Search

Daily milk production and reproductive performance of cows vaccinated with a live double-deleted Bovine Viral Diarrhoea Virus (BVDV) vaccine were compared to those of non-vaccinated cows, cohabitating in endemic BVDV herds. All animals in the treatment group were vaccinated on study day 0 irrespective of lactation or gestation status, while control animals did not receive any treatment. 1463 animals were enrolled in the study from four different farms in three different countries (UK, Italy, France). Endemic presence of BVDV in study herds was demonstrated by the detection of BVDV in the bulk tank milk, and seroconversion was evaluated at the beginning of the study. For individual animals, the day of calving was taken to be the start of lactation for the calculation of days in milk (DIM). The standard lactation period of 305 days was divided into three periods: early lactation (EL, from DIM 8 to DIM 102), mid lactation (ML, from DIM 103 to DIM 204 and late lactation (LL, from DIM 205 to DIM 305). For each farm and each lactation period, a mixed model statistical analysis was performed with daily milk production as response, and group, day as well as the interaction between those two factors as fixed factors. Chi-square test was used to compare abortion rate and prolonged inter-oestrous interval rate between treatment and control groups. A significant increase in milk production in the vaccinated group was observed in farms 1 (1.023 L/day) and 3 (0.611 L/day) during EL (p<0.001) and in farm 2 (1.799 L/day) during ML (P<0.001). In addition, at farm 2, vaccinated cows produced more milk than non-vaccinated cows starting from 80 DIM. No differences were found between groups in abortion rates or prolonged inter-oestrous interval rates. Data demonstrate that cows in herds endemically infected with BVDV and vaccinated with live double-deleted BVDV vaccine produce more milk; the difference in milk production occurs during early lactation., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Lucy Metcalfe and Christian Guidarini are both employees of Boehringer Ingelheim Vetmedica GmbH. The funder provided support in form of salaries for 2 of the authors (CG and LM) and in sponsoring of the study, but this does not alter our adherence to PLOS ONE policies on sharing data and materials.

To determine the nationwide prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in China, 92 dairy farms with more than 500 animals in 19 provinces of China were surveyed in 2017. At each farm, ear notch samples from calves less than six months old and bulk tank milk (BTM) samples were collected. A total of 901 ear notch samples and 329 BTM samples from 183 tanks were sampled. A total of 20 (20/901, 2.22 %) ear notch samples from 10 (10/92, 10.86 %) farms tested positive for BVDV by IDEXX Antigen Point-of-Care (POC) Test kit and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, 80 of 183 (80/183, 43.7 %) BTM samples from 43 (43/92, 46.7 %) farms were identified as positive by qRT-PCR. The RNA of positive and suspect samples identified by qRT-PCR was subjected to 5'- untranslated region (UTR) amplification by nested RT-PCR and then sequenced. A total of 119 sequences were obtained and phylogenetic analysis of these 5'-UTR sequences revealed the presence of eight different subgenotypes of BVDV-1 including 1a (n = 37, 31.09 %), 1b (n = 5, 4.20 %), 1c (n = 34, 28.57 %), 1d (n = 2, 1.68 %), 1m (n = 25, 21.01 %), 1q (n = 6, 5.04 %), and two unknown subgenotypes which were tentatively typed as "BVDV-1v" (n = 8, 6.72 %) and "BVDV-1w" (n = 2, 1.68 %), respectively. BVDV-1a, 1c, and 1m were the dominant strains, collectively accounting for 80.67 % (96/119) of all sequences. Phylogenetic analysis based on selected N-terminal autoprotease (N pro ) sequences confirmed the classification of the 5'-UTR sequences. In conclusion, the prevalence of BVDV persistent infection in dairy cattle was high and genetic diversity was high and increasing, revealing a serious threat to the health of cattle in China and highlighting the need for BVDV control., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest associated with this report., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Bovine viral diarrhea (BVD) is a viral disease that affects cattle and that is endemic to many European countries. It has a markedly negative impact on the economy, through reduced milk production, abortions, and a shorter lifespan of the infected animals. Cows becoming infected during gestation may give birth to Persistently Infected (PI) calves, which remain highly infective throughout their life, due to the lack of immune response to the virus. As a result, they are the key driver of the persistence of the disease both at herd scale, and at the national level. In the latter case, the trade-driven movements of PIs, or gestating cows carrying PIs, are responsible for the spatial dispersion of BVD. Past modeling approaches to BVD transmission have either focused on within-herd or between-herd transmission. A comprehensive portrayal, however, targeting both the generation of PIs within a herd, and their displacement throughout the country due to trade transactions, is still missing. We overcome this by designing a multiscale metapopulation model of the spatial transmission of BVD, accounting for both within-herd infection dynamics, and its spatial dispersion. We focus on Italy, a country where BVD is endemic and seroprevalence is very high. By integrating simple within-herd dynamics of PI generation, and the highly-resolved cattle movement dataset available, our model requires minimal arbitrary assumptions on its parameterization. We use our model to study the role of the different productive contexts of the Italian market, and test possible intervention strategies aimed at prevalence reduction. We find that dairy farms are the main drivers of BVD persistence in Italy, and any control strategy targeting these farms would lead to significantly higher prevalence reduction, with respect to targeting other production compartments. Our multiscale metapopulation model is a simple yet effective tool for studying BVD dispersion and persistence at country level, and is a good instrument for testing targeted strategies aimed at the containment or elimination of this disease. Furthermore, it can readily be applied to any national market for which cattle movement data is available., (Copyright © 2019. Published by Elsevier B.V.)

In a cross-sectional field study involving 51 cattle herds in Belgium, 3159 serum samples and 557 individual milk samples were collected and tested by four different commercial antibody (Ab) ELISAs on serum and two Ab ELISAs on milk. A virus neutralization test (VNT) was performed on serum samples with discording ELISA results and on all samples from non-vaccinating herds. An epidemiological survey was carried out in the same herds to collect information about herd characteristics, management practices, BVD vaccination and BVD infection status. The objective of the study was to evaluate the performances of the Ab ELISAs relatively to the VNT, to assess the possibility of using pooled samples and to give recommendations regarding serological monitoring of BVD-free herds in the context of the Belgian national BVD eradication program which started early 2015. Depending on the assays, for ELISAs on serum, the diagnostic sensitivity (DSe) was estimated to be between 93.0 and 98.7% and the diagnostic specificity (DSp) between 94.3% and 99.1%. For the two ELISAs on milk, the DSe were 91.3% and 96.7% and the DSp 94.0% and 100% respectively and the Cohen's agreement coefficients between serum and milk samples were 0.75 and 0.85. Positive serum and milk samples diluted in negative samples to mimic different pool sizes were not detected by all ELISAs at dilutions above 1:5 or 1:10, leading to the conclusion that the testing of pooled samples should be used cautiously for serological monitoring and only with ELISAs with high sensitivity. The epidemiological analysis and the seroprevalence study, based on a general estimating equation model, showed that several factors had a significant influence on overall animal seroprevalence and within-herd seroprevalence such as age class, herd size, BVD herd infection status, BVD vaccination of young and/or adult cattle and the number of stables in the farm. This study showed that the best performances obtained with commercial Ab ELISAs are observed on individual serum samples, which should therefore be the preferred matrix to monitor BVD-free herds in the context of the Belgian eradication program. By regularly testing a limited number of samples from young (6-18 months) unvaccinated cattle it is possible to confirm the BVD-free herd status or to detect a recent infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Bovine Viral Diarrhea Virus causes significant economic losses in cattle. BVDV has high genomic diversity, with two species, BVDV-1 and BVDV-2, and at least twenty-one subgenotypes for BVDV-1 and four subgenotypes for BVDV-2. Vaccines are important tools to reduce the economic losses caused by this virus. However, vaccine strains must correspond to the antigenic profile of the viruses present in the region where the vaccine is applied. A restricted phylogenetic study with 14 viruses isolated from cattle between 1993 and 2001 showed that the genetic profile of BVDV in Chile consisted of viruses of both species and sub-genotypes 1a, 1b, 1c (currently 1j) and 2a. To determine more accurately the genetic profile of BVDV in Chile, in this study a larger number of viruses obtained from bovines between 2003 and 2007 were typed., Results: The study was performed using partial sequences from the 5' noncoding region (5'UTR) and E2 coding region of the viral genome of thirty-five Chilean viruses isolated from geographic regions that have 84.6% of the Chilean cattle. All tested viruses belonged to species BVDV-1. Eighteen viruses belonged to BVDV-1j subgenotype (51.4%), twelve belonged to BVDV-1b (34.3%) and five belonged to BVDV-1a (14.3%). The Chilean BVDV-1j viruses showed low genetic diversity, both among themselves and with the BVDV-1j present in other regions of the world. This could be explained by a relatively recent introduction of this viral subgenotype in cattle, which agrees with its low geographical distribution worldwide. Otherwise, Chilean BVDV-1b viruses grouped into a single cluster, different even than the viruses present in Argentina and Brazil, countries geographically close to Chile, a process of local evolution that could generate antigenic differences between the Chilean viruses and the viruses used as vaccine strains., Conclusions: The high presence of viruses of the BVDV-1j subgenotype, which show major antigenic differences with BVDV-1a and BVDV-1b subgenotypes used in the commercial vaccines, suggest that BVDV-1j viruses could be an emergent subgenotype of BVDV in cattle in South America and suggest evaluating an update of the vaccines used in Chile.

Hobi-like viruses (HobiPeV) comprise a novel, recently classified species of bovine pestiviruses, originally identified in commercial fetal bovine serum of Brazilian origin and, subsequently, isolated from diseased animals in several countries. Although frequently isolated from clinical cases, most HobiPeV isolates failed to reproduce overt disease in cattle upon experimental inoculation. Herein, we describe the outcome of experimental infection of four to six months-old seronegative calves with two Brazilian HobiPeV isolates. Calves inoculated intranasally with isolate SV478/07 developed viremia between days 2 and 9 post-inoculation (pi) and shed virus in nasal secretions up to day 11pi. These animals presented hyperthermia (day 7 to 10-11 pi) and lymphopenia from days 4 to 8pi. Clinically, all four calves developed varied degrees of apathy, anorexia, mild to moderate respiratory signs (nasal secretion, hyperemia), ocular discharge and pasty diarrhea in the days following virus inoculation. In contrast, calves inoculated with isolate SV757/15 presented only hyperthermia (days 3 to 10-11 pi) and lymphopenia (days 4-8 pi), without other apparent clinical signs. In these animals, viremia was detected up to day 9 pi and virus shedding in nasal secretions lasted up to day 12-14 pi. Both groups seroconverted to the inoculated viruses, developing virus neutralizing (VN) titers from 320 to 5120 at day 28pi. These results extend previous findings that experimental infections of calves with HobiPeV are predominantly mild, yet they also indicate that field isolates may differ in their ability to cause disease in susceptible animals., (Copyright © 2018. Published by Elsevier Ltd.)

A random effect meta-analysis was performed to estimate the worldwide pooled bovine viral diarrhoea virus (BVDV) prevalences of persistently infected (PI), viraemic (VI) and antibody-positive (AB) animals and herds. The meta-analysis covered 325 studies in 73 countries that determined the presence or absence of BVDV infections in cattle from 1961 to 2016. In total, 6.5 million animals and 310,548 herds were tested for BVDV infections in the global cattle population. The worldwide pooled PI prevalences at animal level ranged from low (≤0.8% Europe, North America, Australia), medium (>0.8% to 1.6% East Asia) to high (>1.6% West Asia). The PI and AB prevalences in Europe decreased over time, while BVDV prevalence increased in North America. The highest mean pooled PI prevalences at animal level were identified in countries that had failed to implement any BVDV control and/or eradication programmes (including vaccination). Our analysis emphasizes the need for more standardised epidemiological studies to support decision-makers implementing animal health policies for non-globally-regulated animal diseases.

Background: Farmed and feral water buffaloes (Bubalus bubalis) populations often coexist with cattle in the Northern Territory of Australia, but their level of exposure to bovine viral diarrhoea virus (BVDV) is unknown., Methods: Water buffalo (n = 245) and cattle (n = 184) serum samples were collected by the NT Government as part of an ongoing disease surveillance scheme at varying intervals between 1993 and 2001. All samples were frozen and stored at -80°C until testing. Water buffalo samples from farming properties were identified as 'farmed' animals and the remaining samples as 'feral' populations. Serum samples were analysed using commercially available ELISAs to test for the presence of BVDV antibodies., Results: Testing of historical water buffalo sera for BVDV antibodies revealed a low level of exposure, with 4.5% (95% CI ± 2.6%) being sero-positive; cattle from the same geographical area and time period had higher levels of exposure at 74.5% (95% CI ± 6.3%)., Discussion: This survey showed that water buffalo are susceptible to infection with BVDV. No persistently infected water buffalo were identified in this study., (© 2016 Australian Veterinary Association.)

The objective of this study was to investigate the impact of oral meloxicam (MEL) and long-distance transportation on cell-mediated immunity (CMI) in preconditioned steers receiving a booster vaccination on arrival. We hypothesized that steers treated with MEL at 1mg/kg body weight, 6h before night-time transport, would be less immunocompromised on arrival (day 0) and after 7days, and that CMI following vaccination with a modified live bovine viral diarrhea virus (BVDV) recall antigen would be increased. Brahman crossbreed steers, 13-17 months of age (n=87), were randomly assigned to one of four treatment groups: MEL, transported (MTR) (n=22), MEL, non-transported (MNT) (n=22), lactose placebo, transported (CTR) (n=21), and lactose placebo, non-transported (CNT) (n=22). MTR and CTR steers were transported for approximately 16h non-stop on a truck from Mississippi to Iowa (approximately 1300km), whereas steers in the MNT and CNT groups remained in Mississippi as non-transported controls. Body weight was measured and jugular blood was collected at -1, 0, and 7days from all steers at the same time, regardless of location. Multi-parameter flow cytometry (MP-FCM) was used to identify T-cell subsets and detect the expression of three activation markers (CD25 [interleukin (IL)-2 receptor], intracellular interferon-gamma [IFNγ], and IL-4) after in vitro stimulation with BVDV recall antigen. Plasma cortisol concentration was measured on day -1, 0, and 7 as a marker of transport-associated stress. Serum antibody titer to BVDV was assessed on day -1 and day 7 post-booster vaccination. Whole-blood samples were analyzed using MP-FCM on days 0 and 7. Results were log transformed and analyzed using repeated measures of analysis of variance. Compared with non-transported controls, transport led to an increase in BVDV-induced expression of CD25, IFNγ, and IL-4 in CD4(+), CD8(+), and γδ(+) T-cell subsets (P<0.05). MEL treatment mitigated the transportation-associated increase in CD25 expression by peripheral blood mononuclear cells (PBMCs), CD4(+), and γδ(+) T cells. CMI outputs for the MTR group were less than those of the CTR group (P<0.05); however, the MTR and NT groups did not differ (P>0.10). A treatment*transport interaction was noted for the increase in IL-4 expression by CD8(+) T cells after transport, with a significant difference between the CTR and MTR groups at day 7. In conclusion, the use of oral MEL prior to transport appears to have inhibitory or homeostatic effects, but further research is needed to validate the effect of MEL treatment on specific T-cell subsets in transported cattle., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Background: Acute infection with bovine viral diarrhoea virus (BVDV) usually causes only mild clinical disease in cattle, but infection of animals of breeding age can result in immune suppression (resulting in an increased incidence and severity of secondary disease) and decreased reproductive performance. If infection occurs during pregnancy, the virus may cross the placenta and either cause abortion, establish immunotolerance and persistent infection (PI) in the fetus or cause congenital deformities. These outcomes depend on the stage of pregnancy at the time of infection., International Perspective: BVDV is recognised as a disease of significant financial impact in a number of countries. As a result, national and regional BVDV control programs are now in place in several regions around the world. In Europe, these programs largely rely on the identification and removal of the PI animals, whereas vaccination has tended to be the chosen method of control in the United States., Bvd in Australia: BVDV is endemic in Australian cattle populations, with more than 80% of herds surveyed showing some level of exposure to the pathogen. The cost to the national industry is estimated to be AUD57.9 million annually. This review identifies and discusses the challenges to BVDV control in Australia, including farmer attitudes, herd size, sheep as a potential reservoir host and diagnostic capabilities. We conclude that systematic BVDV control in Australia is, or soon will be, an option; however, detailed cost-benefit analyses will need to be undertaken., (© 2014 Australian Veterinary Association.)

Enterocytozoon bieneusi known as a causative agent of opportunistic infections instigating diarrhoea in AIDS patients was identified also in a number of immunocompetent patients and in a wide range of animals, including cattle. In the present study we tested if the Bovine Viral Diarrhea Virus (BVDV), the most common pathogen underlying immunosuppressive Bovine Viral Diarrhoea (BVD), can enhance the occurrence of opportunistic infections with E. bieneusi in cattle. Six dairy farms were investigated using ELISA to detect antibodies against or antigens arising from BVDV in collected sera. A total of 240 individual faecal samples from four age groups were examined for the presence of E. bieneusi by nested PCR. Sequence analysis of six E. bieneusi positive samples revealed the presence of the genotype I of E. bieneusi, previously described in cattle. The hypothesis expecting higher prevalence of E. bieneusi in BVDV positive cattle herds was not confirmed in this study; however this is the first description about E. bieneusi in cattle in the Czech Republic., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is associated with a range of economically important diseases of cattle including reproductive disorders and an acute fatal hemorrhagic disease. Neutralizing antibodies that bind to the E2 glycoprotein are important predictors of vaccinal immunity. Neutralization tests using the NADL strain of BVDV and five anti-E2 monoclonal antibodies showed one, Wb163, neutralized the NADL strain of BVDV in an unexpected manner. Its titer was 10,000 compared to <35 as reported previously. The present stock of NADL differed from that of the earlier study in that the amino acid at position 79 of E2 was Valine instead of Glutamic acid. MAb Wb163 may, however, recognize a less important neutralizing epitope than another mAb Wb166, because it was less cross reactive than mAb Wb166, had a neutralizing titer 50-fold lower than Wb166 and was of lower relative affinity than Wb166. Variations in the amino terminus of E2 will be discussed in the context of vaccinal immunity.

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Forty-seven cattle management groups from 36 herds in a regional Bovine viral diarrhea virus (BVDV) eradication program were selected to evaluate serology as a tool to detect herd infection with BVDV. Serum samples were obtained from 5 non-vaccinated sentinel calves ≥ 6 months old in each management group and virus neutralizing (VN) antibody titers against BVDV genotypes 1 and 2 were determined. A herd was considered positive if 2 or more sentinel calves had VN antibody titers ≥ 128 to either genotype. Results were compared to individual animal testing of all available calves by reverse transcription polymerase chain reaction (RT-PCR) on skin biopsy samples. In 1 management group from 1 herd (n = 24), 3 sentinel calves had VN antibody titers ≥ 128. Three ear notch samples from that herd were positive for BVDV on RT-PCR assay. All other management groups were negative for BVDV. In the present study, the herd sensitivity of sentinel serology was 100% (95% confidence interval [CI]: 0.05-1.0) and herd specificity was 100% (95% CI: 0.90-1.0). The κ value for agreement between sentinel serology and RT-PCR was 1.0 (95% CI: 1.0-1.0). Preliminary results suggest that sentinel animal serology can be utilized in a BVDV eradication program to provide an accurate and efficient evaluation of herd status., (© 2011 The Author(s))

Whole blood samples (472) were collected during the period of 2006-2008 from 15 cattle farms in seven districts of the Xinjiang Uygur Autonomous Region, which is a major pastoral area and represents 1/6 of Chinese territory. Bovine viral diarrhea virus (BVDV) was detected by nested reverse transcription polymerase chain reaction assay (RT-nPCR) and sequenced within the 5'-untranslated region of the genome. Approximately 43% (202 samples) were BVDV-positive. Of the positive samples, 24 were chosen for further sequencing, sequence comparisons, and phylogenetic reconstructions. The phylogenetic reconstructions demonstrated that these isolates clustered into either BVDV1b (18 samples) or BVDV1c (six samples) subgenotypes. None of the isolates sequenced showed any cytopathic effect in culture. Unlike previously published studies in Chinese cattle, no BVDV2 genotypes were detected. These results indicate that the predominant subgenotypes in Xinjiang are BVDV1b and BVDV1c. This finding can be useful for further BVDV epidemiological study in China.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Previously, bovine viral diarrhea virus (BVDV) had been found in prolonged testicular infections following acute infection of immunocompetent bulls. The primary purpose of this research was to evaluate the production and maintenance of prolonged testicular infections after exposure to BVDV of seronegative bulls in varying circumstances. The secondary objective was to initiate assessment of the potential for transmission of BVDV via semen of bulls exhibiting a prolonged testicular infection. In total, 10 research trials were conducted. The first trial examined the duration of detectable virus in semen after intranasal inoculation of peri-pubertal bulls. The second to fifth trials examined the potential for prolonged testicular infections resulting from natural exposure of seronegative bulls to persistently infected heifers. In the last five trials, the potential for viral transmission from bulls exhibiting prolonged testicular infections to a small number of exposed animals (n=28) was evaluated. Results of this research demonstrated that prolonged testicular infections could result in detection of viral RNA in semen for 2.75 years with infectious virus grown from testicular tissue 12.5 months after viral exposure. A type 1b strain of BVDV caused prolonged testicular infection after natural exposure of seronegative bulls to a persistently infected heifer. However, transmission of BVDV to susceptible animals was not detected in the final five trials of this research. In conclusion, BVDV can persist in testicular tissue after acute infection for several years, but the potential for viral transmission from these prolonged testicular infections appears to be low.

The objective was to determine the effect of cryopreservation by conventional slow controlled cooling (0.5 degrees C/min) and by vitrification on the presence of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) infectivity associated with frozen-thawed Day 7 bovine embryos. In this study, Day 7 embryos generated by in vitro fertilization (IVF) were exposed in vitro for 1.5h to BVDV (N=393) and BHV-1 (N=242) and subsequently tested before and after cryopreservation for the presence of infectivity. Exposure of embryos to viral agents resulted in 72% of them infected prior to cryopreservation. Stepwise exposure of embryos to cryoprotectants, as well as their removal, substantially reduced the proportion of contaminated embryos (46% vs. 72%, P<0.05). Overall, both freezing methods reduced the percentage of infectious embryos compared with that of embryos similarly exposed to viruses but not cryopreserved (31% vs. 72%, respectively; P<0.001). The percentage of embryos with infectious viruses was not significantly higher after vitrification than after slow cooling (38% vs. 22%). In addition, after cryopreservation, a higher percentage (P<0.002) of embryos exposed to BHV-1 (42%) remained infectious than did embryos exposed to BVDV (24%). In conclusion, cryopreservation reduced the proportion of infected embryos but did not render all of them free from infectious pathogens.

The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.

The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDV associated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.

This study investigates the transmission of bovine viral diarrhoea virus (BVDV) 1f from a persistently infected (PI) lesser Malayan mousedeer to two bovine calves. Different contact routes to two calves were analysed: 1) aerosol contact between two adjacent pens without physical contact; 2) indirect contact by use of common utensils; 3) direct nose-to-nose contact for 30 seconds. One of the calves was infected either by aerosol or indirect contact. The virus sequence in 247 nucleotides in the 5'-UTR was 100% identical in mousedeer and calf. To elucidate the distribution of BVDV within the affected mousedeer family the captive population in a Zoo was analysed. The maternal line of PI animals was maintained, whereas a PI male was able to reproduce and have a non-PI calf. As a consequence of this, six female PI mousedeer were killed; subsequent autopsies did not reveal any lesions. Sequencing mousedeer BVD virus in the E2 region (420 nucleotides) through 4 generations showed only 7 mutations, which were maintained from mother to offspring.

Data derived from both experience and validation exercises on the behaviour and properties of BVDV is presented as applied to recovery of the virus from bovine serum used in the industrial production of animal biologicals. Licensed products both in the EU and the U.S. must use ingredients shown to be free of this agent and both regulatory agencies have published regulations governing this topic (EU: EMEA/CVMP and US: 9 CFR). The two systems differ in some specifics covering cell cultures used, timing of testing within the assays and the use of multiple approaches to the isolation of this virus. It was found that selected cell lines instead of primary cells could be used for BVDV isolation. FA tests, however, needed to be performed weekly for maximum sensitivity instead of once at the end of the test. BVDV interference assay (comparative titration) was found to be an unreliable test for the detection of non-CPE BVDV in cell culture. BVDV neutralization assays, while desirable from an informational standpoint, were determined to be unnecessary in serum that was to be adequately irradiated. Ultracentrifuge concentration of BVDV from serum was found to be a satisfactory method of increasing the sensitivity of assays for this agent, although quantification of the virus was found unnecessary in serum to be irradiated. Recommendations for the harmonization of 9 CFR and EMEA/CVMP into one assay are given.

The purpose of this research was to determine the effects of seropositivity for exposure to bovine leukemia virus (BLV), bovine viral diarrhoea virus (BVDV), Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) on overall and reason-specific culling in Canadian dairy cattle. Serum samples from, approximately, 30 randomly selected cows from 134 herds were tested for antibodies against BLV, MAP and NC using commercially available ELISA test kits, while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhoea virus (BVDV). For analyzing the time (in days) to culling of cows after the blood testing, a two-step approach was utilized, non-parametric (Kaplan-Meier survival graphs) visualization and then semi-parametric survival modelling (Cox proportional hazards model), while controlling for confounding variables and adjusting for within herd clustering. For all reasons of culling, MAP-seropositive cows had a 1.38 (1.05-1.81, 95% C.I.) times increased hazard of culling compared to MAP-seronegative cows. Seropositivity for the other pathogens was not associated with an increased risk of overall culling. Among cows that were culled because of either decreased reproductive efficiency or decreased milk production or mastitis, MAP-seropositive cows were associated with 1.55 (1.12-2.15, 95% C.I.) times increased hazard compared to MAP-seronegative cows. Among cows that were culled because of reproductive inefficiency, NC-seropositive cows had a 1.43 (1.15-1.79, 95% C.I.) times greater hazard than NC-seronegative cows. Among cows that were culled because of decreased milk production, cows in BVDV-seropositive herds had a 1.86 (1.28-2.70, 95% C.I.) times increased hazard compared to cows in BVDV-seronegative herds. BLV-seropositive cows did not have an increased risk of reason-specific culling as compared to BLV-seronegative cows. No significant interaction on culling among seropositivity for the pathogens was detected, but only a limited number of cows tested seropositive for multiple pathogens. Results from our research will help in better understanding the economic impacts of these pathogens and justification for their control.

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.

Objective: To evaluate persistence of bovine viral diarrhea virus (BVDV) in semen after inoculation of postpubertal bulls., Animals: Three 2-year-old bulls and five 6-month-old calves., Procedure: 3 seronegative 2-year-old bulls were inoculated intranasally with BVDV. Serum and semen samples were obtained at regular intervals until 7 months after inoculation. Serum samples were tested for BVDV by use of virus isolation (VI) and reverse transcription-nested polymerase chain reaction (RT-nPCR) tests. Semen samples were tested for virus by use of VI and RT-nPCR tests. Testicular biopsy specimens were obtained 7 months after inoculation and tested for BVDV by use of immunohistochemical analysis and VI and RT-nPCR tests. Semen samples collected from 1 bull immediately before and 5 and 7 months after inoculation were administered IV to seronegative calves, which were monitored for subsequent viremia and seroconversion., Results: Use of VI and RT-nPCR tests detected transient virus in serum of all bulls. The VI test detected BVDV in semen of 2 bulls for < 21 days after inoculation, whereas RT-nPCR assay detected BVDV until 7 months after inoculation. Virus was detected in testicular biopsy specimens of these 2 bulls by use of immunohistochemical analysis and RT-nPCR assay but could only be isolated from the biopsy specimen of 1 bull. Of the calves administered semen IV to detect infectious virus, only the recipient of semen collected 5 months after inoculation of the adult bull was viremic and seroconverted., Conclusions and Clinical Relevance: Bovine viral diarrhea virus can persist in semen of acutely infected bulls for several months after exposure.

This report describes the genetic and antigenic variability of bovine viral diarrhea virus strains isolated in Belgium. Part of the 5' untranslated region and the 5' end of the gp53 (E2) coding sequence were amplified by PCR and sequenced. Phylogenetic analysis showed that most field isolates segregated into genotypes Ib or II. Only one out of 28 field isolates belonged to genotype Ia. Interestingly, some type I strains were equally divergent from types Ia and Ib strains and clustered into additional subtypes within genotype I. Immune sera from young calves experimentally inoculated with field isolates first identified on the basis of their sequences were used in two-way neutralisation experiments. The results clearly differentiated type I from type II strains although some degree of cross-neutralisation was observed. Within type I, the new clusters could not be antigenically differentiated from the more prevalent type Ib strains or from type Ia strain NADL, suggesting that BVDV genotype I is antigenically homogeneous. The isolation of BVDV types I and II strains from cell lines and from a bovine vaccine suggest that molecular epidemiology surveillance is warranted for BVDV.

Bovine viral diarrhea virus (BVDV) can be found in cells and fluids from ovaries collected at the abattoir. On the other hand, immunoglobulins are also found in the fluid of ovarian follicles. Anti-BVDV antibodies in follicular fluid might reduce cross-contamination of COCs at the time of collection or hinder the use of virus isolation to test for the presence of virus. One objective of this study was to determine the frequency with which BVDV could be found in pooled follicular fluid collected during the periodic aspiration of COCs from abattoir-origin ovaries. A second objective was to determine the prevalence and neutralizing activity of anti-BVDV antibodies in these blended samples. We collected samples of pooled follicular fluid (n = 55) over a 20-month period as part of our routine oocyte collection activities. We assayed each sample for BVDV using virus isolation as well as reverse transcription nested polymerase chain reaction (RT-nPCR) procedures. We also tested follicular fluid for antibody that would neutralize four representative strains of BVDV (SD-1, a genotype 1a strain; NY-1, a genotype lb strain; CD-87, a genotype 2 strain, and PA-131, a divergent genotype 2 strain). We detected no BVDV by virus isolation, but we did identify the virus by RT-nPCR in one of the 55 samples of follicular fluid. Automated dye terminator nucleotide sequencing of the amplified portion of the viral genome indicated a genotype 1 strain that was distinct from any of our laboratory strains. In addition, each of the samples of follicular fluid contained sufficient antibody to neutralize large quantities of each of the four laboratory strains that were used. Finding BVDV in just 1 of 55 samples was consistent with reports of similar studies in which the occurrence of BVDV in abattoir-origin materials ranged from 0.9 to 12%. We presumed that failure to isolate the virus was due to neutralizing antibody in the sample. Thus, the incidence of BVDV contamination of our IVF system at the level of pooling of follicular fluid was low for the 20-month period. The presence of anti-BVDV antibody in pooled follicular fluid provided a coincidental means of neutralizing BVDV when it was introduced in fluid aspirated from infected ovaries.

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.