The role of genetic variability of the host on the resistance to Neospora caninum infection in cattle.

Neospora caninum is one of the most frequently diagnosed abortifacient pathogens in cattle. There is abundant genomic information about the parasite itself, but very little is known about the genetic variability of resistance in the most common intermediate host. The aim of this review was to compile all the available information about the genetic variability associated with the resistance to N. caninum both between and within cattle breeds. We systematically searched for published studies that investigated the influence of genetics of the host on the prevalence of N. caninum and risk of abortion. Beyond the potential confounding effects of feeding systems, management and animal density, some lines of evidence suggest that Holstein, the most popular breed for milk production, has a comparatively higher risk of abortion due to infections by N. caninum, whereas some beef breeds from Continental Europe seem to be more resistant. It is still not clear if different genetic mechanisms of resistance are involved in the two known routes of infection: postnatal ingestion of oocysts or transplacental transmission from the infected dam to the fetus. Genomic information associated with susceptibility to infection and risk of abortion in different cattle breeds is still scarce. The information reported here could be useful to identify new research alternatives and to define novel strategies to deal with this major problem of animal production.

Epidemic abortions due to Neospora caninum infection in farmed red deer (Cervus elaphus).

This study describes for the first time an abortion outbreak caused by Neospora caninum in farmed red deer. During a 5-year period, farmed hinds, naturally mated, were regularly ultrasound monitored to detect reproductive losses over their gestation. During the 4 years previous to the outbreak, abortion rates ranged from 4.7 to 8.6% (average 6.5%), and serology for indirect diagnosis of neosporosis and toxoplasmosis was performed. At the fifth year, the abortion rate increased to 25.3%. During this outbreak, three aborted foetuses and their placentas were recovered and submitted to laboratory for etiological diagnosis. Blood samples were collected from the 81 hinds at the end of the gestational period and the seropositivity rate for N. caninum, Toxoplasma gondii, Brucella abortus, bovine viral diarrhoea virus and bovine alphaherpesvirus type 1 was 66.7%, 67.9%, 0.0%, 8.6% and 0.0%, respectively. Neospora caninum-seropositive hinds (OR = 5.7, P = 0.0271) and hinds with high antibody titres to N. caninum (OR = 7.4, P = 0.0130) were more likely to abort than seronegative hinds. In addition, N. caninum seropositivity rate in the aborted hinds was higher (OR = 5.4, P = 0.033) than the non-aborted hinds. No association was found between T. gondii nor BVDV-seropositivity and abortions. Typical protozoal histopathologic findings (necrotizing non suppurative encephalitis, meningitis, myocarditis, hepatitis, among others) were observed in all foetuses. Neospora caninum was immunolabelled by immunohistochemistry in several tissues from two foetuses, and infection was also confirmed in the three foetuses by serology and/or DNA detection. No other abortifacient agent was detected in the foetuses. Their dams showed high N. caninum antibody titres (≥ 6400). Serologic evidence and epidemiological data recorded suggested a point-source of N. caninum infection before the occurrence of the outbreak, probably related with contaminated feedstuff with oocysts. Moreover, the intensive production system with a high stocking rate could be also considered a factor which might have increased the risk of horizontal N. caninum infection in this herd.

[A comparative study of commercial ELISAs for antibody detection in the diagnostic investigation of Neospora caninum-associated abortion in dairy cattle herds in Uruguay]. / Comparación de ELISAs comerciales para la detección de anticuerpos en la investigación diagnóstica del aborto asociado a Neospora caninum en rodeos lecheros de Uruguay.

Bovine abortion causes considerable economic losses to the livestock industry worldwide and is of concern for public health and food safety, given that many abortigenic infectious agents of cattle are zoonotic. Despite its importance, the etiological diagnosis of abortion in cattle is challenging both for veterinary practitioners and laboratory technicians, partly due to the difficulty in recovering aborted fetuses under extensive field conditions for pathological and microbiological diagnostic investigation, and in the early identification of aborted dams. Neospora caninum is a cosmopolitan protozoon identified as one of the main abortigenic agents in cattle worldwide. In this study we propose a comparative seroepidemiological approach for the diagnosis of abortion by N. caninum in dairy cattle. Samples from 12 to 93 cows/heifers with and without recent history of abortion (cases and controls) in four commercial dairy farms were tested. The ratio of controls to cases tested varied from 1:1 to 4.6:1. All samples (n=230) were analyzed by three commercial ELISA kits for the detection of anti-N. caninum antibodies. In all four dairy farms, the proportion of seropositive cows and/or heifers per kit was significantly higher in the cases than in the controls (Odds Ratios=5.13 to 36, p=0.0002 to 0.0485). The agreement among the three kits varied from weak to strong (Cohens kappa coefficients=0.58 to 0.83). We conclude that, despite the imperfect agreement between these kits, all of them allowed to arrive at similar conclusions regarding the statistical association between N. caninum seropositivity and abortion, thus representing a useful tool for the diagnostic approach at the population level under field conditions.

Immunization with Neospora caninum profilin induces limited protection and a regulatory T-cell response in mice.

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum.

Immunogenicity, safety and dual DIVA-like character of a recombinant candidate vaccine against neosporosis in cattle.

Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 µg rNcSAG1 + 150 µg rAtHsp81.2; B (n = 4): 200 µg rNcSAG1 + 600 µg rAtHsp81.2; C (n = 4): 500 µg rNcSAG1 + 1,500 µg rAtHsp81.2; D (n = 3): 150 µg rAtHsp81.2; E (n = 3):1,500 µg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Parasitemia and Associated Immune Response in Pregnant and Non-Pregnant Beef Cows Naturally Infected With Neospora caninum.

The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (-Preg, n = 4), and seronegative non-pregnant cows (-Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg (p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio (p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting (p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg (p <0.05). Avidity correlated with total IgG and IgG2 (p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Isolation of Neospora caninum from a beef cattle fetus from Argentina: Immunopathological and molecular studies.

Neospora caninum is an important abortifacient agent affecting mainly cattle worldwide. The aim of the present work was to describe the histopathological findings in a naturally infected beef cow and its midterm fetus caused by a genetically defined N. caninum isolate in Argentina. A N. caninum seropositive multiparous Aberdeen Angus pregnant cow and its fetus in the sixth month of gestation were submitted for histopathological, immunohistochemical, serological, and molecular studies and parasite isolation. The cow belonged to a beef herd under extensive management, with a N. caninum seroprevalence of 11%, and low level of annual abortion rate (≤ 5%). The dam had mild lymphocytic infiltrate in CNS, heart and uterus and no parasites were detected by Immunohistochemistry (IHC). No parasitic DNA was detected in the dam's brain, and gamma interferon knockout mice inoculated with brain material did not become infected. Clusters of tachyzoites and parasitic DNA were detected in the placenta by IHC and PCR, respectively. However, isolation from the placenta was unsuccessful. The fetus developed specific antibodies and an inflammatory response was detected in multiple organs. Furthermore, in vitro and in vivo isolation was achieved from gamma interferon knockout mice inoculated with CNS from the fetus. Multilocus-microsatellite typing revealed a genetically defined N. caninum isolate similar to the previously reported as MLG 72. We report the first N. caninum isolate from beef cattle in Argentina.

Seroprevalence of Neospora caninum infection in cattle from Pereira, Colombia ⋆.

There are over 25.6 million cattle heads in Colombia being the fourth-highest herd in Latin America. This study aimed to describe the seroprevalence of Neospora caninum in cattle from 25 rural farms at Pereira municipality, Risaralda Department, which has a total bovine population of 43,508 animals. A cross-sectional observational study was performed in beef and dairy herds during 2017-2018. A total of 325 animals in 25 herds, with 13 animals per herd, were sampled. A commercial competitive ELISA (cELISA) kit was used to detect N. caninum antibodies. Associated risk factors were analyzed in two different levels: individual animals and herds. For all the independent variables, chi-square (χ2) and Fisher tests were used to assess associations and significance. The overall estimated seropositivity was 20.6% (95%CI 16.2%-25.0%). The seroprevalence by herds was 92.0% ranging from 0.0% to 46.2%. Noteworthy, beef herds had significantly (p = 0.0107) higher seropositivity (50% of them above 35% of seropositivity) compared with those for milk purposes (4.8%) (OR = 20.0; 95%CI 1.2-331.0). Other risk factors were not significantly (p ≥ 0.05) associated with N. caninum seropositivity. Bovine neosporosis may be associated with abortions in both beef and dairy Colombian farms. More studies about the epidemiology, associated factors and consequences, as well as on tools for better diagnosis of bovine abortion, including histopathology and other ancillary tests, should be performed.

Microsatellite pattern analysis of Neospora caninum from a naturally infected goat fetus.

Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within myocardiocytes associated with histopathological lesions. Neospora caninum-DNA was detected by PCR in heart, liver, lungs, kidney, and muscle from the fetus, and was negative for T. gondii by PCR. NC-5 and 18 S rRNA gene fragment sequences showed 100% identity with N. caninum. Inoculated mice bled 30 days post-inoculation resulted seronegative to N. caninum and T. gondii by IFAT, and showed no clinical signs. Multilocus-microsatellite genotyping revealed a genetic profile that differed from previously reported N. caninum genotypes, with unique MS21 and MS10 alleles. These findings indicate that N. caninum was efficiently transmitted from the mother to the fetus. We report the first case of direct detection of N. caninum in a goat fetus in Argentina and N. caninum microsatellite genotyping in naturally infected goat.

Safety and immunogenicity of a soluble native Neospora caninum tachyzoite-extract vaccine formulated with a soy lecithin/ß-glucan adjuvant in pregnant cattle.

The global economic impact of Neospora caninum infection in cattle herds has promoted the development of vaccines that can be safely used during pregnancy. The aim of this study was to evaluate the safety and immunogenicity of a vaccine formulated with the soluble fraction of tachyzoite's lysate and a soy-based aqueous adjuvant (sNcAg/AVEC), which was protective in the mouse model and induced strong IFN-γ responses and high avidity antibodies in non-pregnant cattle. Ten pregnant heifers were vaccinated twice during the first trimester of gestation and 8 remained unvaccinated. Anti-N. caninum immune responses were efficiently primed by vaccination, evidenced by a quick induction of IgM serum titers (7dpv) and a prompt switch to high avidity IgG shortly after infection (performed at 78 or 225 days of gestation; n=5 each); while naïve cattle elicited lower IgG titers, with a delayed kinetics. High systemic IFN-γ levels were induced after infection which did not interfere with pregnancy. No local or systemic adverse effects were recorded along the study. Calves were born in term and in good health conditions, showing that the sNcAg/AVEC vaccine was safe when applied to healthy heifers during the first trimester of gestation.

Comparación de ELISAs comerciales para la detección de anticuerpos en la investigación diagnóstica del aborto asociado a Neospora caninum en rodeos lecheros de Uruguay Comparación de ELISAs comerciales para detección de anticuerpos contra N. caninum / A comparative study of commercial ELISAs for antibody detection in the diagnostic investigation of Neospora caninum-associated abortion in dairy cattle herds in Uruguay

Resumen A pesar de su importancia, el diagnóstico etiológico del aborto en bovinos representa un desafío técnico para los veterinarios clínicos y laboratoristas, en parte debido a la dificultad de recuperar los fetos abortados en condiciones extensivas de campo. Dicha dificultad limita la posibilidad de detectar tempranamente los vientres abortados y de efectuar la investigación patológica y microbiológica de los abortos. Neospora caninum es un protozoario cosmopolita identificado como uno de los principales agentes abortigénicos en bovinos. En este estudio proponemos una aproximación seroepidemiológica para diagnosticar abortos por N. caninum en bovinos lecheros usando kits de ELISA comerciales. Se procesaron muestras de entre 12 y 93 vacas y/o vaquillonas con (casos) y sin (controles) historia reciente de aborto en 4 tambos comerciales de Uruguay. La proporción controles:casos analizados varió entre 1:1 y 4,6:1. Las muestras (n=230) fueron analizadas mediante 3 kits comerciales para la detección de anticuerpos IgG anti-N. caninum. En los 4 tambos la proporción de vacas y/o vaquillonas seropositivas por cualquier kit fue significativamente mayor en los casos respecto de los controles (odds ratio=5,13 a 36; p=0,0002 a 0,0485). La concordancia entre los kits varió de débil a fuerte (coeficiente Kappa de Cohen=0,58 a 0,83). Concluimos que, a pesar de la imperfecta concordancia entre estos kits, el empleo de todos ellos permitió arribar a conclusiones similares respecto de la asociación estadísticamente significativa entre seropositividad a N. caninum y aborto, lo que demuestra la utilidad de estos kits para la aproximación diagnóstica del aborto a nivel poblacional en condiciones de campo.

Abstract Bovine abortion causes considerable economic losses to the livestock industry worldwide and is of concern for public health and food safety, given that many abortigenic infectious agents of cattle are zoonotic. Despite its importance, the etiological diagnosis of abortion in cattle is challenging both for veterinary practitioners and laboratory technicians, partly due to the difficulty in recovering aborted fetuses under extensive field conditions for pathological and microbiological diagnostic investigation, and in the early identification of aborted dams. Neospora caninum is a cosmopolitan protozoon identified as one of the main abortigenic agents in cattle worldwide. In this study we propose a comparative seroepidemiological approach for the diagnosis of abortion by N. caninum in dairy cattle. Samples from 12 to 93 cows/heifers with and without recent history of abortion (cases and controls) in four commercial dairy farms were tested. The ratio of controls to cases tested varied from 1:1 to 4.6:1. All samples (n=230) were analyzed by three commercial ELISA kits for the detection of anti-N. caninum antibodies. In all four dairy farms, the proportion of seropositive cows and/or heifers per kit was significantly higher in the cases than in the controls (Odds Ratios=5.13 to 36, p=0.0002 to 0.0485). The agreement among the three kits varied from weak to strong (Cohen´s kappa coefficients=0.58 to 0.83). We conclude that, despite the imperfect agreement between these kits, all of them allowed to arrive at similar conclusions regarding the statistical association between N. caninum seropositivity and abortion, thus representing a useful tool for the diagnostic approach at the population level under field conditions.

Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina.

The purpose of the present work was to study the epidemiology of Neospora caninum in beef and dairy herds in the Humid Pampas of Argentina. The seroprevalence of N. caninum was evaluated in 2414 serum samples of cows from beef and dairy farms. An indirect fluorescent antibody test (IFAT) was used to determine specific antibodies. The sera was screened at a dilution >or=1:200 and >or=1:600 in cows with reproductive disease antecedents and without them, respectively. Cows without history of reproductive diseases from nine beef and fifteen dairy farms were grouped according to the percentage (> or or

The immune enhancement of a novel soy lecithin/ß-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice.

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 µg of sNcAg, and Providean-AVEC, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4µg of sNcAg + Providean-AVEC developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 µg sNcAg) or with either low or high payload Providean-AVEC formulations (0.4 µg and 4 µg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.

An applied printing immunoassay with recombinant Nc-SAG1 for detection of antibodies to Neospora caninum in cattle.

Neospora caninum is a protozoan parasite that causes an important reproductive disease in cattle. Neospora caninum surface antigen 1 (Nc-SAG1) is an immunodominant candidate for the development of a diagnostic reagent for neosporosis. The current study describes the development and evaluation of an antigen print immunoassay (APIA) with recombinant Nc-SAG1 for the detection of specific antibodies to N. caninum in cattle. The concordance between APIA and a commercial enzyme-linked immunosorbent assay (ELISA) was evaluated with 232 serum samples from experimentally and naturally infected cattle. Sixty-one (26.7%) samples were positive for antibodies to N. caninum by ELISA and 58 (25.4%) by APIA. The new assay had a sensitivity of 85% and a specificity of 96%. These results, along with the potential of APIA to evolve into a multiple antigen detection format, suggest that this method would be a reliable diagnostic test for detection of antibodies to N. caninum in cattle.