[Seroprevalence and risk factors associated with Neospora caninum in dairy farms from the Province of Salta, Argentina]. / Prevalencia sérica de Neospora caninum y factores de riesgo asociados a su transmisión en tambos de la provincia de Salta, Argentina.

The objective of this work was to determine the seroprevalence of Neospora caninum in cattle in Valle de Lerma, province of Salta, Argentina, and the risk factors associated with the disease. Serum samples were taken from 40 cows in each dairy herd, which were analyzed by indirect ELISA to detect antibodies against N. caninum. Chronic and acute infections were discriminated by measuring the avidity of these antibodies. All the herds exhibited at least one seropositive animal, the mean being 35.3 ± 14.9% of positive animals. Specific antibodies were also detected in dogs present in 9of the herds, which showed a seropositivity value of 71.7% ± 19.9%. Among the seropositive animals, 56.3% showed acute infections. A negative association was found between seroprevalence and the avidity index of specific antibodies, indicating that the presence of animals with acute infections is associated with higher seroprevalence. Fields with grazing showed more recent infections. These results show for the first time the importance of this parasite in this particular region and the need to promote the development of control programs considering the different risk factors that affect the epidemiological situation of the disease.

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.

Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages.

Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

Kinetics of foot-and-mouth disease vaccine-induced antibody responses in buffaloes (Bubalus bubalis): avidity ELISA as an alternative to the virus neutralization test.

The role of water buffaloes in foot-and-mouth disease (FMD) epidemiology as one of the major hosts of the virus that can develop persistent asymptomatic infection highlights the importance of sustaining surveillance on the antibody response elicited by vaccination in these animals. There is gap in the knowledge on how serological assays that measure antibodies against capsid proteins perform with buffalo samples and which would be the most reliable test to substitute the virus neutralization test (VNT) a cumbersome and low-throughput tool for field surveillance. Alternatively, the liquid-phase blocking sandwich ELISA (LPBE) is commonly used. Previous data from our laboratory demonstrated that the vaccine-induced antibodies assessed by the LPBE yielded low specificity with buffaloes' samples. In contrast, a single-dilution avidity ELISA (AE) aimed to detect high-avidity antibodies against exposed epitopes, combined with an indirect ELISA (IE) to assess IgG levels, produced more reliable results. Here we analyzed for the first time the kinetics of the antibodies induced by vaccination in two different buffalo herds (n = 91) over 120 days using AE, IE, LPBE, and the VNT. Kinetics were similar in the different assays, with an increase of antibodies between 0- and 14-days post-vaccination (dpv) which were maintained thereafter. VNT and AE results were concordant (Kappa value = 0.76), and both assays revealed a decay in the antibody response in calves with maternal antibodies at 90 and 120 dpv, which was not evidenced by the LPBE. These results show that kinetics of antibody responses to FMD vaccination are similar in buffalo and cattle, and support the use of indirect ELISA assays, in particular Avidity ELISA, as alternatives to the VNT for vaccine-immunity monitoring irrespectively of the animal's passive or active immune status.

Serological screening of SARS-CoV-2 infection in companion animals of Buenos Aires suburbs.

The Coronavirus Disease 2019 (COVID-19) is a zoonotic disease caused by the pandemic virus SARS-CoV-2. Domestic and wild animals are susceptible to infection and are potential reservoirs for virus variants. To date, there is no information about the exposure of companion animals in Buenos Aires Suburbs, the area with the largest population in Argentina where the highest number of COVID-19 human cases occurred during the first infection wave. Here we developed a multi-species indirect ELISA to measure antibodies reactive to the SARS-CoV-2 receptor-binding domain (RBD) from several vertebrates constituting the class Mammalia, making it a valuable tool for field serosurveillance. The ELISA cut-off value was estimated by sera from dogs, cats, cattle, and pigs sampled before 2019 (n = 170), considering a 98% percentile and a grey zone to completely exclude any false positive result. Specificity was confirmed by measuring levels of neutralizing antibodies against canine coronavirus, the avidity of specific antibodies, and their capacity to impede the binding of a recombinant RBD protein to VERO cells in an In-Cell ELISA. Sera from 464 cats and dogs sampled in 2020 and 2021 ("pandemic" samples) were assessed using the RBD-ELISA. Information on COVID-19 disease in the household and the animals' lifestyles was collected. In Buenos Aires Suburbs cats were infected at a higher proportion than dogs, seroprevalence was 7.1 and 1.68%, respectively. Confirmed COVID-19 in the caregivers and outdoor lifestyle were statistically associated with seropositivity in cats. The risk of cats getting infected living indoors in COVID-19-negative households was null. The susceptibility of mammals to SARS-CoV-2, the possibility of transmission between animals themselves and humans, together with the free-roaming lifestyle typical of Buenos Aires suburban companion animals, urge pursuing responsible animal care and avoiding human interaction with animals during the disease course. The multi-species RBD-ELISA we developed can be used as a tool for serosurveillance of SARS-CoV-2 infection in mammalians (domestic and wild), guiding further targeted virological analyses to encounter susceptible species, interspecies transmission, and potential virus reservoirs in our region.

In-vivo Activity of IFN-λ and IFN-α Against Bovine-Viral-Diarrhea Virus in a Mouse Model.

Bovine-viral-diarrhea virus (BVDV) can cause significant economic losses in livestock. The disease is controlled with vaccination and bovines are susceptible until vaccine immunity develops and may remain vulnerable if a persistently infected animal is left on the farm; therefore, an antiviral agent that reduces virus infectivity can be a useful tool in control programs. Although many compounds with promising in-vitro efficacy have been identified, the lack of laboratory-animal models limited their potential for further clinical development. Recently, we described the activity of type I and III interferons, IFN-α and IFN-λ respectively, against several BVDV strains in-vitro. In this study, we analyzed the in-vivo efficacy of both IFNs using a BALB/c-mouse model. Mice infected with two type-2 BVDV field strains developed a viremia with different kinetics, depending on the infecting strain's virulence, that persisted for 56 days post-infection (dpi). Mice infected with the low-virulence strain elicited high systemic TNF-α levels at 2 dpi. IFNs were first applied subcutaneously 1 day before or after infection. The two IFNs reduced viremia with different kinetics, depending on whether either one was applied before or after infection. In a second experiment, we increased the number of applications of both IFNs. All the treatments reduced viremia compared to untreated mice. The application of IFN-λ pre- and post-infection reduced viremia over time. This study is the first proof of the concept of the antiviral potency of IFN-λ against BVDV in-vivo, thus encouraging further trails for a potential use of this cytokine in cattle.

Bovine Interferon Lambda Is a Potent Antiviral Against SARS-CoV-2 Infection in vitro.

Interferon lambda (IFN-λ) is an antiviral naturally produced in response to viral infections, with activity on cells of epithelial origin and located in the mucosal surfaces. This localized activity results in reduced toxicity compared to type I IFNs, whose receptors are ubiquitously expressed. IFN-λ has been effective in the therapy of respiratory viral infections, playing a crucial role in potentiating adaptive immune responses that initiate at mucosal surfaces. Human IFN-λ has polymorphisms that may cause differences in the interaction with the specific receptor in the human population. Interestingly, bovine IFN-λ3 has an in silico-predicted higher affinity for the human receptor than its human counterparts, with high identity with different human IFN-λ variants, making it a suitable antiviral therapeutic candidate for human health. Here, we demonstrate that a recombinant bovine IFN-λ (rbIFN-λ) produced in HEK-293 cells is effective in preventing SARS-CoV-2 infection of VERO cells, with an inhibitory concentration 50% (IC50) between 30 and 50 times lower than that of human type I IFN tested here (α2b and ß1a). We also demonstrated the absence of toxicity of rbIFN-λ in human PBMCs and the lack of proinflammatory activity on these cells. Altogether, our results show that rbIFN-λ is as an effective antiviral potentially suitable for COVID-19 therapy. Among other potential applications, rbIFN-λ could be useful to preclude virus dispersion to the lungs and/or to reduce transmission from infected people. Moreover, and due to the non-specific activity of this IFN, it can be potentially effective against other respiratory viruses that may be circulating together with SARS-CoV-2.

Interferon lambda protects cattle against bovine viral diarrhea virus infection.

Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

Evidence of reduced vertical transmission of Neospora caninum associated with higher IgG1 than IgG2 serum levels and presence of IFN-γ in non-aborting chronically infected cattle under natural condition.

Neospora caninum infection of cattle can be vertically transmitted, resulting in abortion or birth of infected calves. Vertical transmission occurs both in acutely or chronically infected cattle. There is little information on the immune response needed to prevent endogenous transplacental transmission, particularly from chronically infected cattle to their offspring in a natural environment. In this study, N. caninum seropositive pregnant cattle from three different farms with high avidity antibodies and low IgM titers were selected and their newborn colostrum-deprived calves were tested for anti-N. caninum antibodies. Based on these results, dams were grouped according to their congenital transmission status. The analysis of the immune profile of the chronically-infected pregnant cattle revealed that higher ratio between IgG1 and IgG2 anti-N. caninum serum titers and higher levels of systemic IFN-γ were associated with diminished vertical transmission rates, compared to dams with the opposite profile. Our results evidenced an association between the immune profile and vertical transmission in non-aborting chronically infected dams, and confirm that vertical transmission, even when not leading to abortion, is related to a defined immune profile. This is important information to accomplish successful vaccine development efforts.

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity.

Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Prevalencia sérica de Neospora caninum y factores de riesgo asociados a su transmisión en tambos de la provincia de Salta, Argentina / Seroprevalence and risk factors associated with Neospora caninum in dairy farms from the Province of Salta, Argentina

Resumen El objetivo de este trabajo fue conocer la seroprevalencia de Neospora caninum en los bovinos de los tambos del Valle del Lerma (Salta, Argentina) y los factores de riesgo asociados a la transmisión de este parásito en esta región. Se tomaron muestras de suero de aproximadamente 40 vacas en cada tambo, que fueron analizadas por ELISA indirecto para detectar anticuerpos contra N. caninum. También se discriminó entre infecciones crónicas y agudas midiendo la avidez de dichos anticuerpos. Todos los tambos presentaron al menos un bovino seropositivoy la media fue de 35,3 ± 14,9% de animales positivos. También se detectaron anticuerpos específicos en caninos presentes en 9 de los 16 tambos, con un valor de seropositi- vidad del 71,7 ± 19,9%. El 56,3% de los bovinos seropositivos cursaban infecciones agudas. Se halló una asociación negativa entre la seroprevalencia y el índice de avidez de los anticuerpos específicos, lo que indica que la presencia de animales con infecciones agudas se asocia a mayor seroprevalencia. Los campos con pastoreo presentaron mayor cantidad de infecciones recien tes. Estos resultados revelan por primera vez la importancia de este parásito en los tambos de la región y la necesidad de propiciar el desarrollo de programas de control considerando los distintos factores de riesgo que afectan la situación epidemiológica de la enfermedad.

Abstract The objective of this work was to determine the seroprevalence of Neospora caninum in cattle in Valle de Lerma, province of Salta, Argentina, and the risk factors associated with the disease. Serum samples were taken from 40 cows in each dairy herd, which were analyzed by indirect ELISA to detect antibodies against N. caninum. Chronic and acute infections were discriminated by measuring the avidity of these antibodies. All the herds exhibited at least one seropositive animal, the mean being 35.3 ± 14.9% of positive animals. Specific antibodies were also detected in dogs present in 9of the herds, which showed a seropositivity value of 71.7% ± 19.9%. Among the seropositive animals, 56.3% showed acute infections. A negative association was found between seroprevalence and the avidity index of specific antibodies, indicating that the presence of animals with acute infections is associated with higher seroprevalence. Fields with grazing showed more recent infections. These results show for the first time the importance of this parasite in this particular region and the need to promote the development of control programs considering the different risk factors that affect the epidemiological situation of the disease.

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.

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.