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The objective of this study was to compare reproductive protection in cattle against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) provided by annual revaccination with multivalent modified-live viral (MLV) vaccine or multivalent combination viral (CV) vaccine containing temperature-sensitive modified-live BoHV-1 and killed BVDV when MLV vaccines were given pre-breeding to nulliparous heifers. Seventy-five beef heifers were allocated into treatment groups A (n=30; two MLV doses pre-breeding, annual revaccination with MLV vaccine), B (n=30; two MLV doses pre-breeding, annual revaccination with CV vaccine) and C (n=15; saline in lieu of vaccine). Heifers were administered treatments on days 0 (weaning), 183 (pre-breeding), 366 (first gestation), and 738 (second gestation). After first calving, primiparous cows were bred, with pregnancy assessment on day 715. At that time, 24 group A heifers (23 pregnancies), 23 group B heifers (22 pregnancies), and 15 group C heifers (15 pregnancies) were commingled with six persistently infected (PI) cattle for 16days. Ninety-nine days after PI removal, cows were intravenously inoculated with BoHV-1. All fetuses and live offspring were assessed for BVDV and BoHV-1. Abortions occurred in 3/23 group A cows, 1/22 group B cows, and 11/15 group C cows. Fetal infection with BVDV or BoHV-1 occurred in 4/23 group A offspring, 0/22 group B offspring, and 15/15 group C offspring. This research demonstrates efficacy of administering two pre-breeding doses of MLV vaccine with annual revaccination using CV vaccine to prevent fetal loss due to exposure to BVDV and BoHV-1., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Programs for control and eradication of bovine viral diarrhea virus (BVDV) are often considered prudent when the expense of a control program within a specified time frame effectively prevents loss due to disease and the expense of control does not exceed the costs associated with infection. In some geographic areas, concerns about animal welfare or desires to reduce antibiotic usage may motivate BVDV control even when control programs are associated with a lack of financial return on investment. In other geographic areas, concerns about financial return on investment may be the key motivating factor in considering implementation of BVDV control programs. Past experiences indicate that systematic, well-coordinated control programs have a clear potential for success, while voluntary control programs in cultures of distributed decision-making often result in notable initial progress that ultimately ends in dissolution of efforts. Segmentation of the cattle industry into cow-calf producers, stocker/backgrounders, and feedlot operators amplifies the distribution of decision-making regarding control programs and may result in control measures for one industry segment that are associated with significant costs and limited rewards. Though the host range of BVDV extends well beyond cattle, multiple eradication programs that focus only on testing and removal of persistently infected (PI) cattle have proven to be effective in various countries. While some individuals consider education of producers to be sufficient to stimulate eradication of BVDV, research surrounding the adoption of innovative health care procedures suggests that the process of adopting BVDV control programs has a social element. Collegial interactions and discussions may be crucial in facilitating the systematic implementation necessary to optimize the long-term success of control programs. Compulsory control programs may be considered efficient and effective in some regions; however, in a nation where individual identification of cattle remains voluntary, the likelihood of effective compulsion to control BVDV within a farm or ranch appears to be very unlikely. While currently available diagnostic tests are sufficient to support BVDV eradication via systematic, well-coordinated programs, the development of a diagnostic procedure to safely and consistently detect the gestation of a PI fetus after 5 months of gestation would be a valuable research breakthrough. This desired testing modality would allow diagnosis of PI calves, while the dam continues to provide biocontainment of the infected fetus. This development could speed the progress of control programs in achieving the goal of BVDV control and eventual eradication.

Prebreeding vaccination should provide fetal and abortive protection against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) but not impede reproduction when administered to cattle before estrus synchronization and breeding. The objective was to assess reproductive performance when naive beef heifers were vaccinated with modified-live viral (MLV) vaccine 2 days after unsynchronized estrus, and then revaccinated with MLV vaccine at 10 or 31 days before synchronized natural breeding. Sixty beef heifers naive to BVDV and BoHV-1 were randomly assigned to one of four treatment groups. Groups A and B (n = 20 per group) were vaccinated with MLV vaccine containing BVDV and BoHV-1 at 2 days after initial detected estrus, and then revaccinated 30 days later, which corresponded to 10 days (group A) or 31 days (group B) before synchronized natural breeding. Groups C and D (n = 10 per group) served as controls and were vaccinated with an inactivated vaccine that did not contain BVDV or BoHV-1 at the same time points as groups A and B, respectively. Estrous behavior was assessed using radio frequency technology. Estrus synchronization was performed, with initiation occurring at revaccination (groups A and C) or 21 days after revaccination (groups B and D). After synchronization, heifers were submitted to a bull breeding pasture for 45 days. At the end of the breeding period, heifers were assessed for pregnancy using ultrasonography. Progesterone concentrations were evaluated at estrus and 10 days after unsynchronized and synchronized estrus, at initial pregnancy check, and at the end of the study. All pregnant heifers in groups A and B and five pregnant heifers in group C were euthanized between 44 and 62 days of gestation and ovarian and conceptus tissues were assayed for BVDV and BoHV-1. Vaccination with MLV vaccine did not result in significant negative reproductive impact based on the duration of interestrus intervals, proportion of heifers exhibiting estrus within 5 days after synchronization, serum progesterone concentrations, pregnancy rates, and pregnancies in the first 5 days of the breeding season. Bovine viral diarrhea virus and BoHV-1 were not detected in luteal tissue, ovarian tissue, or fetal tissues. Use of MLV vaccine did not impede reproduction, when revaccination was performed at 10 or 31 days before synchronized natural breeding., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is an important reproductive pathogen of cattle worldwide. The reproductive outcome of BVDV infection is largely dependent on the immune status of the dam and the stage of gestation at the time of infection. Potential sequelae include failure of conception, abortion, a variety of congenital malformations, and fetal infection. Vaccination is a possible tool in the control of BVDV, and there has been a recently renewed focus on providing fetal protection through vaccination. Consequently, the aim of this study was to evaluate the efficacy of BVDV vaccination to prevent reproductive disease by performing a quantitative synthesis of previously published studies. Pertinent articles to be included in the analysis were identified by performing a search in four relevant scientific databases (PubMed, CAB abstracts, National Agricultural Library catalog, and Web of Science) and examining the reference lists of 10 germane review articles. Inclusion criteria for the meta-analysis mandated that the studies were controlled, primary studies that included necessary data for use in the meta-analysis (e.g., group size, number of abortions). Forty-six studies in 41 separate articles matched the inclusion criteria. Risk ratio effect sizes were used in random effects, weighted meta-analyses to assess the impact of BVDV vaccination on three outcomes: risk of fetal infection, abortion risk, and pregnancy risk. Within each outcome, subanalyses were performed to evaluate the effect of a variety of interventions, including modified live, inactivated, polyvalent and monovalent vaccination, homologous, heterologous, or field challenge, and studies with only bovine subjects. The analysis revealed a decrease in abortions of nearly 45% and a nearly 85% decrease in fetal infection rate in cattle vaccinated for BVDV compared with unvaccinated cohorts. Additionally, pregnancy risk was increased by approximately 5% in field trials of BVDV vaccinates. This meta-analysis provides quantitative support for the benefit of vaccination in the prevention of BVDV-associated reproductive disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recently, in the United States, a dairy bull was diagnosed as the second confirmed case of persistent testicular infection (PTI) with bovine viral diarrhea virus (BVDV). The first objective of this study was to evaluate the testing methodologies currently used by the artificial insemination industry in order to improve the detection of bulls with PTI. This study evaluated the impact of multiple factors ([1] sample tested, [2] sample handling, [3] assay used, and [4] assay methodology) on the sensitivity of detection of BVDV. The second objective of this study was to evaluate the transmissibility of BVDV from the bull through casual or sexual contact. Results from this study indicate that straws of semen should be transported to the diagnostic laboratory in liquid nitrogen dry shippers. PCR proved to be a more sensitive assay than virus isolation; however, certain PCR protocols exhibited greater diagnostic sensitivity than others. Insemination with cryopreserved semen from this infected bull caused viral transmission to a seronegative heifer resulting in viremia and seroconversion. After 42 months of age, the bull appeared to clear the infection. In conclusion, this bull validates that natural exposure to a 1a strain of BVDV can result in a unique PTI causing contamination of semen with detectable infectious virus. Appropriate handling and testing of samples is necessary in order to detect bulls exhibiting PTI. Additionally, PTI with BVDV may potentially be cleared after an extended duration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen capable of causing disease affecting multiple body systems. Previous studies have shown 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) effectively prevents BVDV infection in cell culture. The aim of this project was to assess the efficacy of DB772 for the prevention of acute BVDV infection. Four calves seronegative to BVDV were treated with DB772 and another 4 calves were treated with diluent only on the same dosing schedule. Each calf was subsequently challenged intranasally with BVDV. Virus was isolated consistently from untreated calves on days 4 to 8, while treated calves remained negative by virus isolation during this period. Azotemia was exhibited by all treated calves on day 4 resulting in the euthanasia of 1 calf on day 10 and the death of another on day 13. Virus was isolated from the 2 remaining treated calves on day 14 or 21. On day 21, both remaining treated calves and all 4 untreated calves had anti-BVDV antibody titers > 1:2048. This pilot study indicates that DB772 temporarily prevented acute disease due to BVDV, but carries a significant concern of renal toxicity.

The objective was to determine whether a multivalent modified-live virus vaccine containing noncytopathic bovine viral diarrhea virus (BVDV) administered off-label to pregnant cattle can result in persistently infected fetuses and to assess whether vaccinal strains can be shed to unvaccinated pregnant cattle commingling with vaccinates. Nineteen BVDV-naïve pregnant heifers were randomly assigned to two groups: cattle vaccinated near Day 77 of gestation with modified-live virus vaccine containing BVDV-1a (WRL strain), bovine herpes virus-1, parainfluenza 3, and bovine respiratory syncytial virus (Vx group; N = 10) or control unvaccinated cattle (N = 9). During the course of the study a voluntary stop-sale/recall was conducted by the manufacturer because of the presence of a BVDV contaminant in the vaccine. At Day 175 of gestation, fetuses were removed by Cesarean section and fetal tissues were submitted for virus isolation, and quantitative reverse transcription polymerase chain reaction using BVDV-1- and BVDV-2-specific probes. Nucleotide sequencing of viral RNA was performed for quantitative reverse transcription polymerase chain reaction-positive samples. Two vaccinated and two control heifers aborted their pregnancies, but their fetuses were unavailable for BVDV testing. Virus was isolated from all eight fetuses in the Vx group heifers and from 2 of 7 fetuses in the control unvaccinated heifers. Only BVDV-2 was detected in fetuses from the Vx group, and only BVDV-1 was detected in the two fetuses from the control group. Both BVDV-1 and BVDV-2 were detected in the vaccine. In conclusion, vaccination of pregnant heifers with a contaminated modified-live BVDV vaccine resulted in development of BVDV-2 persistently infected fetuses in all tested vaccinated animals. Furthermore, BVDV was apparently shed to unvaccinated heifers causing fetal infections from which only BVDV-1 was detected., (Published by Elsevier Inc.)

Bovine viral diarrhea virus can maintain prolonged infections within immunoprivileged sites after an otherwise transient infection of a cow, calf, or bull. Various sites provide unique niches for viral replication which are not susceptible to the complete surveillance commonly provided by the bovine immune system. Evidence indicates that pestiviral infections may be significantly prolonged within ovarian tissue, testicular tissue, central nervous system tissue, and circulating white blood cells. Within avascular portions of the ovarian follicle, granulosa cells and oocytes may maintain BVDV infections which cannot be attacked by cell-mediated immunity. When infections occur within seminiferous tubules in testicular tissue, similar protection from the immune system is provided for BVDV by the blood-testes barrier. Likewise, the blood-brain barrier has been hypothesized to provide protection for BVDV in a case involving neuropathology associated with immunohistochemical detection of BVDV. Furthermore, infections of circulating white blood cells may perturb their stimulation of an adaptive immune response and facilitate chronic infection of these cells. Thus, BVDV has demonstrated an ability to maintain prolonged viral infections in immunoprivileged sites within its natural host. The role of chronic infections in maintaining and disseminating BVDV within the cattle population and heterologous host species remains to be fully understood., (Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

The reproductive impact following controlled introduction of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) was evaluated in BVDV-naive heifers. Heifers were randomly allocated into two groups: an unexposed control herd (n = 34) and a herd exposed to five persistently infected (PI) animals for 7 mo, beginning 50 days before the breeding season (n = 34). Initiation of the BVDV-challenge was timed to mimic either direct contact with PI calves born in the previous calving season or accidental introduction of PI herd additions prior to the breeding season. The PI animals represented BVDV Types 1a (n = 3), 1b (n = 1) and 2 (n = 1). Two BVDV-free, seropositive bulls were used in each group for 78 days breeding seasons. In both groups, 33 of 34 heifers became pregnant, with similar distribution of fetal ages. Two heifers in each group aborted (etiology undetermined). In addition, one calf was born dead and one calf died 3 days post-partum in the BVDV-exposed group. One calf in the unexposed group died 4 mo post-partum. No calves, including the stillborn calf and the two calves that died prior to weaning, were persistently infected with BVDV. In summary, introduction of PI cattle to a group of BVDV-naive heifers 50 days prior to the breeding season did not negatively impact reproductive performance. To the contrary, the active immunity that developed following field exposure to BVDV provided effective reproductive and fetal protection during the breeding season and subsequent gestations, despite continuous exposure to PI animals until approximately midgestation. Although BVDV can have potentially devastating reproductive effects, timing of infection is a critical determinant in the outcome of a BVDV infection. A controlled breeding season with introduction of herd additions at less critical reproductive time points can mitigate the negative reproductive health consequences of BVDV., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection., Design: Randomized controlled clinical trial., Animals: 33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves., Procedures: 20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days., Results: After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers., Conclusions and Clinical Relevance: Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.

Background: Bovine viral diarrhea virus (BVDV) strains circulating in livestock herds show significant sequence variation. Conventional wisdom states that most sequence variation arises during acute infections in response to immune or other environmental pressures. A recent study showed that more nucleotide changes were introduced into the BVDV genomic RNA during the establishment of a single fetal persistent infection than following a series of acute infections of naïve cattle. However, it was not known if nucleotide changes were introduce when the virus crossed the placenta and infected the fetus or during the acute infection of the dam., Methods: The sequence of the open reading frame (ORF) from viruses isolated from four acutely infected pregnant heifers following exposure to persistently infected (PI) calves was compared to the sequences of the virus from the progenitor PI calf and the virus from the resulting progeny PI calf to determine when genetic change was introduced. This was compared to genetic change found in viruses isolated from a pregnant PI cow and its PI calf, and in three viruses isolated from acutely infected, non-pregnant cattle exposed to PI calves., Results: Most genetic changes previously identified between the progenitor and progeny PI viruses were in place in the acute phase viruses isolated from the dams six days post-exposure to the progenitor PI calf. Additionally, each progeny PI virus had two to three unique nucleotide substitutions that were introduced in crossing the placenta and infection of the fetus. The nucleotide sequence of two acute phase viruses isolated from steers exposed to PI calves revealed that six and seven nucleotide changes were introduced during the acute infection. The sequence of the BVDV-2 virus isolated from an acute infection of a PI calf (BVDV-1a) co-housed with a BVDV-2 PI calf had ten nucleotides that were different from the progenitor PI virus. Finally, twenty nucleotide changes were identified in the PI virus of a calf born to a PI dam., Conclusions: These results demonstrate that nucleotide changes are introduced into the BVDV infecting pregnant cattle at rates of 2.3 to 8 fold higher then during the acute infection of non-pregnant animals.

Objective: To evaluate onset of protection induced by modified-live virus (MLV) bovine viral diarrhea virus (BVDV) vaccine administered 7, 5, or 3 days before inoculation with type 1b BVDV (strain NY-1). Animals-40 calves., Procedures: Calves were assigned to 4 groups: an unvaccinated control group or groups vaccinated with MLV vaccine containing BVDV types 1a and 2 at 7, 5, or 3 days, before inoculation with NY-1 BVDV. Blood samples were collected for leukocyte counts, serum virus neutralization, and virus isolation (VI); nasal swab specimens (NSSs) were obtained for VI, and rectal temperatures were monitored for 14 days after inoculation., Results: No significant differences in leukocyte counts or rectal temperatures were detected after BVDV inoculation in vaccinated calves. Vaccinated calves had reduced viremia and viral shedding after inoculation, compared with results for unvaccinated calves. On day 5 after inoculation, a higher proportion of calves vaccinated 3 days before inoculation had positive VI from NSSs, compared with NSS VI results for calves vaccinated 5 and 7 days before inoculation. Unvaccinated calves had leukopenia on days 3, 5, and 6 and had higher rectal temperatures on days 7 and 8 after inoculation, compared with temperatures before inoculation. All unvaccinated calves had ≥ 1 positive VI result from NSSs 3 to 11 days after inoculation, and 4 became viremic., Conclusions and Clinical Relevance: MLV BVDV vaccine prevented fever, viremia, and leukopenia in calves challenge inoculated with NY-1 BVDV. A high proportion of calves vaccinated 3 days before inoculation shed BVDV after inoculation.

Bovine viral diarrhea virus (BVDV) is a pestivirus that is enzootic in most cattle populations throughout the world. This virus is present throughout the body of persistently infected (PI) cattle. Previous research has not assessed the cooking temperature at which BVDV in meat from PI cattle can be inactivated. Therefore, muscle tissue from 6 PI cattle was harvested, refrigerated, frozen, and heated to various internal temperatures. The concentration of virus present was determined by virus isolation. Average cell culture infective doses (50% endpoint; CCID(50)) of BVDV per gram of frozen, uncooked meat from PI cattle were 10(5.85) CCID(50)/g of whole cuts and 10(6.02) CCID(50)/g of ground meat. The virus in whole and ground meat was consistently inactivated when cooked to temperatures greater than or equal to 75°C. A second objective of this research was to thoroughly reassess if Vero cells were permissive to BVDV infection in our laboratory to provide further indication of whether primates, including humans, might be susceptible to BVDV. Vero cells were not permissive to infection with any of 43 different strains of BVDV that readily replicated in Madin Darby bovine kidney cells. In conclusion, this bovine pathogen, which is not considered to be a human pathogen, can be inactivated by cooking ground or whole cuts of meat to 75°C or higher. Care should be taken to ensure that susceptible hosts such as pigs are not fed improperly cooked meat, meat by-products, or waste food originating from PI cattle.

Background: Animals persistently infected (PI) with bovine viral diarrhoea virus (BVDV) are a key source of viral propagation within and among herds. Currently, no specific therapy exists to treat PI animals. The purpose of this research was to initiate evaluation of the pharmacokinetic and safety data of a novel antiviral agent in BVDV-free calves and to assess the antiviral efficacy of the same agent in PI calves., Methods: One BVDV-free calf was treated with 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) once at a dose of 1.6 mg/kg intravenously and one BVDV-free calf was treated three times a day for 6 days at 9.5 mg/kg intravenously. Subsequently, four PI calves were treated intravenously with 12 mg/kg DB772 three times a day for 6 days and two PI control calves were treated with an equivalent volume of diluent only., Results: Prior to antiviral treatment, the virus isolated from each calf was susceptible to DB772 in vitro. The antiviral treatment effectively inhibited virus for 14 days in one calf and at least 3 days in three calves. Subsequent virus isolated from the three calves was resistant to DB772 in vitro. No adverse effects of DB772 administration were detected., Conclusions: Results demonstrate that DB772 administration is safe and exhibits antiviral properties in PI calves while facilitating the rapid development of viral resistance to this novel therapeutic agent., (© 2012 International Medical Press)

Bovine viral diarrhea viruses (BVDV) are ubiquitous viral pathogens of cattle with a high degree of sequence diversity amongst strains circulating in livestock herds. The driving force behind change in sequence is not well established but the inaccurate replication of the genomic RNA by a viral RNA polymerase without proof-reading capabilities as well as immune pressure on immunodominant proteins are thought to play major roles. Additionally, it is not clear when the majority of changes are introduced, whether during acute infections with exposure to innate and adaptive immune responses or in establishment of persistent infections (PI) in utero. To examine which generates greater sequence diversity, two groups of viruses were compared. The first was six isolates of a single strain of BVDV-2 that were isolated over greater than a year's time. These viruses caused a series of severe acute (SA) BVD outbreaks over a large geographic area. Changes in nucleotide sequence were determined by comparison of the sequence of each strain to the six virus consensus sequence. The second group was composed of six BVDV strains isolated from PI calves whose dams were exposed to PI cattle. Changes were identified by comparison of the sequence of the progenitor PI virus to that of the progeny viruses from the single in vivo 'passage'. The open reading frames (ORF) of the six SA isolates were >99% identical at the nucleotide level with 30% of the changes being nonsynonymous changes. The amount of genetic change increased with time and distance from the original outbreak. Similarly, the PI viruses isolated from single passage PI calves had >99% identity with the progenitor virus. The number of nucleotide changes in these viruses was equal to or greater than that observed in the SA viruses. The majority of the nonsynonymous changes were found in the structural proteins, with 65% of these occurring in the immunodominant E2 protein. Antigenic mapping studies using a monoclonal antibody panel specific for the BVDV E2 protein showed no antigenic differences amongst the six SA viruses, nor between the progenitor and progeny type 1a and type 2 persistent viruses. However, antigenic differences were observed in the two type 1b progeny viruses that possessed the greatest number of amino acid changes. Two antibodies were found to have altered staining patterns. These results suggest that the establishment of a single persistent infection results in more rapid generation of genetic diversity in BVDV strains than a series of acute infections and may contribute to antigenic change in the absence of an immune response., (Published by Elsevier B.V.)

Identifying reservoirs and transmission routes for bovine viral diarrhea virus (BVDV) are important in developing biosecurity programs. The aim of this study was to evaluate BVDV transmission by the hematophagous horn fly (Haematobia irritans). Flies collected from four persistently infected cattle were placed in fly cages attached to principal (n = 4) and control (n = 4) BVDV-naïve calves housed individually in isolation rooms. Flies were able to feed on principal calves, but a barrier prevented fly feeding from control calves. Flies were tested for BVDV by RT-PCR and virus isolation at time of collection from PI cattle and after 48 h of exposure on BVDV-naïve calves. Blood samples were collected from calves and tested for BVDV infection. Virus was isolated from fly homogenates at collection from PI animals and at removal from control and principal calves. All calves remained negative for BVDV by virus isolation and serology throughout the study. Bovine viral diarrhea virus may be detected in horn flies collected from PI cattle, but horn flies do not appear to be an important vector for BVDV transmission.

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Eighty crossbred beef heifers were randomly allocated to four groups to evaluate the efficacy of vaccination in preventing development of calves persistently infected with bovine viral diarrhea virus (BVDV). Group 1 (n=11) was non-vaccinated controls, whereas three groups were vaccinated with commercially available multivalent BVDV vaccines at weaning (approximately 7 mo of age), 28 d post-weaning, approximately 1 y of age, and 28 d later. Groups 2 (n=23) and 3 (n=23) were given a modified-live BVDV vaccine, whereas Group 4 was given an inactivated BVDV vaccine. Heifers were bred by AI and subsequently exposed to two bulls. At 61 d after AI, 70 heifers were pregnant (n=10 for Group 1 and n=20/group for Groups 2, 3, and 4). Three cattle persistently infected with BVDV were commingled with the pregnant heifers (in an isolated pasture) from 68 to 126 d after AI. Thereafter, viremias were detected in pregnant heifers from Groups 1, 3, and 4 (10/10, 1/20, and 10/20, respectively), but not in pregnant heifers from Group 2 (0/20). Resulting calves were assessed for persistent infection using serum PCR, ear notch antigen capture-ELISA, and immunohistochemistry. Persistently infected calves were only produced in Group 1 (10/10) and Group 4 (2/18). In conclusion, commercial vaccines provided effective fetal protection despite prolonged natural exposure to BVDV. Given that viremias were detected in 11 vaccinated heifers after BVDV exposure, and two vaccinated heifers gave birth to persistently infected calves, there is continued need for biosecurity and diagnostic surveillance, in addition to vaccination, to ensure effective BVDV control., ((c) 2010 Elsevier Inc. All rights reserved.)

Cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, are an important source of viral transmission to susceptible hosts. Persistent BVDV infections have been identified in white-tailed deer (Odocoileus virginianus), the most abundant free-ranging ruminant in North America. As PI deer shed BVDV similarly to PI cattle, maintenance of BVDV within white-tailed deer populations may be possible. To date, intraspecific transmission of BVDV in white-tailed deer has not been evaluated, which prompted this study. Six pregnant white-tailed deer were captured in the first trimester of pregnancy and cohabitated with a PI white-tailed deer. Cohabitation with the PI deer resulted in BVDV infection in all does, as indicated by seroconversion. All does gave birth to live fawns and no reproductive losses were observed. At birth, evidence of BVDV infection was identified in two singlet fawns, of which one was determined to be PI by repeated serum reverse transcription nested PCR, whole blood virus isolation and immunohistochemistry. This study demonstrates for the first time that BVDV transmission may occur among white-tailed deer. The birth of a PI fawn through contact to a PI white-tailed deer indicates that under appropriate circumstances, BVDV may be maintained in white-tailed deer by congenital infection., (INRA, EDP Sciences, 2009)

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.

Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers.

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7d post-fertilization were exposed (for 2h) to 2 x 10(5-7) cell culture infective dose (CCID(50))/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was

Two reverse transcription-nested polymerase chain reaction tests, 1 quantitative (qRT-nPCR) and 1 standard (RT-nPCR), were evaluated to assess sensitivity for detection of bovine viral diarrhea virus (BVDV) of a single positive serum sample in a pool of 30. The RT-nPCR and qRT-nPCR each detected 95 of 100 known positives. The RT-nPCR was used to estimate the prevalence of BVDV in adult beef cows. Serum samples were obtained from the US Department of Agriculture brucellosis testing laboratories in 3 Midwestern states. Samples originated from auction markets and private treaty sales throughout the 3 states. A total of 2,990 serum samples were collected and randomly pooled into 100 pools for testing. Two of the 100 pools of field samples were positive, and each positive pool had a single positive individual sample upon confirmation. The estimate of BVDV prevalence in adult cows in this study was 0.07%. This study estimates the diagnostic sensitivity of RT-nPCR for BVDV and confirms that it is a useful diagnostic tool for pools of 30 serum samples and that prevalence of BVDV in adult cattle from auction markets is low.

The objective of this research was to evaluate reactivation of bovine viral diarrhea virus (BVDV) following dexamethasone treatment in 4 bulls that had previously been inoculated with BVDV, 3 of which had been demonstrated to have a localized testicular infection. Bulls were housed in an isolated pasture with in-contact steers. Beginning on day 0 of this study, all bulls received a daily dose of 0.1 mg/kg body weight (BW) of dexamethasone intravenously for 5 consecutive days. Blood was collected from the in-contact steers and semen, blood, and cerebrospinal fluid were collected from the bulls during and following dexamethasone treatment. Samples were assayed for BVDV using virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR). Serum was assayed for antibody using standard virus isolation. Virus was not isolated from blood, cerebrospinal fluid, or semen from any of the 4 bulls during the study period. One of the bulls was positive for BVDV in semen by RT-nPCR throughout the study period. The BVDV was not recovered from any in-contact control steers during the 28-day study period, nor did any of the in-contact control steers seroconvert to BVDV. Raw semen from 1 bull that was RT-nPCR positive was intravenously inoculated into 7 seronegative steers based upon the Cornell Semen Test. The BVDV could not be recovered from the steers and none of them seroconverted to BVDV. The results indicated that reactivation of BVDV in bulls with a localized testicular infection is unlikely; however, further research is necessary to determine the full potential for BVDV transmission from bulls with a localized testicular infection.

Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.

Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.

Artificial insemination and embryo transfer are used commonly in cattle production and exchange of germplasm between populations of cattle. If properly monitored, assisted reproductive techniques can be used to prevent the spread of infectious agents. However, these techniques potentially represent unnatural routes for transmission of diseases. Bovine viral diarrhea virus (BVDV) is broadly distributed among the world's populations of cattle. Fluids, gametes and somatic cells from infected animals are likely contaminated with the virus. Thus, use of semen or embryos from infected animals could result in spread of BVDV. This paper provides an overview of the risks of transmitting this virus by AI or production and transfer of embryos and summarizes the precautions needed to prevent such transmissions of disease from occurring.

Currently, a variety of tests are used to detect bovine viral diarrhea virus (BVDV) in persistently infected (PI) cattle. These tests include immunohistochemical staining (IHC), antigen capture enzyme-linked immunosorbent assay (ACE), virus isolation (VI), and reverse transcription-polymerase chain reaction (RT-PCR). However, a lack of methods standardization could compromise the ability to consistently identify animals infected with BVDV. This study evaluated the diagnostic proficiency of current methods for detecting BVDV in infected cattle using intra- and interlaboratory comparisons. Samples were collected from 4 animals more than 7 months of age (2 BVDV negative animals, a PI animal, and a PI animal that previously lacked detectable virus in serum as determined by VI). Samples were submitted to 23 participating diagnostic laboratories using the respective laboratory's standard submission protocol. Samples collected for submission included: 1) serum for ACE, RT-PCR, and VI; 2) whole blood for RT-PCR and VI; and 3) skin biopsies for ACE and IHC. The ACE performed on skin provided the greatest consistency in detecting positive samples and a perfect level of agreement among laboratories. Reverse transcription-polymerase chain reaction and IHC performed well by correctly identifying > or = 85% of samples positive for BVDV. Virus isolation performed on serum yielded the lowest consistency in detecting positive samples and the lowest level of agreement. The level of agreement between laboratories for detecting BVDV in persistently infected cattle ranged from perfect to less than expected by chance. The variation between laboratories suggests a need for training opportunities in standardized laboratory protocols and proficiency testing.

Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.

The objectives of this research were to evaluate the risk of prolonged testicular infection as a consequence of vaccination of peri-pubertal bulls with a modified-live, noncytopathic strain of BVDV and to assess vaccine efficacy in preventing prolonged testicular infections after a subsequent acute infection. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with an approximate minimum immunizing dose or a 10x standard dose of modified-live, noncytopathic BVDV or were maintained as unvaccinated controls. Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry and the identity of viral strains was determined by nucleotide sequencing of PCR products. The vaccine strain of BVDV was detected in testicular tissue of vaccinated bulls as long as 134 days after immunization. Prolonged testicular infections with the challenge strain were detected only in unvaccinated bulls as long as 85 days after challenge. Whereas vaccination caused prolonged testicular infection in some bulls, it did prevent subsequent infection of testicular tissue with the challenge strain. This research demonstrates that subcutaneous vaccination of naïve, peri-pubertal bulls with a noncytopathic, modified-live strain of BVDV can result in prolonged viral replication within testicular tissue. The risk for these prolonged testicular infections to cause venereal transmission of BVDV or subfertility is likely to be low but requires further investigation.

A commercial vaccine containing modified-live bovine viral diarrhea virus (BVDV; types 1 and 2) was administered to one group of 22 peripubertal bulls 28 days before intranasal inoculation with a type 1 strain of BVDV. A second group of 23 peripubertal bulls did not receive the modified-live BVDV vaccine before intranasal inoculation. Ten of 23 unvaccinated bulls--but none of the vaccinated bulls--developed a persistent testicular infection as determined by immunohistochemistry and polymerase chain reaction. Results of this study indicate that administration of a modified-live vaccine containing BVDV can prevent persistent testicular infection if peripubertal bulls are vaccinated before viral exposure.

Although BVDV-free offspring have been produced from persistently infected bulls and heifers via advanced reproductive techniques, embryos and semen can potentially transmit the virus. Due to this potential for transmission, appropriate testing is necessary to ensure freedom of semen and embryos from BVDV. In the future, less constraining quality control measures may ensure freedom of embryos and semen from BVDV. These quality control measures require additional research to be validated.

Bovine viral diarrhea virus (BVDV) is a significant pathogen that can be shed in the semen of infected bulls. Thus, screening for BVDV in semen of bulls is recommended prior to their entry into an artificial insemination center. No previous research has compared the analytical sensitivity of reverse transcription-nested polymerase chain reaction (RT-nPCR) and virus isolation assays for detection of BVDV in semen from an infected bull. Therefore, the goals of this research were to compare the analytical sensitivity of RT-nPCR and virus isolation assays for BVDV in semen and to apply these assays to determine the prevalence in the Southeastern United States of bulls that lack viremia yet shed BVDV in semen. Semen collected from a bull that was persistently infected with BVDV was serially diluted (1/10) in semen from uninfected bulls and frozen in liquid nitrogen as raw, partially extended or fully extended semen. Subsequently, samples of semen were assayed by virus isolation and RT-nPCR. Viral detection was more sensitive in extended semen samples than in raw semen samples and more sensitive by RT-nPCR than virus isolation. After this evaluation of analytical sensitivity, serum and semen were collected from 558 post-pubertal bulls in our region. These samples were tested for BVDV by virus isolation. Partially extended semen was also assayed for BVDV by RT-nPCR. All samples were negative by all assays for BVDV. The application of analytically sensitive assays reveals a very low prevalence (

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.