Bovine Herpesvirus Type Research Articles

A large-scale epidemiological model of BoHV-1 spread in the Irish cattle population to support decision-making in conformity with the European Animal Health Law

We present a new modelling framework to address the evaluation of national control/surveillance programs planned in line with the European Animal Health Law (AHL) for livestock diseases. Our modelling framework is applied to the cattle sector in Ireland where there is need for policy support to design an optimal programme to achieve bovine herpesvirus type 1 (BoHV-1) free status under the AHL. In this contribution, we show how our framework establishes a regional model that is able to mechanistically reproduce the demography, management practices and transport patterns of an entire cattle population without being dependent on continuous livestock registry data. An innovative feature of our model is the inclusion of herd typing, thereby extending these beyond the categories of dairy, beef and mixed herds that are frequently considered in other regional modelling studies. This detailed representation of herd type-specific management facilitates comparative assessment of BoHV-1 eradication strategies targeting different production types with individual strategy protocols. Finally, we apply our model to support current discussions regarding the structure and implementation of a potential national BoHV-1 eradication programme in Ireland.

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid. The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD. These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

Efficacy of Different Treatment Modalities for Eye Cancer in Bovines

Background: Bovine ocular squamous cell carcinoma also called cancer eye, represents the most economically important neoplasm in large animals. Hereditary factors, environmental factors (e.g: latitude, altitude, exposure to sunlight), lack of eyelid pigmentation, age and dietary habits have all been reported to play a role in the etiopathogenesis of bovine ocular squamous cell carcinoma. In addition, in cattle the etiology has been linked to a number of viral agents, especially bovine papilloma virus and bovine herpes virus type 1 and 5. Nevertheless, ultraviolet light, viruses and circumocular pigmentation are the major epidemiologic risk factors for the development of the tumor. The efficacy of different treatment modalities for eye cancer in bovines was studied.Methods: All the animals were divided in to four groups of six animals each. Surgical excision, intra lesion BCG vaccine, surgery with auto vaccine and surgery with mitomycin was the treatment protocols followed. Immunohistochemical studies were conducted to know the rate of proliferation of bovine ye cancer. Immunopositive reaction was observed against Vascular Endothelial Growth Factor and Epidermal Growth Factor Receptor in all the cases.Result: The benefits of different treatment modalities depended on nature, type, location and extensiveness of tumor. Early detection and aggressive treatment were essential in the successful management of these tumors. A multimodal treatment approach was recommended with surgery, immunotherapy and chemotherapy in providing 100% disease free interval.

DEVELOPMENT OF A CRISPR-BASED ANTIVIRAL SYSTEM AGAINST BOHV-1

<h3>Background</h3> Bovine Herpesvirus type 1 (BoHV-1) infection has no cure and infected animals become lifelong carriers, greatly impacting health and productivity of herds worldwide. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas (CRISPR associated) tool has been widely explored for the development of antiviral strategies due to its ability to precisely edit genetic sequences. In this manner, we have designed a CRISPR-based antiviral system against BoHV-1 through the tetracycline-inducible expression of the Cas9 nuclease and a guide RNA (gRNA) homologous to a viral sequence in genetically modified bovine cells. <h3>Methods</h3> The in silico design of the antiviral system was based on the literature. BoHV-1 and bovine (Bos taurus taurus) genome were obtained from GenBank and basic molecular vectors from Addgene. Sequence analysis of appropriate targets were performed with Snapgene and Serial Cloner. Primers for cloning were obtained using the NCBI tool Primer BLAST. The gRNA for bovine DNA cleavage was selected from literature considering cleavage efficiency and potential off-target effects. The gRNA for the viral genome cleavage was designed using the CRISPR gRNA design tool from ATUM and CHOPCHOP. <h3>Results</h3> The developed molecular vector allows the delivery of the necessary machinery to perform a cleavage in the chromosome 25 of the bovine DNA between the FSCN1 and the ACTB genes, where the sequence of the antiviral mechanism will be inserted by homologous recombination (HR). The cleavage site has already been reported as safe for the insertion of sequences into the bovine genome. The HR fragment has the Cas9 nuclease sequence and a tetracycline-inducible expression system capable of conditioning the expression of the nuclease gene to the presence of the antibiotic. In addition, the fragment has a sequence for the expression of a gRNA targeting an essential sequence for virus replication, the UL52 gene. The expressed Cas9 enzyme and gRNA form a ribonucleoprotein complex capable of cleaving only the virus genome leading to its inactivation and preventing its replication in the genetically modified cells stimulated by tetracycline. <h3>Conclusion</h3> The mechanism and elements developed explore new applications of the CRISPR-Cas tool to combat viral diseases that currently have no cure. The ease of altering the gRNA target makes the proposed system highly flexible and can possibly be applied to different diseases caused by viruses.

Identification of Bovine Herpesvirus type 5 in nasal swabs of naturally infected calves by Polymerase Chain Reaction

In this article, the results of molecular-genetic analyses of nasal swabs are presented. The experimental studies were carried out in the dairy farms of Vologda region suspected to the respiratory infections. The samples were obtained from the asymptomatic calves and from calves with cough and nasal discharges. The laboratory studies were conducted in the laboratory of virology of the FSC VIEV. The genome of the virus was detected by a polymerase chain reaction. The comparative analyses of identified nucleotide sequences of the virus with the reference strains of the International Nucleotide sequence database has shown the similarity with Bovine herpesvirus-5. Accordingly, as a result of our studies, the Bovine herpesvirus type 5 was identified in the nasal swabs of calves in farms of Vologda regions.

Recombinant bovine IL17A acts as an adjuvant for bovine herpesvirus vaccine

The Bovine herpes virus type 5 glycoprotein D (gD) is essential for viral penetration into host permissive cells. The Herpes virus gD glycoprotein has been used for bovine immunization, being efficient in reduction of viral replication, shedding and clinical signs, however sterilizing immunity is still not achieved. Recombinant subunit vaccines are, in general, poorly immunogenic requiring additional adjuvant components. Interleukin 17A (IL17A) is a pro-inflammatory cytokine produced by T helper 17 cells that mediate mucosal immunity. IL17 production during vaccine-induced immunity is a requirement for mucosal protection to several agents. In this study, we investigated the potential of a recombinant IL17A to act as an adjuvant for a recombinant BoHV-5 glycoprotein D vaccine in cattle. Three cattle groups were divided as: group 1) rgD5 + alumen + rIL-17A; 2) rgD5 + alumen; and 3) PBS + alumen. The cattle (3 per group) received two doses of their respective vaccines at an interval of 21 days. The group that received rIL17 in its vaccine formulation at the 7th day after the prime immunization had significant higher levels of specific rgD-IgG than the alumen group. Addition of rIL17 also led to a significant fold increase in specific anti-rgD IgG and neutralizing antibodies to the virus, respectively, when compared with the alumen group. Cells stimulated with rIL17A responded with IL17 transcription, as well IL2, IL4, IL10, IL15, Bcl6 and CXCR5. Our findings suggest that the rIL17A has adjuvant potential for use in vaccines against BoHV-5 as well as potentially other pathogens of cattle.

Bacillus Toyonensis BCT-7112T Spores as Parenteral Adjuvant of BoHV-5 Vaccine in a Murine Model.

Bacterial spores of the genus Bacillus are being evaluated as adjuvant molecules capable of improving the immune response to vaccines. In this study, we investigate whether subcutaneously administered spores of B. toyonensis BCT-7112T could enhance a vaccine immune response in mice. Three groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: 40µg of recombinant glycoprotein D (rgD) from bovine herpesvirus type 5 (BoHV-5) adsorbed in 10% aluminum hydroxide (alum) without B. toyonensis spores (group 1) and B. toyonensis (1 × 106 viable spores) + 40µg of rgD adsorbed in 10% alum (group 2); and B. toyonensis (1 × 106 viable spores) without rgD (group 3). Group 2 showed significantly higher titers (P < 0.05) of total specific serum IgG, IgG2a, and neutralizing antibodies, when compared with the groups 1 and 3. A significantly higher (P < 0.05) transcription level of cytokines IL-4, IL-12, and IFN-γ was observed in splenocytes from mice that received the B. toyonensis spores in the vaccine formulation. In addition, stimulation of the macrophage-like cell line RAW264.7 with spores of B. toyonensis markedly enhanced the cell proliferation and mRNA transcription levels of IL-4, and IL-12 cytokines in these cells. Our findings indicated that the subcutaneous administration of B. toyonensis BCT-7112T spores enhanced the humoral and cellular immune response against BoHV-5 in mice.

BoHV-1-Vectored BVDV-2 Subunit Vaccine Induces BVDV Cross-Reactive Cellular Immune Responses and Protects against BVDV-2 Challenge.

The bovine respiratory disease complex (BRDC) remains a major problem for both beef and dairy cattle industries worldwide. BRDC frequently involves an initial viral respiratory infection resulting in immunosuppression, which creates a favorable condition for fatal secondary bacterial infection. Current polyvalent modified live vaccines against bovine herpesvirus type 1(BoHV-1) and bovine viral diarrhea virus (BVDV) have limitations concerning their safety and efficacy. To address these shortcomings and safety issues, we have constructed a quadruple gene mutated BoHV-1 vaccine vector (BoHV-1 QMV), which expresses BVDV type 2, chimeric E2 and Flag-tagged Erns-fused with bovine granulocyte monocyte colony-stimulating factor (GM-CSF) designated here as QMV-BVD2*. Here we compared the safety, immunogenicity, and protective efficacy of QMV-BVD2* vaccination in calves against BVDV-2 with Zoetis Bovi-shield Gold 3 trivalent (BoHV-1, BVDV types 1 and 2) vaccine. The QMV-BVD2* prototype subunit vaccine induced the BoHV-1 and BVDV-2 neutralizing antibody responses along with BVDV-1 and -2 cross-reactive cellular immune responses. Moreover, after a virulent BVDV-2 challenge, the QMV-BVD2* prototype subunit vaccine conferred a more rapid recall BVDV-2-specific neutralizing antibody response and considerably better recall BVDV types 1 and 2-cross protective cellular immune responses than that of the Zoetis Bovi-shield Gold 3.

Evolution of the Seroprevalence of Pestivirus and Respiratory Viral Infections in Spanish Feedlot Lambs.

Simple SummaryThe presence of viruses, such as bovine parainfluenza type 3, bovine herpesvirus type 1, bovine respiratory syncytial virus and pestiviruses, has been widely demonstrated in sheep in the past, but it is unknown how these infections may affect the lambs during their fattening period under commercial conditions. In the present study, the exposure to the aforementioned viruses was studied in 120 feedlot lambs throughout the whole fattening period. Seroprevalences were measured by enzyme-linked immunosorbent assays (ELISA), relating their presence to health and production parameters in the studied lambs. During the studied period, the lambs seroconverted against 3 out of the 4 viruses analysed and only 10.8% of the lambs were seronegative for all the tested viruses throughout the entire study. Finally, no associations were found between the seroconversion to one or more viruses and the presence of respiratory clinical signs or lung lesions at the slaughterhouse. In addition, no disturbances were observed in the performance of the lambs, except in the case of pestivirus infections, for which the presence of antibodies in the animals was associated with reduced final weight at the end of the fattening period.The presence of respiratory viruses and pestiviruses in sheep has been widely demonstrated, and their ability to cause injury and predispose to respiratory processes have been proven experimentally. A longitudinal observational study was performed to determine the seroprevalence of bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and pestiviruses in 120 lambs at the beginning and the end of the fattening period. During this time, the animals were clinically monitored, their growth was recorded, and post-mortem examinations were performed in order to identify the presence of pneumonic lesions in the animals. Seroconversion to all viruses tested except BHV-1 was detected at the end of the period. Initially, BPIV-3 antibodies were the most frequently found, while the most common seroconversion through the analysed period occurred to BRSV. Only 10.8% of the lambs showed no detectable levels of antibodies against any of the tested viruses at the end of the survey. In addition, no statistical differences were found in the presentation of respiratory clinical signs, pneumonic lesions nor in the production performance between lambs that seroconverted and those which did not, except in the case of pestiviruses. The seroconversion to pestiviruses was associated with a reduction in the final weight of the lambs.

Development of a reliable bovine neuronal cell culture system and labeled recombinant bovine herpesvirus type-1 for studying virus-host cell interactions

Bovine herpesvirus type 1 (BoHV-1) is the viral causative agent of infectious bovine rhinotracheitis and a component of the bovine respiratory complex commonly referred to as shipping fever in calves. BoHV-1 is also responsible for losses of aborted calves and reductions in dairy productivity. BoHV-1 belongs to the neurotropic alphaherpesviruses which have a predilection to infect and establish latency in sensory neurons. Neuronal cell cultures provide a useful platform for experiments investigating neuronal entry, retrograde and anterograde transport, and the establishment of latency. Rodent neuronal cell lines and primary rabbit neuronal cells have been utilized for BoHV-1, though a reliable host-specific neuronal cell culture system has not been developed. In this study, BoHV-1 readily infected bovine-derived immortalized neuronal progenitor cells (FBBC-1) differentiated in cell culture producing neurite-like projections and exhibiting neuronal cell markers NeuN and L1CAM. FBBC-1 cells expressed both nectin-1 and nectin-2 alphaherpesvirus receptors on their cell surfaces, however, nectin-2 was detected in much greater abundance than nectin-1. To facilitate investigations of BoHV-1 infection, a recombinant BoHV-1 virus expressing the green fluorescent protein (GFP) cloned into a bacterial artificial chromosome (BAC) was used to generate an mCherry-VP26 fusion protein. The BoHV-1 GFP expressing VP26mCherry labeled virus infected differentiated FBBC-1 cells as evidenced by the production of infectious virions and the expression of both GFP and mCherry fluorophores. Time-lapse live cell microscopy revealed the presence of mCherry fluorescent capsids in neuronal projections immediately after virus entry moving retrograde in a saltatory manner. Proximity ligation assays (PLA) using MDBK cells demonstrated that BoHV-1 glycoprotein D (gD) interacted more efficiently with nectin-1 than nectin-2. However, the gD interaction with nectin-2 predominated in differentiated FBBC-1 cells in comparison to the gD nectin-1 interaction. The efficiently differentiated FBBC-1 neuronal cell line and fluorescently labeled BoHV-1 virions will assist experimentation aiming to elucidate specific mechanisms of virus entry and transport in a homologous bovine neuronal cell culture system.

A serological investigation of Bovine enterovirus-1, Bovine herpesvirus-1, Bovine viral diarrhea virus, and Parainfluenza-3 infections in camelsin Western Turkey.

Camels (Camelus dromedarius) are bred in Western Turkey, particularly in the province of Aydin, for touristic, social and cultural purposes. Bovine enterovirus‑1 (BEV‑1), Bovine herpesvirus type‑1 (BHV‑1), Bovine viral diarrhea virus (BVDV), and Parainfluenza‑3 (PI‑3) virus infections are significant causes of health and/or economic concerns in several animal species. These agents have not been investigated in the camel population in Turkey. The objective of this study was to serologically investigate the presence and infection rates of these viruses in camels in Aydin province, Western Turkey. Ninety‑two serum samples were taken from clinically healthy camels that were kept in private farms or brought to the local slaughterhouses. Serum neutralization test was performed to assess the presence and the titers of specific antibodies against BEV‑1, BHV‑1, BVDV, and PI‑3 virus in camel sera. Of the 92 camels tested, 30 (32.61%), 2 (2.17%), 54 (58.7%), and 20 (21.74%) were seropositive for BEV‑1, BHV‑1, BVDV, and PI‑3, respectively. These results suggest that, except for BHV‑1, these viral infections are common among camels in Western Turkey. To our knowledge, this the first comprehensive, large‑scale study investigating these viral infections in camels in Turkey.

Predicting bovine respiratory disease outcome in feedlot cattle using latent class analysis.

Bovine respiratory disease (BRD) is the most significant disease affecting feedlot cattle. Indicators of BRD often used in feedlots such as visual signs, rectal temperature, computer-assisted lung auscultation (CALA) score, the number of BRD treatments, presence of viral pathogens, viral seroconversion, and lung damage at slaughter vary in their ability to predict an animal’s BRD outcome, and no studies have been published determining how a combination of these BRD indicators may define the number of BRD disease outcome groups. The objectives of the current study were (1) to identify BRD outcome groups using BRD indicators collected during the feeding phase and at slaughter through latent class analysis (LCA) and (2) to determine the importance of these BRD indicators to predict disease outcome. Animals with BRD (n = 127) were identified by visual signs and removed from production pens for further examination. Control animals displaying no visual signs of BRD (n = 143) were also removed and examined. Blood, nasal swab samples, and clinical measurements were collected. Lung and pleural lesions indicative of BRD were scored at slaughter. LCA was applied to identify possible outcome groups. Three latent classes were identified in the best model fit, categorized as non-BRD, mild BRD, and severe BRD. Animals in the mild BRD group had a higher probability of having visual signs of BRD compared with non-BRD and severe BRD animals. Animals in the severe BRD group were more likely to require more than 1 treatment for BRD and have ≥40 °C rectal temperature, ≥10% total lung consolidation, and severe pleural lesions at slaughter. Animals in the severe BRD group were also more likely to be naïve at feedlot entry and the first BRD pull for Bovine Viral Diarrhoea Virus, Bovine Parainfluenza 3 Virus, and Bovine Adenovirus and have a positive nasal swab result for Bovine Herpesvirus Type 1 and Bovine Coronavirus. Animals with severe BRD had 0.9 and 0.6 kg/d lower overall ADG (average daily gain) compared with non-BRD animals and mild BRD animals (P < 0.001). These results demonstrate that there are important indicators of BRD severity. Using this information to predict an animal’s BRD outcome would greatly enhance treatment efficacy and aid in better management of animals at risk of suffering from severe BRD.

Genome-wide analysis of codon usage pattern in herpesviruses and its relation to evolution

The preferential use of a specific codon, out of a group of synonymous codons encoding the same amino acid, in a gene transcript results from the bias in codon choice. Various evolutionary forces namely mutation pressure and natural selection influence the pattern of codon usage i.e. distinct for each gene/genome. We investigated the pattern of codon usage of eight human herpesvirus genomes and compared them with two other herpesvirus genomes namely murine herpesvirus 68 and bovine herpesvirus type 1.1 to elucidate its compositional features, pattern of codon usage across the genomes and report the differences of codon usage pattern of human herpesviruses from that of other two other viruses. We also identified the similarity of the codon usage of human herpesviruses with its host (human). The genes were found to be CG rich in HHV2, HHV3, HHV4, HHV6, HHV7 and BH genomes while TA rich in HHV1, HHV5, HHV8 and MH genomes. The codon usage bias (CUB) of genes was low. A highly significant correlation was found among compositional contents depicting the role of mutational pressure along with natural selection in framing CUB. Several more frequently used codons as well as less frequently used codons were identified to be similar between each human virus and its host (human), while murine herpesvirus 68 and bovine herpesvirus type 1.1 genomes did not possess similar adaptation strategy as human herpesviruses to human (host), thus we could conclude that viral CUB might have been shaped as per their host’s nature for better surveillance. Neutrality plot revealed mutational pressure mostly influenced the CUB of HHV1, HHV8 and MH viruses, while natural selection had a major impact in the CUB of HHV2, HHV3, HHV4, HHV5, HHV6, HHV7 and BH genomes.

Association of Bacillus toyonensis spores with alum improves bovine herpesvirus 5 subunit vaccine immune response in mice

Spores of the genus Bacillus are molecules capable of increasing the vaccine adjuvanticity. Bovine herpesvirus type 5 (BoHV-5) is responsible for meningoencephalitis that causes important economic losses in cattle. BoHV-5 glycoprotein D (gD) is a target of vaccine antigen and plays an important role in host cell penetration. The present study aimed to evaluate the adjuvanticity of Bacillus toyonensis (B.t) spores, live and heat-killed, associated with a vaccine formulated with aluminum hydroxide (alum) and the recombinant BoHV-5 glycoprotein D (rgD) in an experimental murine model. Six experimental groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: rgD (40 µg) + live spores (2 × 109 CFU); rgD + killed spores; rgD + live spores + alum (2.0 mg); rgD + killed spores + alum; rgD + alum, and rgD + PBS. Mice from rgD + live spores group showed an increase in rgD IgG titers from the 21st day until the end of the experiment. The groups of live and killed spores, associated to alum, had similar levels of IgG titers with no significant difference between each other; however, by the 14th and 28th day until the end of the experiment, presented higher IgG titers in comparison to the rgD + alum group. Moreover, increased serum levels of IgG1, IgG2a, and IgG2b were detected in mice that received spores in the vaccine formulation. The spores associated with alum groups showed neutralizing BoHV-5 antibodies and high mRNA transcription of the cytokines IFN-γ (66-fold), IL-17 (14-fold), and IL-12 (2.8-fold). In conclusion, our data demonstrated that the B. toyonensis spores, live or killed, associated with alum increased the adjuvanticity for BoHV-5 rgD in mice, suggesting the use of B. toyonensis spores as a promising component for vaccine formulations.

Assessment of replication of bovine herpesvirus type 4 in human glioblastoma and breast cancer cells as a potential oncolytic virus.

Oncolytic viruses have been extensively used in cancer treatment due to their tropism, selective replication only in tumor cells, and possible synergic interaction with other therapeutics. Different researchers have demonstrated that bovine herpesvirus 4 (BoHV-4), a member of the gammaherpesviridae family, has oncolytic potential in some human-origin cancer cell lines like glioma through the selective replication strategy. Using four apoptosis detection methods, namely MTT, LDH, TUNEL, and Annexin V assays, we evaluated the apoptotic effect of BoHV-4 Movar33/63 reference strain along with a recombinant BoHV-4 expressing EGFP in U87 MG cells (human glioblastoma cell line), MDA MB-231 (human breast cancer cell line), and MCF10a (non-tumorigenic human mammary epithelial cell line). Our findings indicate that this virus can replicate and induce apoptosis in these cell lines and hinder in vitro proliferation in a dose-dependent manner. In conclusion, BoHV-4 has in vitro potential as a novel oncolytic virus in human cancer therapy. However, its replication potential in the MCF10a cells as a non-tumorigenic human mammary epithelial cell line is a concern in using this virus in cancer therapy, at least against human mammary tumors. Further studies must therefore be conducted to examine the specific apoptotic pathways induced by this virus to move on to further experiments.

Epidemiology of age-dependent prevalence of Bovine Herpes Virus Type 1 (BoHV-1) in dairy herds with and without vaccination

Many studies report age as a risk factor for BoHV-1 infection or seropositivity. However, it is unclear whether this pattern reflects true epidemiological causation or is a consequence of study design and other issues. Here, we seek to understand the age-related dynamics of BoHV-1 seroprevalence in seasonal calving Irish dairy herds and provide decision support for the design and implementation of effective BoHV-1 testing strategies. We analysed seroprevalence data from dairy herds taken during two Irish seroprevalence surveys conducted between 2010 and 2017. Age-dependent seroprevalence profiles were constructed for herds that were seropositive and unvaccinated. Some of these profiles revealed a sudden increase in seroprevalence between adjacent age-cohorts, from absent or low to close to 100% of seropositive animals. By coupling the outcome of our data analysis with simulation output of an individual-based model at the herd scale, we have shown that these sudden increases are related to extensive virus circulation within a herd for a limited time, which may then subsequently remain latent over the following years. BoHV-1 outbreaks in dairy cattle herds affect animals independent of age and lead to almost 100% seroconversion in all age groups, or at least in all animals within a single epidemiological unit. In the absence of circulating infection, there is a year-on-year increase in the age-cohort at which seroprevalence changes from low to high. The findings of this study inform recommendations regarding testing regimes in the context of contingency planning or an eradication programme in seasonal calving dairy herds.

Indirect detection of respiratory viruses responsible for respiratory disease in sheep

Respiratory disease in sheep is an important disease in sheep flocks, in which viruses are categorized as primary etiological agents. It is responsible for elevated economic losses in sheep production and new data about its occurrence and the etiological agents are necessary to understand its situation in Brazilian flocks. This research aimed to detect antibodies against Bovine Viral Diarrhea Virus, Bovine Herpesvirus type 1, Bovine Respiratory Syncytial Virus (BRSV) and Bovine Parainfluenza type 3 (bPI-3) virus in healthy and pneumonic sheep in relation to clinical signs of respiratory disease. Ninety-nine male and female sheep from the states of São Paulo (SP) and Rio de Janeiro (RJ) were enrolled in this study. They were classified as healthy and pneumonic using physical examination. Serum samples were obtained, and they were used to determine the presence of antibodies against respiratory viruses using serum neutralization test. The association between the microorganisms, clinical status, and clinical signs of respiratory disease in sheep was evaluated. Antibodies against Bovine Parainfluenza type 3 virus were mainly detected (52.5%), followed by Bovine Respiratory Syncytial Virus (48.5%). Data analysis did not reveal any association between the presence of antibodies and clinical signs of respiratory disease in sheep. Respiratory disease is an important issue in sheep production in which different bacterial and viral etiologic agents may be involved in its development. BRSV and bPI-3 are circulating in sheep flocks from São Paulo and Rio de Janeiro.

A Phylogenetic and Genotyping Study of Bovine Herpesvirus Type 4 (BHV-4) in Turkey

Infertility and reproductive infections are the huge problems for the diary management throughout the world. Bovine herpesviruses act an enormous role in these complicated problems. Bovine Herpesvirus Type 1 (BHV-1) is the most outstanding herpesvirus causing genital and uterine tracts infections among other reproductive viral agents, however, Bovine Herpesvirus Type 4 (BHV-4) is also responsible in terms of similar symptoms and diseases. The main aims of the study are both to investigate the underlying potential presence of BHV-4 in subclinical uterine tract infection, and both to perform molecular and recombination analyses. A herd including 25 repeat breeder cows were investigated by BHV-4. Two out of them were determined BHV-4 infected after a series of Polymerase Chain Reactions (PCRs) tests which able to amplify partial Glycoprotein B (gB) and Thymidine Kinase (TK) gene regions. Obtained sequences were analyzed by using phylogenetic and recombinational software, and two Maximum Likelihood (ML) tree have been constructed. To results, novel Turkish BHV-4 sequences fell into Genotype I in both constructed Maximum Likelihood (ML) phylogenetic trees, however, no recombination evidence has been observed in relevant software. This report is the first genotyping study of BHV-4 from Turkey. This study showed that Turkish BHV-4 substantially originated from European strains and might be observed in different clinical presentations. This suggests that BHV-4 should be deeply investigated by further molecular techniques and included in diagnostic panels for reproductive diseases viruses.

Effects of Parenteral Supplementation with Minerals and Vitamins on Oxidative Stress and Humoral Immune Response of Weaning Calves.

We aimed to evaluate the effects of injectable mineral and vitamin supplementation on weaning calves subjected to a low-stress (fence-line) weaning system. Seven-month-old Aberdeen Angus female calves (n = 40, 152 ± 11 kg body weight) from a selenium-deficient area of extensive cattle production on natural grass were randomly assigned to two groups (n = 20 each). One group received subcutaneous supplementation with copper, zinc, selenium, manganese and vitamins A and E (SG), and the other was given sterile saline solution (CG). The animals were supplemented twice, seven days before weaning (day -7) and on the day of weaning (day 0), and they were evaluated 30 (+30) and 60 (+60) days after weaning. Total antioxidant status (TAS), selenium-dependent glutathione peroxidase (GPx) activity, body weight, and average daily gain (ADG) were evaluated. Additionally, antibody titers were assessed prior to and after each immunization with a vaccine containing bovine herpes virus type 1 (BoHV-1). On day +30, body weight (p = 0.03) was higher in SG, whereas TAS (p = 0.02) and GPx (p = 0.0038) activity were lower in CG and remained constant in SG. Antibody titers increased in SG and CG following immunization, being higher in SG on days +30 and +60 (p < 0.05). In conclusion, parenteral supplementation of minerals and vitamins with antioxidant effects in a low-stress weaning system prevented the decrease in TAS and GPx activity, improved antibody response and had positive effects on body weight.

Toxocara canis infection may impair bovine herpesvirus type 5 immunization

Helminths have developed complex mechanisms to suppress the host immune response. These mechanisms may impair the host vaccine response. This study aimed to evaluate the effect of Toxocara spp. infection on the vaccine immune response to bovine herpesvirus type 5 (BoHV–5). First, 30 heifers received two doses of an experimental BoHV–5 vaccine. At 42nd days after the primo vaccination the vaccine efficacy was evaluated, and the presence of anti-Toxocara antibodies. Second, 20 Balb/c mice were divided into two groups, one infected with T. canis and the other without infection. After infection, both groups received two doses of vaccine. The vaccine immune response was assessed by BoHV-5 serum neutralization and splenic cytokines transcription by qPCR. All heifers positive for Toxocara spp. (40%) showed BoHV-5 SN titer ≤1:32, whereas heifers negative for Toxocara spp. (60%) had BoHV-5 SN titer ≥1: 128. Infected T. canis mice showed BoHV-5 SN titer ≤1:2, whereas mice not infected with T. canis BoHV-5 SN titer ≥1:8. Splenocytes from control mice stimulated with BoHV-5 had a significant (p < .05) mRNA transcription for the cytokines IL-12, IL-17, and IL-23, whereas the same cytokines were down-regulated in T. canis infected mice. These results suggest that Toxocara spp. infection may impair BoHV-5 immunization and should be considered for efficient herd immunization.