Characterization of the first bovine gammaherpesvirus 4 strain isolated from an aborted bovine fetus in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly believed to be responsible for several disorders of the bovine reproductive tract. The first characterization of BoHV-4 in Argentina was from samples from an aborted fetus. Argentinean isolates are highly diverse and are phylogenetically grouped in three genotypes. In this study, we describe the isolation of BoHV-4 from a bovine fetus with a gestational age of 8 months and without macroscopic lesions. Genetic analyses revealed that the isolated strain belongs to genotype 2. This is the first report on the presence of infectious BoHV-4 in tissues from an aborted bovine fetus.

Delayed type hypersensitivity induced by intradermal inoculation of a Neospora caninum tachyzoite antigen in previously exposed cattle.

The aim of this study was to evaluate delayed type hypersensitivity (DTH) induced by the intradermal inoculation of a Neospora caninum tachyzoite soluble lysate in cattle previously exposed with the protozoa. Four experimental groups were selected according to the prior exposure to N. caninum antigen. All cows were intradermally injected with a N. caninum tachyzoite soluble lysate and skinfold thickness growth at the inoculation sites was measured at 0, 24, 48, 72 and 96 h post inoculation (hpi). Additionally, specific antibodies and IFN-γ production were assessed. Cows experimentally infected with live N. caninum tachyzoites and cows naturally exposed to N. caninum developed skin reactions compatible with DTH between 24 and 96 hpi (p < 0.05). Moreover, cows inoculated with an experimental N. caninum vaccine and cows without evidence of exposure to N. caninum did not show a significant increase in skin thickness (p > 0.05). Furthermore, serological status of the animals was not modified due to the intradermal inoculation. The highest IFN-γ production was observed at 15 days after intradermal inoculation (p < 0.05). Therefore, these results suggest that cattle previously exposed to N. caninum develop a reaction compatible with DTH which could be useful as in vivo cell mediated immunity parameter for assessed bovine neosporosis.

Dual natural infection with bovine viral diarrhea virus -1 and -2 in a stillborn calf: tissue distribution and molecular characterization.

Dual infections with both bovine viral diarrhea virus (BVDV)-1 and -2 seem to be unusual. The aim of this study was to describe an infection with both BVDV genotypes in a stillborn calf. Virus isolation and phylogenetic analyses of the 5´UTR and NS5B regions confirmed the presence of BVDV-1b and -2b in spleen and lung, whereas BVDV-2b was also detected in brain, heart, liver, kidney and, fluid of cavities. These results confirm that dual infections with both BVDV-1 and BVDV-2 species can occur naturally and their tissue distribution can be different.

Interaction of bovine viral diarrhea virus with bovine cumulus-oocyte complex during IVM: Detection in permissive cells.

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus-oocyte complexes were exposed to 10(7) ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription-polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P<0.001). Levels of antibodies against rNcGRA7 were statistical higher in group A animals when compared with groups B and C at weeks 5 and 16 (P>0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation.

Concomitant infection of Neospora caninum and Bovine Herpesvirus type 5 in spontaneous bovine abortions / Infecção simultânea de Neospora caninum e Herpesvirus bovino tipo 5 em casos espontâneos de aborto bovino

Bovine Herpesvirus type 5 (BoHV-5) has not been conclusively demonstrated to cause bovine abortion. Brain lesions produced by Neospora caninum and Bovine Herpesvirus type 1 (BoHV-1) exhibit common features. Therefore, careful microscopic evaluation and additional diagnostic procedures are required to achieve an accurate final etiological diagnosis. The aim of the present work was to investigate the occurrence of infections due to BoHV-1, BoHV-5 and N. caninum in 68 cases of spontaneous bovine abortions which showed microscopic lesions in the fetal central nervous system. This study allowed the identification of 4 (5.9%) fetuses with dual infection by BoHV-5 and N. caninum and 33 (48.5%) cases in which N. caninum was the sole pathogen identified. All cases were negative to BoHV-1. The results of this study provide evidence that dual infection by BoHV-5 and N. caninum occur during pregnancy in cattle; however, the role of BoHV-5 as a primary cause of bovine abortion needs further research. Molecular diagnosis of BoHV-5 and N. caninum confirmed the importance of applying complementary assays to improve the sensitivity of diagnosing bovine abortion.

Não está demonstrado até ao momento, que o Herpesvírus bovino tipo 5 (BoHV-5) seja um agente causal de aborto bovino. Uma vez que as lesões cerebrais tanto de Neospora caninum como de Herpesvírus bovino tipo 1(BoHV-1) têm características similares, é necessária uma avaliação microscópica cuidadosa, bem como exames laboratoriais adicionais, para obter um diagnóstico final preciso. O objetivo do presente trabalho foi investigar a presença de infeções por BoHV-1, BoHV-5 e N. caninum em 68 casos de aborto espontâneo, nos quais se verificaram lesões microscópicas no sistema nervoso central. Foram encontrados 4 (5,9%) fetos com infeção simultânea de BoHV-5 e N. caninum e 33 (48,5%) casos com infeção exclusiva de N. caninum. Todos os casos foram negativos a BoHV-1. Os resultados deste estudo demonstram que a infeção dual por BoHV-5 y N. caninum está presente durante a gestçao dos bovinos. Apesar disso, o papel de BoHV-5 como agente primário causal de aborto, carece de mais investigaçao. O diagnóstico molecular de BoHV-5 e N. caninum confirmou a importância de se aplicar ensaios complementares para melhorar a sensibilidade do diagnóstico de aborto bovino.

Bovine viral diarrhea virus genomic associations in mucosal disease, enteritis and generalized dermatitis outbreaks in Argentina.

The objective of the present work is the description outbreaks caused by bovine viral diarrhea virus (BVDV) in commercial beef cattle ranches in Argentina. Genetic affiliation and their association with the clinical manifestation were carried out with five BVDV isolates from an outbreak of mucosal disease (MD) (Outbreak #1), acute enteritis (Outbreaks #2 and #3) and generalized dermatitis (Outbreaks #4 and #5). Upon genetic analysis CP BVDV isolate of Outbreak #1 clustered to closely to BVDV Oregon (Genotype 1). BVDV isolates from the outbreaks of generalized dermatitis (Outbreaks #4 and #5) were located close to BVDV Osloss within Genotype 1. The identification by immunohistochemistry of BVDV in exudative dermatitis indicates the epithelial cell tropism of the virus. Phylogenic characterization of BVDV from Outbreaks #2 and #3 locate them as BVDV-2. 5'UTR sequence of these viruses revealed a homology of 88 and 90% to BVDV-890 (Genotype 2) and a 77 and 75% to BVDV-SD1 (Genotype 1), respectively. The association of BVDV-2 with severe disease indicates the presence of highly virulent strains. Data from natural outbreaks where BVDV-1 and BVDV-2 were isolated revealed that pathology overlaps and not clearly allows the differentiation between genotypes based on gross or microscopic lesions. Thus, for a definitive diagnosis, further virology and molecular studies are necessary. Additionally, the results of this work focused on the origin and consequences of genetic variations of BVDV with regard to pathogenesis and suggest the association between genotype and a defined clinical syndrome.