Search

Introduction: Bovine viral diarrhea virus (BVDV), a positive-sense single-stranded RNA virus, causes significant economic losses in the cattle industry. Current diagnostic methods for BVDV exhibit variable sensitivity and specificity, underscoring the need for more rapid and accurate detection approaches. Here, we developed a novel competitive ELISA (cELISA) to detect antibodies against the BVDV E2 protein., Methods and Results: We generated three monoclonal antibodies (mAbs)-3E6, 2D5, and 5B9-by immunizing mice with purified BVDV E2 protein expressed in Expi293F cells. Among these, mAb 3E6 displayed superior competitive binding abilities to the E2 protein, enabling effective differentiation between BVDV positive and negative sera. Remarkably, mAb 3E6 exhibited pan-genotypic recognition of various BVDV strains, including BVDV-1a, -1b, -1c, -1m, -1p, -1v, and -2a, while showing no cross-reactivity with the classical swine fever virus (CSFV). Computational modeling using AlphaFold 3 identified domain B of the E2 protein as the primary binding site for mAb 3E6. Building upon these findings, we established a cELISA employing mAb 3E6 and recombinant E2 protein. Receiver-operating characteristic (ROC) analysis revealed outstanding diagnostic performance, achieving a sensitivity of 99.26% and specificity of 98.99%. Further tests confirmed the cELISA's specificity for detecting BVDV-specific antibodies, with no cross-reactivity with antisera from animals infected or immunized against BCoV, BHV-1, BRV, AKAV, LSDV, BLV, and CSFV. Consistency was observed between results from the BVDV E2 cELISA and traditional virus neutralization test (VNT), demonstrating high sensitivity for monitoring antibody dynamics. In performance evaluations, the established cELISA exhibited high concordance with VNT in assessing 160 vaccinated sera and 190 clinical samples., Discussion: The BVDV E2 cELISA, utilizing mAb 3E6 to target domain B of the BVDV E2 protein, represents a reliable and effective serological diagnostic tool for the detection of antibodies against both BVDV-1 and BVDV-2. This methodology holds significant promise for applications in clinical diagnosis and the evaluation of vaccine efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qi, Wang, Le, Sun, Chang, Jiang, Yin and Pang.)

Introduction: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route., Methods: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID 50 , RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor., Results: Our immunoperoxidase staining, RT-qPCR, and TCID 50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells., Conclusion: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Dassanayake, Menghwar, Bickel, Holthausen, Ma, Diaz-San Segunda, Rodriguez-Calzada, Medina, Attreed, Falkenberg, Kanipe, Sacco, De Los Santos and Casas.)

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Background: Pigs are susceptible to several ruminant pathogens, including Coxiella burnetti, Schmallenberg virus (SBV) and bovine viral diarrhea virus (BVDV). These pathogens have already been described in the pig population, although the dynamics of the infection and the impact on pig farms are currently unclear. The aim of this work was to evaluate the presence of these infections in the pig population of the Campania region, southern Italy, and to evaluate the risk factors associated with a greater risk of exposure., Results: A total of 414 serum samples belonging to 32 herds were tested for the presence of antibodies against SBV, Coxiella, and BVD using commercial multispecies ELISA kits. SBV (5.3%) was the most prevalent pathogen, followed by Coxiella (4.1%) and BVD (3%). The risk factors included in the study (age, sex, province, farming system, ruminant density and major ruminant species) had no influence on the probability of being exposed to BVD and Coxiella, except for the location, in fact more pigs seropositive to Coxiella were found in the province of Caserta. However, the univariate analysis highlighted the influence of age, location, and sex on exposure to SBV. The subsequent multivariate analysis statistically confirmed the importance of these factors. The presence of neutralizing antibodies for SBV and BVDV, or antibodies directed towards a specific phase of infection for Coxiella was further confirmed with virus-neutralization assays and phase-specific ELISAs in a large proportion of positive samples. The presence of high neutralizing antibody titers (especially for SBV) could indicate recent exposures. Twelve of the 17 positive samples tested positive for antibodies against Coxiella phase I or II antigens, indicating the presence of both acute and chronic infections (one animal tested positive for both phases antibodies)., Conclusions: Our study indicates a non-negligible exposure of pigs from southern Italy to the above pathogens. Further studies are necessary to fully understand the dynamics of these infections in pigs, the impact on productivity, and the public health consequences in the case of Coxiella., (© 2024. The Author(s).)

Bovine viral diarrhea virus (BVDV), is widely spread, poses a considerable risk of infection in the majority of dairy farms, causing respiratory, gastrointestinal, and reproductive problems. The aim of this study was to determine the seroprevalence and the risk variables associated with the seroprevalence of BVDV infection in cattle in four Egyptian governorates. A total of 680 blood samples were collected from cattle and examined for the presence of antibodies against BVDV using indirect ELISA (iELISA). Reproductive and management factors were considered, and epidemiological surveys were conducted. The total seroprevalence of BVDV in cattle was 18.24% (124/680) and it was significantly higher in females 19.66% (116/590), cattle older than 8 years 22.14% (62/280), dairy animals 22.65% (94/514), introduction of new animals to herd 21.39% (89/416), breeding with artificial insemination 28.46% (74/260), animals with history of abortion 28.76% (49/357), or during lactation stage 23% (89/387). The present findings suggest that BVD is prevalent in Egyptian dairy cattle and has an impact on farm productivity and production. Therefore, older, lactating, and aborted animals should also be identified for the disease, pose a risk of infection, and be handled appropriately., (© 2024 Japanese Society of Animal Science.)

Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR . However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV-host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA , with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR ), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Background: Between 2007 and 2011 several thousands of calves died from bovine neonatal pancytopenia (BNP), a bleeding syndrome triggered by vaccine induced alloantibodies from the dams. Following withdrawal of the involved bovine viral diarrhoea virus (BVDv) vaccine, the incidence of this condition rapidly decreased, with no reported cases in the last 5 years. Here, we report a recent immune-mediated pancytopenia in three calves from two different suckler herds, clinically indistinguishable from BNP., Case Presentation: Three Belgian Blue suckler calves from two different farms, aged around two weeks, showed multiple bleedings disseminated on the skin and petechiae and ecchymoses on the mucosae. Blood examination confirmed anaemia, leukopenia and thrombocytopenia. BVDv infection was excluded. Despite blood transfusion and cortisone therapy, all three animals died. Necropsy and histology confirmed bone marrow depletion. Binding of IgG from the dams on leukocytes of the calves was demonstrated by flow cytometry. Two calves, originating from the same farm, received colostrum from the same dam. None of the calves were given colostrum replacers or colostrum supplements. No link with the BNP causing BVDv vaccine could be evidenced. However, dams had been vaccinated against bovine herpesvirus 1, parainfluenza-3 virus, bovine respiratory syncytial virus and bluetongue virus serotype 8., Conclusions: Alloimmune mediated pancytopenia was evidenced in three animals, clinically and pathologically indistinguishable from BNP. Whether this disease is again vaccine mediated remains to be determined., (© 2021. The Author(s).)

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

Bovine viral diarrhea (BVD) is a major worldwide disease with negative economic impact on cattle production. Successful control programs of BVD require the identification and culling of persistently infected (PI) animals with bovine viral diarrhea virus (BVDV). A variety of diagnostic tests are available to detect BVDV, but no comparison has been performed among those tests in Argentina. Sera collected from 2864 cattle, belonging to 55 herds from three Argentinean provinces, were analyzed by nested RT-PCR (RT-nPCR) to detect BVDV for diagnostic purposes. Additionally, this study evaluated the agreement of the RT-nPCR along with virus isolation, antigen-capture ELISA, and real-time RT-PCR for BVDV detection in archived bovine serum samples (n = 90). The RT-nPCR was useful for BVDV detection in pooled and individual serum samples. BVDV was detected in 1% (29/2864) of the cattle and in 20% (11/55) of the herds. The proportion of BVDV-positive sera was not statistically different among the tests. In addition, comparisons showed high agreement levels, with the highest values between both RT-PCR protocols. The frequency of BVDV infection at individual and herd level was lower than the reported values worldwide. Since follow-up testing was not performed, the frequency of PI cattle was unknown. Also, this study demonstrated that the four diagnostic tests can be used reliably for BVDV identification in individual serum samples. Further epidemiologically designed studies that address prevalence, risk factors, and economic impact of BVDV in Argentina will be necessary to implement effective control programs.

Background: Bovine Viral Diarrhea virus (BVDV) is one of important diseases of cattle worldwide causing economic losses to the cattle industry primarily due to increased premature culling and decreased reproductive performance. The virus can cross the placenta during early pregnancy and result in the birth of persistently infected (PI) calves that are efficient transmitters of BVDV and serving as the primary reservoirs for BVDV. Relatively few studies have focused on understanding BVDV seroprevalence, virus detection, genotyping and its distribution in Africa. Most BVDV research in Ethiopia has involved serologic surveys in adult cattle, rather than the identification of PI calves, despite their role in viral shedding and recurring infections. A cross-sectional study was undertaken in three different livestock production systems of Ethiopia with the objective to estimate the prevalence of bovine abortion, calf mortality, and BVDV persistently infected calves., Results: Ear notch samples (882) collected from calves in 349 households were tested for BVDV antigen using antigen capture enzyme-linked immunosorbent assay (ACE). All samples tested were negative for BVDV antigen. The overall animal level crude abortion and calf mortality prevalence were 4.0% (95% CI: 2.9-5.2) and 9.2% (95% CI: 7.7-11.0) respectively. The lower BVDV PI prevalence may be due to a lower effective contact rate between cattle reared in small-scale extensive production systems in Ethiopia., Conclusions: This is the first report of BVDV Ag test in Ethiopia and no PI was detected in calves in the study areas. Since BVDV is a disease of great economic importance, this study finding must be interpreted with care since absence of evidence is not evidence of absence and even a single BVDV infected animal can serve as source of infection and contribute to the persistent spread of the virus. Greater attention needs to be given to screening for PI animals through testing large number of animals and culling positive animals. Hence, future research should focus on regions and production systems with high BVDV seroprevalence followed by antigen ELISA or BVDV real-time PCR to detect persistently infected and acutely viremic animals.

Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to d ifferentiate i nfected from v accinated a nimals (DIVA principle). Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing E rns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses "Ra", "Pro", and "RaPro" contained E rns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the "Pro" chimera replicated to high titers, while replication of the "Ra" chimera was limited. The "RaPro" chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV E rns antibody ELISA. In conclusion, the E rns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.

Bovine Viral Diarrhea Virus (BVDV) is an important pathogen that plays a significant role in initiating Bovine Respiratory Disease Complex (BRDC) in cattle. The disease causes multi-billion dollar losses globally due to high calf mortality and increased morbidity leading to heavy use of antibiotics. Current commercial vaccines provide limited cross-protection with several drawbacks such as safety, immunosuppression, potential reversion to virulence, and induction of neonatal pancytopenia. This study evaluates two prototype vaccines containing multiple rationally designed recombinant mosaic BVDV antigens for their potential to confer cross-protection against diverse BVDV strains. Genes encoding three novel mosaic antigens, designated E2 123 , NS2-3 1 , and NS2-3 2 , were designed in silico and expressed in mammalian cells for the formulation of a prototype protein-based vaccine. The mosaic antigens contain highly conserved protective epitopes from BVDV-1a, -1b, and -2, and included unique neutralizing epitopes from disparate strains to broaden coverage. We tested immunogenicity and protective efficacy of Expi293 TM -expressed mosaic antigens (293F-E2 123 , 293F-NS2-3 1 , and 293F-NS2-3 2 ), and baculovirus-expressed E2 123 (Bac-E2 123 ) mosaic antigen in calves. The Expi293 TM -expressed antigen cocktail induced robust BVDV-specific cross-reactive IFN-γ responses, broadly neutralizing antibodies, and following challenge with a BVDV-1b strain, the calves had significantly ( p < 0.05) reduced viremia and clinical BVD disease compared to the calves vaccinated with a commercial killed vaccine. The Bac-E2 123 antigen was not as effective as the Expi293 TM -expressed antigen cocktail, but it protected calves from BVD disease better than the commercial killed vaccine. The findings support feasibility for development of a broadly protective subunit BVDV vaccine for safe and effective management of BRD., (Copyright © 2020 Sangewar, Hassan, Lokhandwala, Bray, Reith, Markland, Sang, Yao, Fritz, Waghela, Abdelsalam, Chase and Mwangi.)

Background: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored., Results: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals., Conclusions: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

This experiment evaluated the impacts of administering a bovine appeasing substance (BAS) to beef calves at weaning on their performance, physiological responses, and behavior during a 42-d preconditioning program. Eighty calves (40 heifers and 40 steers; 90% British × 10% Nellore) were weaned at 233 ± 2 d of age (day 0); ranked by sex, weaning age, and body weight (BW); and assigned to receive BAS (IRSEA Group, Quartier Salignan, France; n = 40) or placebo (diethylene glycol monoethyl ether; CON; n = 40). Treatments (5 mL) were topically applied to the nuchal skin area of each animal following dam separation. Within treatment, calves were allocated to one of eight drylot pens (four pens per treatment; pen being the experimental unit) and received a free-choice total mixed ration (TMR) from day 0 to 42, intake of which was assessed daily. Live behavior observations were conducted on days 1, 2, 4, 8, 16, and 32. Temperament was assessed and blood samples were collected via jugular venipuncture on days -21, 0, 3, 7, 14, 28, and 42. Hair samples were collected from the tail switch on days 0, 14, 28, and 42. Calves were vaccinated against bovine respiratory disease viruses on days -21 and 0. Average daily gain from day 0 to 42 did not differ between treatments (P = 0.57) but was greater (P = 0.05) in BAS vs. CON calves from day 0 to 28. Intake of TMR was greater (P = 0.05) during the first week for BAS vs. CON calves (treatment × week; P = 0.08). The mean proportion of calves feeding simultaneously and performance of social and play behaviors were greater (P ≤ 0.05) for BAS vs. CON calves. Escape attempts were greater (P < 0.01) for BAS vs. CON calves on day 1 (treatment × day; P = 0.03). Exit velocity was greater (P = 0.04) for CON vs. BAS calves on day 14 and tended (P = 0.10) to be greater for CON vs. BAS calves on day 7 (treatment × day; P = 0.03). Mean plasma concentrations of haptoglobin were greater (P = 0.02) in CON vs. BAS calves. Hair cortisol concentrations were greater (P = 0.05) in CON vs. BAS calves on day 14 (treatment × day; P = 0.03). Mean serum concentrations of antibodies against bovine viral diarrhea virus were greater (P = 0.02) in BAS vs. CON calves. Collectively, BAS administration to beef calves at weaning alleviated stress-induced physiological reactions, improved temperament evaluated via chute exit velocity, enhanced humoral immunity acquired from vaccination, and appeared to have accelerated adaptation to novel management scheme and environment., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2 -ΔΔCq ; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.

Bovine coronaviruses (BoCVs) have been found in respiratory tissues in cattle and frequently associated with bovine respiratory disease (BRD); however, pathogenesis studies in calves are limited. To characterize the pathogenesis and pathogenicity of BoCV isolates, we used 5 different BoCV strains to inoculate colostrum-deprived calves, ~ 2-5 wk of age. Later, to determine if dual viral infection would potentiate pathogenicity of BoCV, calves were inoculated with BoCV alone, bovine viral diarrhea virus (BVDV) alone, or a series of dual-infection (BVDV-BoCV) schemes. A negative control group was included in all studies. Clinical signs and body temperature were monitored during the study and samples collected for lymphocyte counts, virus isolation, and serology. During autopsy, gross lesions were recorded and fixed tissues collected for histopathology and immunohistochemistry; fresh tissues were collected for virus isolation. Results suggest increased pathogenicity for isolate BoCV OK 1776. Increased body temperature was found in all virus-inoculated groups. Lung lesions were present in calves in all dual-infection groups; however, lesions were most pronounced in calves inoculated with BVDV followed by BoCV inoculation 6 d later. Lung lesions were consistent with mild-to-moderate interstitial pneumonia, and immunohistochemistry confirmed the presence of BoCV antigen. Our studies demonstrated that BVDV-BoCV dual infection may play an important role in BRD pathogenesis, and timing between infections seems critical to the severity of lesions.

Background: Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein., Methods: A recognition site for the viral proteinase 3C pro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain., Results: The rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0., Conclusion: A new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

The objective of this study was to provide a screening scheme of persistently infected (PI) cattle on dairy herds by combining reverse-transcription polymerase chain reaction (RT-PCR) to detect bovine viral diarrhea virus (BVDV) in milk tanker samples and commercial enzyme-linked immunosorbent assay to detect BVDV antibodies in bulk tank milk. We conducted a pilot survey and regional survey targeting all dairy farms in Ibaraki Prefecture by using milk tanker and bulk tank milk samples to screen PI cattle. Farms with positive samples underwent a follow-up test to identify PI cattle. In the pilot study, all virus-positive samples in bulk tank milk were included in the positive milk tanker samples. The RT-PCR assay successfully detected BVDV at dilutions of 1:1,600 by using two PI cows' milk. In the regional survey, 5 of 79 milk tanker samples were virus-positive. The virus was detected in three PI lactating cows and one PI calf on three farms. Antibody screening using bulk tank milk samples revealed 15 of 363 samples were positive, and 12 of 348 farms were BVDV antibody-positive. Follow-up tests on one farm identified three PI calves. Thus, eight PI cattle on five farms were identified in this study. In conclusion, combining BVDV detection using milk tanker samples and antibody detection using bulk tank milk is a feasible and economical method to efficiently screen PI cattle and confirm the PI-free status among dairy herds.

Members of the Pestivirus genus (family Flaviviridae ) cause severe and economically important diseases in livestock. Serological studies have revealed the presence of pestiviruses in different cervid species, including wild and semi-domesticated Eurasian tundra reindeer. In this retrospective study, serum samples collected between 2006 and 2008 from 3339 semi-domesticated Eurasian reindeer from Finnmark County, Norway, were tested for anti-pestivirus antibodies using an enzyme linked immunosorbent assay (ELISA) and a subset of these by virus neutralization test (VNT). A seroprevalence of 12.5% was found, varying from 0% to 45% among different herding districts, and 20% in western Finnmark, as compared to 1.7% in eastern Finnmark. Seroprevalence increased with age. Pestivirus-specific RNA was not detected in any of the 225 serum samples tested by real-time RT-PCR. Based on VNT results, using a panel of one bovine viral diarrhea virus (BVDV) strain and two border disease virus (BDV) strains, the virus is most likely a reindeer-specific pestivirus closely related to BDV. A characterization of the causative virus and its pathogenic impact on reindeer populations, as well as its potential to infect other domestic and wild ruminants, should be further investigated., Competing Interests: The authors declare no conflict of interest.

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Our previous study reported that persistently infected (PI) cattle of bovine viral diarrhea virus (BVDV) have co-infected with BVDV/END - and /END + that promote and inhibit host's type-I interferon (IFN) production, respectively. However, the relationship between co-infection of immunologically distinct BVDVs and persistent infection as well as the biological significance of END - viruses remains unknown. Experiments using cultured cells revealed that END + virus, which is unable to propagate in situations where the host's immune response is induced by IFN-α addition, is able to propagate under those conditions when co-infecting with END - virus. These results indicate that BVDV/END - can coexist with BVDV/END + and that co-infection with END - viruses supports the propagation of END + viruses. Our in vitro experiments strongly suggest that co-infection with END - virus is involved in the maintenance of persistent infection of BVDV., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effect of late-gestation vaccination of beef heifers with 2 doses of a killed-virus (KV) vaccine containing bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus 1 (BVDV-1), and bovine viral diarrhea virus 2 (BVDV-2) on the serum concentrations of antibody against BoHV-1, BVDV-1, and BVDV-2 in heifers and their calves and on the IgG concentration in the calves. Of the 47 pregnant beef heifers selected, 26 received 2 doses of the vaccine at 6.5 to 8 mo of gestation (at pregnancy check), and 21 received 2 doses of saline. The mean log 2 serum titers of neutralizing antibody against BoHV-1, BVDV-1, and BVDV-2 before vaccination did not differ significantly between the treatment groups; however, at calving all 3 mean titers were significantly greater ( P < 0.05) in the vaccinated heifers than in the control heifers. At 24 h after birth the mean serum IgG levels in the calves did not differ significantly between the 2 groups, at 30.18 and 32.28 g/L, respectively ( P < 0.05); however, the mean log 2 serum titers of antibody to all 3 viruses were greater in the calves nursing colostrum from the vaccinated heifers than in the calves nursing colostrum from the nonvaccinated heifers and significantly so for BoHV-1 and BVDV-1 ( P < 0.001 and P = 0.009, respectively). Thus, late-gestation vaccination of beef heifers could result in a greater and more consistent deposition of specific antibodies in colostrum, reducing the variability of initial titers in calves and increasing the duration of maternal immunity., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Smallholder large ruminant production in Lao People's Democratic Republic (Laos) is characterised by low reproductive efficiency. To determine if common abortifacient bovid infectious diseases are involved, a serological investigation was conducted. Sera was collected from stored and fresh cattle (n = 390) and buffalo (n = 130) samples from 2016-18 from, and then examined for associations in a retrospective risk factor study of 71 herds. The sera were assayed for antibodies to Neospora caninum, bovine viral diarrhoea virus (BVDV), Leptospira interrogans serovar Hardjo and Brucella abortus using commercially available enzyme-linked immunosorbent assay kits. These pathogens were detected in buffalo samples at 78.5% (95% CI 71.4-85.6), 0%, 2.3% (95% CI 0-4.9) and 0%, respectively, and in cattle at 4.4% (95% CI 2.4-6.4), 7.7% (95% CI 3.1-12.3), 12.8% (95% CI 9.5-16.1) and 0.26% (95% CI 0-0.8), respectively. Exposure of buffalo to N. caninum was positively associated with buffalo age, with a predicted seropositivity at birth of 52.8%, increasing to 97.2% by 12 years of age (p = 0.037). Exposure of cattle to L. interrogans serovar Hardjo was more prevalent in females compared to males, was associated with higher titres of BVDV, and was more prevalent in the wet season compared to the dry season. Exposure of cattle to BVDV was more prevalent in males compared to females, the wet and dry seasons were comparable, and was associated with rising antibody titres against N. caninum and L. interrogans serovar Hardjo. The risk factor survey identified that the probability of herds being N. caninum positive increased with farmer age, if farmers believed there were rodents on farm, and if farmers weren't aware that canids or rodents could contaminate bovid feed on their farm. The probability of a herd being positive to L. interrogans serovar Hardjo increased on farms where multiple cows shared the same bull, where farmers had lower husbandry knowledge, and on farms that used water troughs. The probability of a herd being BVDV seropositive increased with increasing herd size and increasing titres to N. caninum. The benchmarking of bovid exposure to emerging abortifacient pathogens and identification of their risk factors potentially informs disease prevention strategies, supporting efforts to establish a biosecure beef supply for enhanced smallholder livestock productivity, public health and food security in Laos and surrounding countries., Competing Interests: The authors have declared that no competing interests exist.

Determining the Bovine Viral Diarrhoea (BVD) infection status of cattle herds is a challenge for control and eradication schemes. Given the changing dynamics of BVD  virus (BVDV) antibody responses in cattle, classifying herds based on longitudinal changes in the results of BVDV antibody tests could offer a novel, complementary approach to categorising herds that is less likely than the present system to result in a herd's status changing from year to year, as it is more likely to capture the true exposure dynamics of the farms. This paper describes the dynamics of BVDV antibody test values (measured as percentage positivity (PP)) obtained from 15,500 bovines between 2007 and 2010 from thirty nine cattle herds located in Scotland and Northern England. It explores approaches of classifying herds based on trend, magnitude and shape of their antibody PP trajectories and investigates the epidemiological similarities between farms within the same cluster. Gaussian mixture models were used for the magnitude and shape clustering. Epidemiologically meaningful clusters were obtained. Farm cluster membership depends on clustering approach used. Moderate concordance was found between the shape and magnitude clusters. These methods hold potential for application to enhance control efforts for BVD and other infectious livestock diseases.

The goal of this study was to compare the cell-mediated immune responses of highly commingled, sale-barn origin calves (STR; n = 10) to those of single source calves that had been weaned for 60 d (UNS; n = 10). Peripheral blood mononuclear cells and neutrophils (PMNs) were isolated from jugular venous blood of each calf. Peripheral blood mononuclear cells were stimulated with Concanavalin A (ConA), BVDV-1, BVDV-2, BHV-1, Mannheimia haemolytica, and Pasteurella multocida and evaluated for clonal proliferation and secretion of IL-8 into cell culture supernatants. The native functional capacities of PMNs were evaluated in response to stimulation with heat-killed Escherichia coli and Staphylococcus aureus. Complete blood counts and serum biochemical profiles were performed for each animal at the time of sample collection. Compared with STR calves, UNS calves had greater lymphocyte proliferative responses following stimulation BVDV1 (P = 0.041), BVDV2 (P = 0.002), BHV-1 (P = 0.001), M. haemolytica (P = 0.016), and P. multocida (P = 0.049). In addition, PMNs isolated from UNS calves had a greater ability to phagocytose E. coli (P = 0.001) and S. aureus (P = 0.003) when compared with STR calves. Serum nonesterified fatty acids were higher in STR calves (P < 0.001). Serum β-hydroxybutyrate was lower in STR calves (P < 0.003). These data suggest that immunologic and physiologic differences exist between STR and UNS calves. Although the underlying mechanisms for these differences are not clear, it is possible that combinations of energy imbalances, stress-induced immunosuppression, and general immune naiveté may predispose STR calves to an increased risk of morbidity and mortality due to bovine respiratory disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies ( p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer's patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 ( p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

This is a cross-sectional study to assess the presence of antibodies in ruminants against selected pathogens associated with reproductive disorders in cattle in four Brazilian states, including the zoonotic agent Coxiella burnetii. The used tests were Virus Neutralization Assay for IBR and BVD, Microscopic Agglutination Test for Leptospira spp., Indirect Fluorescent Antibody Test (IFAT) for C. burnetii and Toxoplasma gondii, and Enzyme-Linked Immunosorbent Assay for Neospora caninum and Trypanosoma vivax. Seropositivity for C. burnetii was 13.7% with titers from 128 to 131,072; 57.8% for BoHV-1, with titers between 2 and 1,024; 47.1% for BVDV-1a, with titers from 10 to 5,120; 89.2% for N. caninum; 50% for T. vivax; and 52.0% for Leptospira spp., with titers between 100 to 800 (the following serovars were found: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona and Icterohaemorrhagiae); 19.6% for T. gondii with titer of 40. This is the first study that has identified C. burnetii in cattle associated with BoHV and BVDV, N. caninum, Leptospira spp., T. gondii and T. vivax. Thus, future studies should be conducted to investigate how widespread this pathogen is in Brazilian cattle herds.

Recombinant herpesvirus vaccine vectors offer distinct advantages in next-generation vaccine development, primarily due to the ability to establish persistent infections to provide sustainable antigen responses in the host. Recombinant bovine herpesvirus-4 (BoHV-4) has been previously shown to elicit protective immunity in model laboratory animal species against a variety of pathogens. For the first time, we describe the induction of antigen-specific immune responses to two delivered antigens in the host species after intranasal nebulization of recombinant BoHV-4 expressing the chimeric peptide containing the bovine viral diarrhea virus (BVDV) glycoprotein E2 and the bovine herpesvirus 1 (BoHV-1) glycoprotein D (BoHV-4-A-CMV-IgK-gE2gD-TM). In this study, four cattle were immunized via intranasal nebulization with the recombinant BoHV-4 construct. Two of the cattle were previously infected with wild-type BoHV-4, and both developed detectable serologic responses to BVDV and BoHV-1. All four immunized cattle developed detectable viral neutralizing antibody responses to BVDV, and one steer developed a transient viral neutralizing response to BoHV-1. Approximately one year after immunization, immunosuppressive doses of the glucocorticoid dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle., Competing Interests: The authors have declared that no competing interests exist.

The aims of this study were to describe when and how vaccines are administered during the production cycle in cow-calf herds in western Canada, as well as the factors that influence vaccine usage as reported by producers. The most commonly used vaccines were bovine viral diarrhea virus/infectious bovine rhinotracheitis (BVDV/IBR) in adult animals and clostridial vaccines in calves. While there has been improvement in usage of reproductive and respiratory viral vaccines since previous studies, there are still several areas in which uptake could be improved. Only 72% of herd owners vaccinated their bulls for at least 1 disease. Not all producers are vaccinating their calves for clostridial diseases, and 15% of producers did not vaccinate their calves for respiratory disease before weaning. One goal of increasing vaccine use is to obtain better infection prevention and control and decrease antimicrobial use in cow-calf herds. Two areas in which antimicrobials are commonly used, but vaccine uptake is limited, are foot rot in adult cows and diarrhea in calves.

Background: No comprehensive studies have been carried out on the infectious causes of abortion in Kenyan dairy cattle herds. A survey was carried out to determine the seroprevalence of antibodies against Bovine Viral Diarrhoea Virus (BVDV), Brucella abortus (BA) and Neospora caninum (NC) among dairy cattle herds in Nakuru County, a major dairying area in Kenya. A prospective sero-epidemiological study was also undertaken to investigate the effects of BVDV, BA and NC on the occurrence of bovine abortion in dairy cattle herds, where monthly rectal palpations for pregnancy were performed, and monthly serum samples were tested for antibodies to the 3 pathogens., Results: In the 398 randomly selected cattle on 64 dairy herds, the seroprevalences of antibodies to BVDV, NC and BA were 79.1, 25.6 and 16.8%, respectively. Of the cattle seropositive to NC, 83.3% were also seropositive to BVDV and 13.7% to BA. Of the cattle seropositive to BVDV, 17.1% were also seropositive to BA. Among 260 monitored pregnant dairy cattle on the same 64 dairy farms, an incidence risk for abortion of 10.8% (28/260) was identified, while the incidence of other foetal losses was 1.1% (3/260). The incidence rates of sero-conversion for NC, BVD and BA were 1.1, 0.06 and 0.5 new infections/100 cow-months at risk, respectively. The foetal losses were mainly observed in animals less than 96 months old and occurred in mid-gestation. Neospora caninum was associated with most cases (29.0%) of foetal losses, followed by mixed infections of NC and BVDV (12.9%), BVDV (9.9%) and co-infections of BA and NC (6.5%)., Conclusions: This is the first study to document the substantial incidence risk of BVDV and NC abortions in dairy cattle in Kenya, and demonstrates the relative importance of BA, BVDV and NC infections in dairy cattle in Kenya. Kenya laboratories should offer diagnostic tests for BVDV and NC to help farmers determine their roles in abortions on their farms. A comprehensive policy on the control of these important diseases should also be put in place by government with the involvement of all stakeholders in the dairy cattle industry.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5-10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

OBJECTIVE To evaluate cell-mediated and humoral immune responses of calves receiving 2 doses of a dual-adjuvanted vaccine containing inactivated bovine herpesvirus type 1 (BHV1) and bovine viral diarrhea virus types 1 (BVDV1) and 2 (BVDV2) before and after exposure to BHV1. ANIMALS 24 Holstein steers negative for anti-BHV1 antibodies and proliferative cell-mediated immune responses against BHV1 and BVDV. PROCEDURES Calves were randomly assigned to 3 groups. The vaccinated group (n = 10) received 2 doses of vaccine on days 0 and 21. Control (n = 10) and seeder (4) groups remained unvaccinated. Calves were commingled during the study except for the 3-day period (days 53 to 55) when seeders were inoculated with BHV1 (1.04 × 10 7 TCID 50 , IV) to serve as a source of virus for challenge (days 56 through 84). Rectal temperature and clinical illness scores were monitored, and blood and nasal specimens were obtained for determination of clinicopathologic and immunologic variables. RESULTS After BHV1 challenge, mean rectal temperature and clinical illness scores were lower for vaccinates than controls. In vaccinates, antibody titers against BHV1 and BVDV2, but not BVDV1, increased after challenge as did extracellular and intracellular interferon-γ expression, indicating a T helper 1 memory response. Additional results of cell marker expression were variable, with no significant increase or decrease associated with treatment. CONCLUSIONS AND CLINICAL RELEVANCE Calves administered 2 doses of a killed-virus vaccine developed cell-mediated and humoral immune responses to BHV1 and BVDV, which were protective against disease when those calves were subsequently exposed to BHV1.

The antibody seroprevalence of young stock can be a useful indicator of recent or current infection in a herd. We examine the factors that contribute to the assessment of herd exposure to disease, via spot testing for antibody, using bovine viral diarrhoea virus (BVDv) as an example. A statistical distribution of seroprevalences for BVDv in beef herds identified three groups of herds: low, intermediate and high within-herd BVDv antibody seroprevalence. We tested two assumptions -the intermediate seroprevalence group of herds is assumed to be negative for BVDv at the herd level and alternatively if this group is assumed to be positive. We found that: The herd-level sensitivity and specificity are sensitive to the assumption regarding the herds with intermediate seroprevalence. If an appropriate cut-point is chosen, reducing the sample size from ten to five does not produce a large drop in herd-level test performance. Increasing the cut-point may be valuable at the outset of an eradication programme. Increasing the sample size and decreasing the cut-point is advantageous towards the end of an eradication programme, to minimise the risk of positive herds being misclassified. The framework presented here illustrates how seroprevalence screening may be understood and assessed.

Bovine viral diarrhea virus (BVDV) is the etiological agent of BVD causes substantial economic losses and endemic in world-wide cattle population. Mucosal immunity plays an important role in protection against BVDV infection and Lactobacillus casei is believed as an excellent live vaccine vector for expressing foreign genes. In this study, we have constructed a novel recombinant L. casei/pELX1-E2 strain expressing the most immunogenic E2 antigen of BVDV; using growth phage dependent surface expression system pELX1. The expression of E2 protein was verified by SDS-PAGE, Western blotting, and Immunofluorescence microscopic analysis. The immune responses triggered by the E2 producing recombinant L. casei were evaluated in BALB/c mice revealed that oral and intranasal (IN) administration of the recombinant strain was able to induce a significantly higher level of specific anti-E2 mucosal IgA and serum IgG as well as the greater level of cellular response by IFN-γ and IL-12 than those of intramuscular (IM) and control groups of mice. However, IN inoculation was found the most potent route of immunization. The ability of the recombinant strain to induce serum neutralizing antibody against BVDV and reduced viral load after viral challenge indicated better protection of BVDV infection. Therefore, this recombinant L. casei expressing E2 could be a safe and promising mucosal vaccine candidate against BVD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) 1b was isolated from tissues of a term bovine fetus with petechial hemorrhages noted throughout the body and placenta at autopsy. Fresh lung, kidney, thymus, and liver tissues were examined by direct fluorescent antibody testing and were positive for BVDV antigen and negative for bovine herpesvirus 1 antigen. An organ pool of fresh tissues was positive for noncytopathic (NCP) BVDV-1 by virus isolation. BVDV-1b was identified by sequencing of the 5'-UTR region of the genome. Fixed brain, placenta, thymus, lymph node, lung, kidney, skeletal muscle, liver, and bone marrow were positive for BVDV antigen by immunohistochemistry. Although BVDV hemorrhage and/or thrombocytopenia has been associated historically with NCP strains of BVDV-2, this case adds to more recent reports of BVDV-1 infections and hemorrhage in cattle. This BVDV-1b isolate should be investigated for its potential to cause hemorrhage in postnatal cattle.

Pestiviruses are enveloped viruses that bud intracellularly. They have three envelope glycoproteins, E rns , E1, and E2. E2 is the receptor binding protein and the main target for neutralizing antibodies. Both E rns and E2 are retained intracellularly. Here, E2 of the bovine viral diarrhea virus (BVDV) strain CP7 was used to study the membrane topology and intracellular localization of the protein. E2 is localized in the ER and there was no difference between E2 expressed alone or in the context of the viral polyprotein. The mature E2 protein was found to possess a single span transmembrane anchor. For the mapping of a retention signal CD72-E2 fusion proteins, as well as E2 alone were analysed. This confirmed the importance of the transmembrane domain and arginine 355 for intracellular retention, but also revealed a modulating effect on retention through the cytoplasmic tail of the E2 protein, especially through glutamine 370. Mutants with a strong impact on retention were tested in the viral context and we were able to rescue BVDV with certain mutations that in E2 alone impaired intracellular retention and lead to export of E2 to the cells surface.

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

The objective of this study was to determine if precolostral blood samples are useful to detect apparent fetal infections with bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses. A convenience sample of 317 sera from 50 Canadian herds was used in the study. Antibody level was measured using 2 commercial IBR and BVD ELISA kits. Precolostral status of sera was confirmed on 304 samples using serum gamma-glutamyl transferase activity. Postcolostral serum samples yielded a higher proportion of positive results to IBR (OR = 86; 95% CI: 17.8 to 415.7) and BVD (OR = 199.3; 95% CI: 41.7 to 952.3) than did precolostral samples. All positive precolostral serum samples ( n = 7 of 304) originated from calves born to vaccinated cows. Postcolostral positive serum samples ( n = 11 of 13) originated mostly (60%) from calves born to non-vaccinated cows. Precolostral serum sampling can detect apparent fetal infections in a herd.

Effective control and the eventual eradication of Bovine viral diarrhea virus (BVDV) from cattle populations depend on the accurate identification of infected animals. Although typically a disease agent of cattle, BVDV is known to infect a wide variety of nonbovine species, including sheep. However, validation of serologic tests in these nonbovine species, particularly sheep, is lacking. We analyzed 99 sheep sera (57 samples from Pestivirus-naive sheep, and 42 samples from BVDV-inoculated sheep) in order to investigate 3 serologic tests: the agarose gel immunodiffusion (AGID) and 2 commercial enzyme-linked immunosorbent assays (ELISAs) for detection of BVDV antibodies. At the manufacturer's cutoff thresholds, the AGID performed with 95.2% diagnostic sensitivity; ELISA-A performed with sensitivity of 90.5% and ELISA-B with 69.1%. All 3 tests performed with 100% diagnostic specificity. Two-graph receiver operating characteristic analysis showed that performance characteristics were optimized, such that both diagnostic sensitivity and diagnostic specificity were >95% for both ELISAs, if the thresholds were altered to 34.9% inhibition for ELISA-A and 63.5 signal-to-noise ratio for ELISA-B.

Four species of ruminant pestivirus are currently circulating in the United States: Bovine viral diarrhea virus 1 and 2 (BVDV-1, -2; predominant host: cattle), Border disease virus (BDV; predominant host: sheep), and pronghorn virus (sporadically detected in wild ruminants). A third bovine pestivirus called HoBi-like virus has been detected in cattle and water buffalo in South America, Asia, and Europe. To date, no isolations of HoBi-like viruses from U.S. cattle have been reported. To assess exposure, 2,000 cattle sera, collected between 2014 and 2015 as part of the U.S. brucellosis surveillance program, were tested for antibodies against BVDV-1, BVDV-2, and HoBi-like viruses. In addition, RNA was extracted and tested by reverse transcription-polymerase chain reaction for the presence of pestiviruses; all samples tested negative. The percent of VN-positive samples was 91.3% for BVDV-1, 89.3% for BVDV-2, and 84.9% for HoBi-like viruses. Because the 3 bovine pestiviruses are antigenically cross-reactive, the comparative level of antibody against each pestivirus species was determined. Based on comparative titers, samples were segregated into 6 categories: no titers (7.6%), titers clearly higher against BVDV-1 (22.2%), titers substantially higher against BVDV-2 (9.1%), BVDV-1 and BVDV-2 titers equivalent but substantially higher than HoBi titers (25.7%), titers substantially higher against HoBi-like viruses (0%), and equivocal (35.4%). Titers tended to be higher against BVDV-1 than BVDV-2. However, the overall percentage of animals with titers below levels considered protective against acute bovine pestivirus infection were ~11% for BVDV-1, 12% BVDV-2, and 18% for HoBi-like virus.

Background: This paper examines the use of Bulk Milk antibody (BM Ab), Youngstock (YS) serology (Check Tests) and Bulk Milk PCR (BM PCR) for determining the presence or absence of animals persistently infected (PI) with Bovine Viral Diarrhoea Virus (BVDV) within a herd. Data is presented from 26 herds where average herd sizes were 343 and 98 animals for dairy and beef units respectively. Seventeen herds had sufficient data to analyse using Receiver Operating Characteristic (ROC) and probability curves enabling calculation of the sensitivity and specificity of BM Ab and YS Check tests for determining the presence of PI animals within herds in this dataset., Results: Using BM Ab to screen a herd for the presence of PI animals, achieved a herd level sensitivity and specificity of 80.00 % (44.39-97.48 %) and 85.71 % (42.13-99.64 %) respectively (95 % confidence intervals quoted). Sensitivity and specificity of YS Check Tests at a cut off of 3/10 Ab positive YS were 81.82 % (48.22-97.72 %) and 66.67 % (22.28-95.67 %) respectively (95 % confidence interval). These results were achieved by comparing the screening tests to whole herd PI searches that took place 1-19 months after the initial screen with a mean interval of 8 months. Removal of this delay by taking BM samples on the day of a whole herd test and simulating a YS Check Test from the herd test data produced improvements in the reliability of the Check Tests. BM Ab sensitivity and specificity remained unchanged. However, the Check Test sensitivity and specificity improved to 90.9 % (58.72-99.77 %) and 100 % (54.07-100 %) respectively (95 % confidence interval) at a cut of off 2.5/10 Ab positive animals. Our limited BM PCR results identified 5/23 dairy farms with a positive BM PCR result; two contained milking PIs, two had non-milking PIs and another had no PIs identified., Conclusions: Delaying a PI search following an initial herd screen decreased the diagnostic accuracy and relevance of our results. With careful interpretation, longitudinal surveillance using a combination of the techniques discussed can successfully determine farm status and therefore allow changes in BVDV status to be detected early, thus enabling prompt action in the event of a BVDV incursion.

Antigen enzyme-linked immunosorbent assay (ELISA) is used for the detection of Bovine viral diarrhea virus persistently infected (BVDV PI) cattle; however, colostrum-derived antibodies may interfere with antigen detection in serum from young PI calves. Our study aimed to assess serum pretreatment methods for reducing such interference. Dilution of PI serum with serum containing specific antibody showed that antibody levels equivalent to those observed in colostrum-fed calves were able to eliminate all antigen signals in a serum sample. Serum was treated with ethylenediamine tetra-acetic acid at pH 4.5, 5.5, 6.5, and 7.5, then boiled, centrifuged, and the supernatant-recovered. BVDV antibody was undetectable by ELISA in supernatants from treated samples, and the antigen ELISA signal was improved. Maximum antigen signal recovery of >90% was achieved at pH 5 ± 0.5. When this optimal treatment method was applied to field samples from 3 PI calves (which were negative in the antigen-capture ELISA without treatment), the antigen signal improved and gave a positive result in each case. Pretreatment may provide an improvement in the detection of young PI calves., (© 2016 The Author(s).)

The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family.

Classical swine fever (CSF) is a highly contagious systemic hemorrhagic viral disease of pigs. Wild boar plays a crucial role in the epidemiology of CSF. Between 2010 and 2014, samples were collected nationwide from 6,654 wild boars hunted in South Korea. Anti-CSF antibodies were identified in 0.59% (39 of 6,654) of the wild boar samples using a virus neutralization test and were primarily detected in wild boars living close to the demilitarized zone and the area of the Taebaek Mountains surroundings. The CSF virus (subgroup 2.1b) was isolated from two wild boars captured in a nearby border area. The criteria used to define high-risk areas for targeted CSF surveillance in South Korea should be further expanded to include other regions nationwide.

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5'-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.

Objective: To evaluate the efficacy of 4 commercially available multivalent modified-live virus vaccines against clinical disease, viremia, and viral shedding caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BHV1) in early-weaned beef calves., Animals: 54 early-weaned beef steers (median age, 95 days)., Procedures: Calves were randomly assigned to 1 of 5 groups and administered PBSS (group A [control]; n = 11) or 1 of 4 commercially available modified-live virus vaccines that contained antigens against BHV1, BVDV types 1 (BVDV1) and 2 (BVDV2), parainfluenza type 3 virus, and bovine respiratory syncytial virus (groups B [11], C [10], D [11], and E [11]). Forty-five days after vaccination, calves were exposed simultaneously to 6 cattle persistently infected with BVDV and 8 calves acutely infected with BHV1 for 28 days (challenge exposure). For each calf, serum antibody titers against BVDV and BHV1 were determined before vaccination and before and after challenge exposure. Virus isolation was performed on nasal secretions, serum, and WBCs at predetermined times during the 28-day challenge exposure., Results: None of the calves developed severe clinical disease or died. Mean serum anti-BHV1 antibody titers did not differ significantly among the treatment groups at any time and gradually declined during the study. Mean serum anti-BVDV antibody titers appeared to be negatively associated with the incidence of viremia and BVDV shedding. The unvaccinated group (A) had the lowest mean serum anti-BVDV antibody titers. The mean serum anti-BVDV antibody titers for group D were generally lower than those for groups B, C, and E., Conclusions and Clinical Relevance: Results indicated differences in vaccine efficacy for the prevention of BVDV viremia and shedding in early-weaned beef calves.

Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 μg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 μg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213-500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.

A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.