Julie A. Gard

Comparison of Tests for Detection of Bovine Viral Diarrhea Virus in Diagnostic Samples

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 laboratorys 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 ≤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.

Epidemiology of prolonged testicular infections with bovine viral diarrhea virus.

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) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

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 <or=6.62+/-1.57 copies/5 microL; 90% of the contaminated embryos had <or=4.64+/-1.57 viral copies/5 microL of embryo-associated virus, using tolerance intervals (P<0.05). The SEM was 0.33 and the mean of averages was 1.12/5 microL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was <or=3.44+/-0.89 copies/5 microL; 90% of the contaminated embryos had <or=2.40+/-0.89 viral copies/5 microL of embryo-associated virus using tolerance intervals (P<0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 microL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo―derived bovine embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo-derived bovine embryos (n=10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID(50))/mL. Additionally, control heifers received 1.5 x 10(6) CCID(50) BVDV/.5 mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.

Effect of exercise and environmental terrain on development of the digital cushion and bony structures of the bovine foot.

OBJECTIVE To determine whether exercise on alternative terrain affects the development of the digital cushion and bony structures of the bovine foot. ANIMALS 20 weaned bull calves. PROCEDURES Two-month-old calves were randomly allocated to an exercise or control group. For 4 months, the control group was maintained in grass paddocks, and the exercise group was maintained in a 0.8-km lane with a mixed terrain of dirt, stones (0.32- to 0.95-cm pea gravel and 5-cm crusher run), and grass. Water and food for the exercise group were located at opposite ends of the lane; calves were fed twice daily, which ensured they walked 3.2 km/d. Pedometers were applied to all calves to measure distance traveled. All calves were slaughtered at 6 months of age. The right forefeet and hind feet were harvested for MRI and CT evaluation. RESULTS Control calves walked a mean of 1.1 km daily, whereas the exercised calves walked a mean of 3.2 km daily. Mean digital cushion volume and surface area were 25,335 mm(3) and 15,647 mm(2), respectively, for the exercised calves and 17,026 mm(3) and 12,745 mm(2), respectively, for the control calves. When weight was controlled, mean digital cushion volume and surface area for the exercise group were increased by 37.10% and 18.25%, respectively, from those for the control group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that exercise on alternative terrain increased the volume and surface area of the digital cushion of the feet of dairy calves, which should make them less susceptible to lameness.

Efficacy of four commercially available multivalent modified-live virus vaccines against clinical disease, viremia, and viral shedding in early-weaned beef calves exposed simultaneously to cattle persistently infected with bovine viral diarrhea virus and cattle acutely infected with bovine herpesvirus 1.

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

119 ASSESSMENT OF OVARIAN FOLLICULAR DYSPLASIA UTILIZING ULTRASOUND AND HISTOLOGIC EXAMINATION

A slaughterhouse study commissioned by Florida Cattlemans Association in 2007 identified ovarian follicular dysplasia (OFD) as a primary cause of infertility in Florida beef cows. Ovaries with OFD have progressive bilateral development of solid clustered follicles containing multiple Call-Exner bodies that originate in the rete ovarii and the hilar region, and progress into the cortex to eventually form bilateral Sertoli-type granulosa theca cell tumours (GTCT). The objectives of this study were to assess the distribution of OFD in cull animals and to evaluate utilisation of ultrasound for diagnosis of OFD in cattle. Ultrasound images of the right and left ovaries from 390 cull cows and heifers representing 4 Florida ranches were made with 5-MHz linear probes (Aloka, Ibex). Then, 10 to 12 females per ranch were followed to slaughter the proceeding day for collection of reproductive tracts. The fixed ovaries were measured, sectioned para-sagittally through the hilus, photographed, and arranged in histology cassettes for complete examination of the cut surface. Large ovarian structures including corpus luteum, Graafian follicles, atretic follicles, dysplastic follicles, rete ovarii, dysplastic follicles, and tumours were counted and measured for each ovary. Ovaries with OFD were graded I to IV. Grade I OFD contained small individual dysplastic follicles with diameter less than 200µm mostly limited to the rete ovarii and medulla. Grade II OFD possessed dysplastic follicles greater than 200µm diameter that were present in the medulla and cortex. Grade III OFD had extensive multi-sized dysplastic follicles scattered throughout the entire cortex of the ovary and Grade IV OFD had Sertoli-type GTCT. Grade II-IV often had dystrophic mineralization of dysplastic follicles. Gross morphology of fixed sagittal sections and ultrasound images were blindly compared against OFD grade in 40 individual ovaries. The OFD was identified at slaughter in 29/41 cows and in 1/5 of heifers. The distribution of OFD for 30 affected females was Gr I 16/30, Gr II 9/30, Gr III 4/30, and Gr IV 1/30. Characteristics that could be detected by routine ultrasound included increased size and length, increased hyperechogenicity and decreased number of fluid-filled follicles. Hyperechogenic shadows were evident in higher grade OFD. The study demonstrated that Grade III and IV OFD can be observed by routine ultrasound but Grade I and II may require higher resolution ultrasound probes, imaging analysis software, or Doppler ultrasound.

Shaping the norms that regulate international commerce of embryos

As various embryo technologies in livestock were developed and evolved to a state of usefulness over the past 40 years, scientists with a specific interest in infectious diseases sought to determine the epidemiologic consequences of movement, especially international movement, of increasing numbers of embryos. Many of the foundational studies in this area were reported in Theriogenology, beginning in the 1970s and especially throughout the 1980s and 1990s. Unquestionably, Theriogenology has been a widely used venue for dissemination of basic information on this subject, which ultimately led to the development of the now universally accepted techniques for certification of embryo health. Today it is well-recognized that movement in commerce of embryos, especially in vivo-derived embryos, is a very low-risk method for exchange of animal germ plasm. This paper chronicles the evolution of strategies for health certification of embryos. An overview is provided of the calculated efforts of practitioners, scientists, and regulators to organize, forge necessary partnerships, stimulate needed research, provide purposeful analysis of the results, and, through these processes, guarantee the universal acceptance of efficient protocols for certifying the health of embryos intended for movement in international commerce.