal mortality were more likely to be seropositive to N. caninum (OR = 3.1) or have N. caninum titers ≥400 (OR = 27.4) than hinds that weaned a fawn. No statistical associations were detected for T. gondii seropositivity and reproductive losses. The pregnancy rate was not affected by N. caninum or T. gondii infection, while the serological evidence of N. caninum causing postnatal mortality was marginal. Based on serological evidence, N. caninum would be a potential abortigenic agent in red deer hinds.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in pigs in the State of Pará, Brazil.

We investigated the occurrence of Toxoplasma gondii and Neospora caninum antibodies in pigs raised in the Northeast of Pará, Brazil. At Study I, convenience sampled 151 pigs at two slaughterhouses, with and without state inspection; and Study II, which assessed 159 pigs with probabilistic sampling from nine pig farms. Serological analysis was performed using indirect fluorescent antibody test for T. gondii and N. caninum with a cutoff of 64 and 50, respectively. Overall, 6.77% pigs were seropositive for T. gondii and 5.16% for N. caninum. In Study I, pigs slaughtered with and without state inspection presented similar occurrence for both coccidia (p>0.05). Study II found an association between N. caninum seropositivity and sludge discarded into the soil, feeding pigs with animal-based protein, subsistence system, and absence of nipple drinkers. No association was found for T. gondii. Pigs from Pará are a potential source of T. gondii infection to humans. To our best knowledge, this is the first study to report anti-N. caninum antibodies in the serum of pigs in Pará State, Brazilian Amazon.

Risk factor analysis associated with Neospora caninum in dairy cattle in Western Brazilian Amazon.

Neospora caninum is considered to be one of the main causes of abortion among cattle. The present survey was conducted in the municipality of Rolim de Moura, Rondônia State, Brazil. A questionnaire that investigates the epidemiological aspects of neosporosis was used in the analysis of risk factors associated with the animal-level and herd-level prevalence in dairy cattle. A total of 416 bovine blood samples were collected from 30 farms, and N. caninum antibody levels were measured by Indirect Fluorescent Antibody Test (IFAT). Analysis of dairy cattle serum samples revealed the presence of anti-N. caninum antibodies to be 47.36% (n = 197). Risk factors associated with N. caninum infection were the management system and access locations of dogs. The results of the present survey indicated that infection of dairy cattle with N. caninum is widespread in the studied region of Western Amazon, which has implications for prevention and control of neosporosis in this region. Therefore, integrated control strategies and measures are recommended to prevent and control N. caninum infection in dairy cattle. In addition, direct contact between dairy cattle, dogs and wild animals, which can influence the epidemiology of neosporosis, should be investigated further.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Experimental infection of tachyzoites of the NC1 strain of Neosporacaninum in female swine.

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 107 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.