A COMPARISON OF PCR WITH AND WITHOUT RNA EXTRACTION AND VIRUS ISOLATION FOR DETECTION OF BVD VIRUS IN YOUNG CALVES

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1 A COMPARISON OF PCR WITH AND WITHOUT RNA EXTRACTION AND VIRUS ISOLATION FOR DETECTION OF BVD VIRUS IN YOUNG CALVES D. Deregt,* P.S. Carman, R.M. Clark, K.M. Burton, W.O. Olson, and S.A. Gilbert. Animal Diseases Research Institute, Canadian Food Inspection Agency, Lethbridge Laboratory, Lethbridge, Alberta, and Animal Health Laboratory, University of Guelph, Guelph, Ontario. Previously, we described a multiplex RT-PCR assay for detection and typing of bovine viral diarrhea (BVD) virus from blood of persistently infected cattle which could be used with or without RNA extraction (J. Clin. Microbiol. 1999, 37: ). In the present study, the PCR assay was evaluated for its ability to detect BVD virus in young calves as a screening tool for detection of persistent infections. Both methods, PCR after RNA extraction (rpcr) and the direct method without RNA extraction (dpcr) were applied and compared with virus isolation (VI) using diagnostic specimens. From 450 whole blood samples from Ontario calves, 47 and 39 samples were positive by rpcr and VI, respectively. From the 47 samples positive by rpcr, 45 (96 %) were also positive by dpcr when samples were tested both undiluted and diluted 1/10. In comparison to VI, the relative sensitivities of both PCR assays were 100%. The results indicate that both PCR assays can be used for screening calves for persistent infection with BVD virus.

2 COMPARISON OF SENSITIVITY FOR BVD VIRUS ISOLATIONS FROM LEUKOCYTES VERSUS SERUM Greg Nitzel*, Wendy Kneiss, Matt Ricker, Mark Goodyear, Jay Thompson, Steve Muir and Mike Huether Veterinary Medicine, Pfizer Global Research and Development, Groton, CT Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle that is distributed worldwide. BVDV, a single stranded RNA virus, is classified as a member of the genus Pestivirus in the family Flaviviridae. BVD virus occurs in two biotypes, non-cytopathic and cytopathic. BVD virus can be readily isolated from tissues, blood and serum, although antibody may interfere with isolation from serum in calves due to interference or neutralization. The objective of this study is to compare assay sensitivity in detecting viremia in calves with type 1 and 2 BVDV infection and to evaluate its relationship to clinical disease and temperature, as specified in 9CFR Two dose titration challenge studies were conducted in order to establish a suitable dose for BVD type 1 and 2 viruses administered to 7 to 9 month-old calves. BVD type 2 virus was isolated more frequently and for a longer duration from calves, as compared to the BVD type 1 virus. Furthermore, virus isolation for each biotype was more sensitive from leukocytes than isolations conducted using serum.

3 LABORATORY DIAGNOSIS OF BVDV BY USING ELISA FOR ANTIGEN AND ANTIBODY DETECTION G. Holmquist 1 *, R. Toomik 1, S. Rodgers 2, J. Lawrence 2 and A. Ballagi 1 1 IDEXX Scandinavia AB, Österbybruk, Sweden, 2 IDEXX Laboratories Inc., Westbrook, Maine The IDEXX HerdChek BVDV test family consists of a new BVDV antibody detection ELISA and two different BVDV antigen detection ELISAs. The new BVDV antibody test is an indirect ELISA, detecting antibodies for all pestivirus viral proteins. The test kit can be used to test bovine serum, plasma and milk samples. The results can be used qualitatively or quantitatively. The test has been evaluated on positive and negative samples from different geographies. Test results were also compared to serum neutralization, the golden standard for antibody detection. The ELISA results correlated well with the serum neutralization data. Furthermore, the test could be shown to have good specificity testing serum and milk from negative populations. Of the BVDV antigen ELISAs, one detects pestiviral antigens in peripheral blood leukocytes, tissues and cell culture material. The other antigen test can be used to test serum, plasma and whole blood samples. While the leukocyte test detects all the different antigenic epitopes of BVDV, the serum test is specific for the E rns glycoprotein. The BVDV Ag ELISAs were extensively evaluated. The serum test clearly detects all of the 15 different Type I and II strains tested and correlated perfectly with virus isolation data from PI animals of different geographies. It also showed to be surprisingly little affected by the presence of maternal antibodies..

4 DEVELOPMENT OF AN ENZYME LINKED IMMUNOSORBENT ASSAY TO DETECT BOVINE VIRUS DIARRHOEA VIRUS IN BLOOD, PLASMA, SERUM AND LEUCOCYTES G. van de Wetering, C. Weesepoel, L. Jacobs* Institute of Animal Science and Health (ID-Lelystad), Cedi-Diagnostics BV, Lelystad Bovine virus diarrhoea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae. Both cytopatic and noncytopatic virus strain can be distinguished. BVDV infection during pregnancy may result in an infection of the fetus. The outcome of the fetal infection depends on the age of the fetus but can result in persistently infected calves. Persistently infected cattle are the main source for virus transmission and control of BVDV must concentrate on the identification and removal of persistently infected animals. At our institute an ELISA was developed to detect BVDV antigen originating from strain Ia, Ib and II in blood plasma serum and leucocytes. The specificity was evaluated by testing negative blood field samples, plasma samples and leucocyte concentrate samples and was calculated to be 98.4 %. The sensitivity was evaluated by testing 560 positive blood field samples, 220 positive plasma field samples and 231 leucocyte concentrate samples collected from BVDV infected animal/bvdv carrier animals and was calculated to be 99.5 %. However validation was mainly done using samples from Dutch origin. At this moment the ELISA is validated in several countries. Results will be discussed

5 EVALUATION OF SERUM VIRUS ISOLATION, RT-PCR, AND IMMUNOHISTOCHEMISTRY TO DETECT BVDV PI ANIMALS Claudia Muñoz-Zanzi 1, Randall Singer 1*, Sharon Hietala 3, E. J. Ehrhart 1, Gail Scherba 1, and Richard Wallace 2 1 Department of Veterinary Pathobiology and 2 Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign. 3 California Animal Health and Food Safety Laboratory, University of California, Davis. A key component of BVDV control programs is the detection and elimination of PI animals with a reliable, rapid, and cost-efficient diagnostic method. Various diagnostic tests are currently available for detecting PI animals, including serum virus isolation, RT-PCR on whole blood, and IHC on skin biopsies. In order to evaluate the performance of these 3 tests under representative field conditions, we sampled a total of 970 animals throughout the state of Illinois, 131 from 5 beef herds and 839 from 31 dairy herds. Based on a perfect agreement between the RT-PCR and the IHC, 14 of the animals tested positive for BVDV PI, all from beef herds. Three additional beef calves tested positive by RT-PCR but negative on IHC. On retest 45 days later, these calves were negative by RT-PCR, thus indicating acute infection. The results of the serum virus isolation were not consistent with the RT-PCR and IHC, primarily when the animals were less than 3 months of age. When we included the impact of other factors, such as test cost and turnaround time, we concluded that the IHC appears to be a reliable and efficient test for large scale testing and detection of BVDV PI animals.

6 BOVINE VIRAL DIARRHEA VIRUS DIAGNOSIS USING RT-PCR SK Hietala 1 *, MC Thurmond 2, CA Munoz-Zanzi 2 California Animal Health and Food Safety Laboratory System 1, Dept. of Medicine and Epidemiology 2. University of California, Davis. Reliable detection of BVDV is critical to effective diagnosis of acute BVDV outbreaks, individual and herd-level screening for BVDV persistent infection (PI), and pre-purchase testing for herd biosecurity. Molecular diagnosis, using RT-PCR to specifically amplify genetically conserved regions in the 5=UTR of the BVDV genome is a reliable and cost-effective diagnostic tool that can be used to test serum, whole blood, or tissue samples. RT-PCR can be used in a multiplex format to detect and differentiate BVDV type I from type II, and recent innovations in real-time PCR have decreased assay time from hours or days, to less than 30 minutes. BVDV PCR has been used for over 5years in routine diagnostic testing, on more than 20,000 samples, at the California Animal Health and Food Safety laboratory. RT-PCR has advantages over standard virus isolation, microplate isolation, and tissue immunohistochemistry in that PCR is not negatively impacted by BVDV antibody or poor sample quality, as occurs with fetal tissues. PCR also lends itself to pooled-sample testing, so that large numbers of animals or entire herds can be screened cost-effectively for BVDV PI. PCR has equal sensitivity to the punch-biopsy technique for PI detection, with neither showing false positive reactions. Examples of BVDV diagnosis, including real-time PCR will be presented.

7 DILUTION OF EXTRACTS OF FRESH SKIN BIOPSIES FOR AN ANTIGEN- CAPTURE ELISA (ACE) FOR DETECTION OF BVDV Bruce W. Brodersen* 1, Roy Huchzermeier 2,, Judi Galeota 1, David R. Smith 1 David J. Steffen 1. 1.University of Nebraska, Lincoln, NE 2.Syracuse Bioanalytical, Inc., Ithaca, NY The E0 antigen of BVDV is present in the skin of cattle persistently infected (PI) with BVDV. The E0 antigen can be consistently demonstrated in skin of PI cattle by IHC with advantages of ease of specimen collection, high sensitivity, and rapid turn-around time. Fewer laboratory resources are required to perform ACE compared to IHC. Specimens utilized in this study originated from five body locations of a calf demonstrated to be PI with BVDV by virus isolation from peripheral blood leukocytes and by IHC on skin. Skin samples were either frozen (-80C) or not frozen in either PBS or tissue diluent buffer (TDB, provided by the ACE kit manufacturer). Extracts were prepared by brief vigorous agitation of skin in the buffer followed by a 10 minute incubation at ambient temperature. Extracts were tested as per ACE kit manufacturer=s instructions at dilutions of 1:1, 1:5, 1:25, and 1:125. Results obtained with ACE testing on the specimens diluted at 1:1 were in perfect agreement with IHC results. Mean normalized OD differed significantly by each dilution and was greatest for tissues diluted 1:1. Accounting for the effect of different tissues, the mean normalized OD from frozen samples was significantly higher than non-frozen tissues (all at a 1:1 dilution).

8 UTILIZATION OF AN ANTIGEN-CAPTURE ELISA ON EXTRACTS OF FRESH SKIN (ACEsk) SAMPLES FOR DETECTION OF BVDV. Bruce W. Brodersen* 1, Roy Huchzermeier 2, Edward J. Dubovi 3, Jeremiah T. Saliki 4. 1.University of Nebraska, Lincoln, NE 2.Syracuse Bioanalytical, Inc., Ithaca, NY 3.Cornell University, Ithaca, NY 4.Oklahoma State University, Stillwater, OK. The E0 protein of BVDV is expressed in skin of cattle persistently infected with BVDV. Detection of this protein in skin by IHC has advantages of ease of specimen collection, high sensitivity, and rapid turn-around time. Results of ACEsk extracts were compared against five reference methods, IHC, PCR performed on peripheral blood leukocytes (PBL), virus isolation on PBL (VI) or serum (IPMA), and ACE performed on serum. Specimens utilized originated from New York, Nebraska, and Oklahoma. Ages of animals tested ranged from one day to greater than three months. One hundred animals were tested. Thirty-nine were positive by ACEsk and at least one of the five reference methods. Sixty-one were negative by ACEsk and at least one of the reference methods. One of the specimens positive by ACEsk had discordant results compared to IPMA, but was positive by PCR. That serum had an SN titer of 1:1536 and originated from a calf that was approximately 10-days-old. The ACEsk has sensitivity equal to the reference methods PCR, IHC, and VI and appears to have a greater sensitivity than IPMA. Utilization of ACEsk has the potential for reduced reporting time in Diagnostic laboratory settings without compromise of sensitivity and specificity.

9 SERUM AS A DIAGNOSTIC SPECIMEN FOR BVDV Jeremiah T. Saliki Oklahoma State University, Stillwater, OK Required attributes of diagnostic specimens include: analyte viability/stability, ease of collection, and minimal processing before testing. Serum as a diagnostic sample for BVDV exhibits all these characteristics. Stability of BVDV in serum was evaluated by storing BVDV-positive sera under conditions mimicking normal laboratory practice. Compared to storage at B20EC, two adverse conditions (room temperature for 7 days or heat inactivation at 56EC for 1 hour) reduced virus titer only by about 2 logs from 5 logs/ml. This was deemed to be satisfactory for specimens from persistently infected (PI) animals, which typically have ~5 logs/ml. The usefulness of serum for BVDV-PI diagnosis was further evaluated using two diagnostic tests: monolayer ELISA (M-ELISA) and commercial antigen capture ELISA (ACE). A total of 408 sera were tested using both methods. The M-ELISA detected BVDV in 172 sera while ACE was positive on 161 samples. There were 18 confirmed PI animals and both tests were positive on these samples. Using IPMA as gold standard, ACE yielded relative sensitivity and specificity values of 93.6% and 100%, respectively. Both parameters were 100% when only PI animals were considered. Because of its diagnostic attributes, serum is an ideal sample for detection of BVDV- PI animals. It is highly desirable that ELISA-based BVDV antigen detection tests be able to use serum as the test specimen. Besides being faster, antigen detection tests, unlike M-ELISA, might be applicable on samples that do not contain live virus.

10 BULK MILK TANK TESTING FOR BVDV Edward J. Dubovi, Sung Kim, and Randall Renshaw Animal Health Diagnostic Laboratory Cornell University The main assumption that motives control programs for BVDV is that through the elimination of persistently infected (PI) animals one will eventually eradicate BVDV from the bovine population. Given the low prevalence of the PI animal, testing strategies necessitate the screening of every animal in the herd. For a testing strategy to be utilized, it must be cost effective and compatible with the farm management. Testing of bulk milk samples as a method for detecting PI animals in the lactating herd fits the above criteria. During the year of 2001, 775 bulk milk samples were tested by PCR and virus isolation. Of these, 6% were positive for BVDV. If we assume that the average sample represented 150 animals, bulk milk testing saved the producer over $350,000 in testing costs plus the cost savings for sample collection. The challenge of bulk milk testing is not at the laboratory level; it resides at the farm level where animal movement must be accurately documented.

11 DETECTION OF FETAL BOVINE VIRAL DIARRHEA VIRUS INFECTION BY PERCUTANEOUS COLLECTION OF FETAL FLUIDS *RJ Callan 1, JA Schnackel 2, H Van Campen 1, RG Mortimer 1, JL Cavender 3, and ES Williams 3 1 Colorado State University, Fort Collins, CO, 2 Fort Dodge Animal Health, Fort Dodge, IA 3 University of Wyoming, Laramie, WY Control of bovine viral diarrhea virus (BVDV) requires effective screening for the introduction of viral infection in addition to other herd biosecurity and vaccination practices. Pregnant cattle that are acutely infected between days of gestation can introduce BVDV into a herd through their persistently infected (PI) fetus. Standard testing of the pregnant dam cannot determine the status of the fetus. In order to identify PI fetuses, virus isolation was performed on fetal fluid collected between 175 and 246 days of gestation in 169 pregnant cattle with documented natural BVDV exposure. Right flank percutaneous fetal fluid collection was performed using an 18g 3-inch spinal needle. Fetal fluid was obtained from all 169 animals. BVDV was isolated from the fetal fluid of one fetus whose dam was also persistently infected. Calves were subsequently tested following birth and no false negative or false positive test results were identified. No complications were observed in the cows immediately following the procedure. Fourteen animals either aborted or delivered premature calves within 3 weeks of the procedure. Late term percutaneous fetal fluid collection may be useful for the determination of fetal BVDV infection and could be utilized in biosecurity programs. However, the potential risks to fetal viability must be considered.

12 INDIRECT TRANSMISSION OF BOVINE VIRAL DIARRHOEA VIRUS VIA FOETAL FLUIDS, UTERINE LOCHIAS AND CALVING PENS A. A. Lindberg *1, R. Niskanen 2, M. Stokstad 3, T. Løken 3, and S. Alenius 2 B. 1 Swedish Dairy Association, R&D, Uppsala, Sweden 2 Dept. of Ruminant Medicine and Veterinary Epidemiology, SLU, Uppsala, Sweden 3 Department of Large Animal Clinical Sciences, NVH, Oslo, Norway This study investigated if foetal fluids and/or discharge of uterine lochias from dams delivering calves persistently infected (PI) with BVDV can transmit the agent, and if susceptible cattle in close contact with them could become infected. Four trials were carried out. In trial I B III, 14 seronegative calves were actively exposed to fluids collected from dams delivering PI calves. There were 8 exposures to samples collected on the calving day and twelve exposures to samples collected on days 1 to 14 after calving. One calf, exposed to a calving day sample, seroconverted. Virus isolation was attempted on samples from day 0 and 1, and succeeded on the sample that caused seroconversion. Antibody analyses of the fluids showed a rapid increase in antibody levels already 1 day post partum. In trial IV, 6 seronegative calves were in direct contact with, or housed close to cows that had delivered PI calves. Two calves seroconverted. There seems to be a limited, but factual risk of indirect BVDV transmission via fetal fluids and uterine lochia and/or the calving environment. However, this risk is rapidly reduced after parturition. In foetal fluids, virus is probably neutralised by maternal antibodies.

13 BVDV PERSISTS IN SEMEN AFTER ACUTE INFECTION OF POST-PUBERTAL, IMMUNOCOMPETENT BULLS *M. Daniel Givens, Allen M. Heath, Kenny V. Brock and Mylissa S. D. Edens Departments of Clinical Sciences and Pathobiology College of Veterinary Medicine, Auburn University, AL Recent reports revealed the potential for BVDV to produce persistent, localized, testicular infections in post-pubertal, non-viremic bulls. This research was designed to evaluate the use of a reverse transcription-nested polymerase chain reaction (RT-nPCR) to detect BVDV in semen after acute infection of bulls. Three 2-year-old bulls (a, b and c) were intranasally inoculated with 5 x 10 5 CCID 50 of BVDV strain SD-1. Semen and serum were obtained from these bulls on days 0, 4, 6, 8, 10, 13, 17, 21, 28, 35, 42, 52, 56, 73, 88, 120, 150, and 186. Antibodies to SD-1 were not detected by virus neutralization in any bull on day 0, but were present in all bulls on days 28, 88 and 186 (all titers > 1:1,280). Virus was detected in serum of all bulls by virus isolation (until day 10) and by RT-nPCR (until days 10, 17 and 21, respectively). Virus was detected in semen of the latter 2 bulls by virus isolation (until days 10 and 21, respectively) and by RT-nPCR (consistently to day 186). Thus, viral RNA of BVDV was detected in semen of post-pubertal bulls longer than 6 months after acute infection.

14 TYPE-SPECIFIC RESPONSE TO PESTIVIRUSES IN BALB/C MICE IMMUNIZED WITH A POOL OF DNA PLASMIDS EXPRESSING STRUCTURAL GENES FROM BVDV 1, BVDV 2 AND PESTIVIRUS TYPE3 Ausama Yousif, Sandra M. Watkins, Lyle J. Braun, Darin Peterson, David J. Hurley * and Christopher C. L. Chase Veterinary Science Department and Department of Biology & Microbiol. * South Dakota State University, Brookings, SD Control of pestivirus infections in large animals is complicated by genetic variation between pestivirus types, significant genetic variation within pestiviruses, and inter-species transmission. To broaden the capacity for immune response across several different types of pestiviruses, we constructed a series of mammalian expression plasmids containing genes from BVDV 1, BVDV 2 and pestivirus type 3. The BVDV 1 and BVDV 2 constructs expressed the C and E rns genes of Singer and A125, respectively. The type 3 constructs expressed the C protein. In addition, all constructs included the nonstructural protein N pro for proper processing of downstream structural proteins. Groups of 30 BALB/c mice were immunized with: 1) a pool of pestivirus mammalian expression plasmids, 2) a commercial inactivated BVDV type 1 and 2 vaccine, or 3) the parent plasmid. The lymphoproliferative response to pestiviruses (1x10 4 and 0.5X10 4 TCID 50 /ml of Singer, A125 or ID211/CB5 in mixture, Type 1, 2 and 3 respectively) was measured on weeks 1, 2 and 3 following priming, and weeks 2, 3 and 4 weeks following booster immunization. DNA vaccinates exhibited a mild primary response to Singer when stimulated with 10 4 virus per ml the 1 st and 2 nd week post priming. The 3 rd week after the booster immunization, a secondary response was observed. The primary response to A125 peaked the 3 rd week after priming and was observed the 3 rd and 4 th week after boosting. The peak responses to the inactivated commercial vaccine were lower than those observed with DNA pool vaccination. However, the pattern of response was similar. The lymphoproliferative response to the type 3 viruses [ID211/CB5] at 10 4 /ml was high 1 week post priming, and gradually declined to the control level over 2 weeks. There was only small increase in the response to type 3 viruses on the 3 rd and 4 th week post booster and only in response to the higher virus concentration. Antibodies to the pestivirus strains, Singer, 125, ID211 and CB5 were also produced. Neutralizing antibodies were produced to BVDV 1 and BVDV 2 but not BDV. We propose conducting challenge experiments in cattle to test the protective response induced by vaccination with this set of DNA plasmids as an approach to establish broad-spectrum protection.

15 FIELD EFFICACY OF MLV VACCINES IN PREVENTING BVDV INFECTION Mark Thurmond *1, Sharon K. Hietala 2 1 Department of Medicine and Epidemiology, 2 California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California, Davis, CA Efficacy of 2 commercial BVDV MLV vaccines in preventing BVDV infection was assessed for 200 calves on 2 dairies (A and B). On dairy A, one group remained unvaccinated and one was vaccinated with a product containing both BVDV type I and II strains. On dairy B, one group was vaccinated with a product containing only BVDV type I and one was vaccinated with the product used on dairy A. Serum was collected every 30 days for 7 months and tested for SN antibodies to BVDV type I and type II. A >3-fold seroconversion >30 days after vaccination, with titers sustained >1:512, was interpreted as evidence of infection. No difference was found in type I (0.04 vs 0.04; p=0.96) or type II (0.14 vs 0.08, p=0.80) infection rates between the 2 groups in herd A. For dairy B, no difference was found in type I infection rates (0.37 vs 0.35; p=0.69), but the type II infection rate for those vaccinated with both type I and type II strains was lower (0.27) than for those vaccinated only with type I (0.55)(p=0.001). Comparative efficacy of the vaccine containing both types I and II in preventing type II infection on dairy B was 64%. Results suggest that, depending on genotypes and strains present in the vaccine and in the herd, BVDV MLV vaccination may not necessarily adequately prevent BVDV infection in calves.

16 MODIFIED LIVE TYPE 1A BOVINE VIRAL DIARRHEA VIRUS (BVDV) PROVIDES FETAL PROTECTION AGAINST CHALLENGE WITH A TYPE 2 BVDV *Schnackel, John 1 and Van Campen, Hana 2 1 Fort Dodge Animal Health, Fort Dodge, IA, 2 Colorado State University, Fort Collins, CO Twenty-one heifers vaccinated with a MLV type 1a BVDV (Singer) vaccine (Pyramid MLV 4) and eight unvaccinated control heifers were bred between 28 to 53 days post vaccination. The heifers were inoculated intranasally with 4.6 to 4.9 log 10 TCID 50 of a noncytopathic (ncp) type 2 BVDV once between days 74 to 85 of gestation. Whole blood was obtained daily from inoculation through day 21 post-inoculation (p.i.). Fetuses were recovered by C- section 21 days p.i. Pooled fetal tissues, allantoic and amniotic fluids were assayed by virus isolation (VI). Virus isolation was performed on WBC lysates prepared from whole blood samples. Serum neutralizing antibody titers to both type 1a and type 2 BVDV were determined on p.i. day 0, 21 and 35. BVDV was not isolated from the 21 fetuses (0%) of the vaccinated heifers, but was isolated from 5 of 7 fetuses (71%) from the control heifers. All control heifers with VI positive fetuses, were viremic and seroconverted to type 2 BVDV. Six of 21 vaccinated heifers were also viremic on at least one day and 12 of 21 (57%) seroconverted to type 2 BVDV. These results suggest that vaccination with MLV type 1a BVDV vaccine can provide fetal protection should the dam be exposed to an antigenically unrelated BVDV.

17 VIRUS ISOLATION AND SEQUENCING - VALUABLE DIAGNOSTIC TOOLS John F. Anderson 1, Julia F. Ridpath 2, Kurt Rossow 3 1 Valley Vet. Consulting, Cannon Falls, MN,,2 NADC/ARS/USDA, Ames, IA, 3 Vet. Diag. Lab. Univ. Minn., St. Paul, MN Frequently herd expansion results in mixing of cattle from a variety of sources. Unfortunately mixing of cattle populations also means mixing of the diseases they carry. This study involved a herd expansion which combined two existing dairy herds. Cow numbers were inadequate and outside sourcing of animals was necessary. One hundred pregnant heifers were purchased from Canada. Additional cows and heifers were obtained from Minnesota dairies. Isolation, within the limits of practicality, was provided with concurrent vaccination procedures. Label directions were closely followed in vaccination administration and timing. In the fall of 1997, 40 additional late gestation heifers were purchased. These animals were housed at a separate off-site facility to observe a quarantine period and vaccination. The vaccination history of these animals was not available and a blood sample from each animal was taken to evaluate immune status. Titers against BVDV were low (majority of animals <1:4) in these animals. These animals were not screened for persistent infection. The vaccination program in effect was based on a BVDV1a modified live vaccine. In the season following the addition of the 40 late gestation heifers 48 cows either aborted or experienced early embryonic death. The BVDV isolated from the fetal abortions belonged to the BVDV1b genotype.

18 CORRELATION OF A BVDV-1 SEROLOGICAL POTENCY TEST IN GUINEA PIGS TO THE HOST ANIMAL M. Goodyear*, G. Nitzel, W. Burdett, M. Huether Veterinary Medicine, Pfizer Global Research and Development, Groton, CT BVDV is considered to be one of the most important viral pathogens of cattle. Prophylactic vaccination is commonplace, with a variety of modified live and killed virus vaccines commercially available. Domestically, killed BVDV vaccines are tested for potency by either a host animal serology assay (9CFR ), or by an in vitro assay such as ELISA. The EU, in general, favors potency testing killed vaccines in laboratory animals. The purpose of this project was to develop a laboratory animal model for assessing the potency of EU vaccine serials containing killed BVDV-1. The critical parameters evaluated in the development of the assay were: 1. vaccine dosage in guinea pigs; 2. time of blood collection; 3. minimum immunizing dose (MID) in a host animal efficacy study; 4. correlation of guinea pig and host animal serology. The minimum immunizing dose in cattle was determined using vaccines prepared at four different antigen levels. The serologic response at day 35 from both species was determined. The geometric mean titers of each treatment group was log 2- transformed and the correlation between the guinea pig and bovine titers was determined to be The MID was within the sensitive region of the dose response curve in the guinea pig assay. The guinea pig potency test was shown to be a valid means of assessing the potency of inactivated BVDV-1 vaccines.

19 CAMPAIGN TESTING OF MODIFIED LIVE VIRUS VACCINES FOR EXTRANEOUS BVDV P. Foley* and L. Wilbur Center for Veterinary Biologics-Laboratory, USDA, APHIS, VS, Ames, IA USA The Mammalian Virology Section (MVS) of the CVB-L instituted a campaign testing program to examine modified live mammalian viral vaccines for bovine viral diarrhea virus (BVDV) contamination. The agent, BVDV, was selected due to the recent and historical occurrence of contamination of both food animal and companion animal mammalian virology products. The Supplemental Assay Method for the Detection of Extraneous Bovine Viral Diarrhea Virus in Modified-Live Vaccines (SAM 108), was revised to increase sensitivity, allow blockage, and encompass all modified live mammalian products. A survey of licensed product outlines was conducted and the cell lines used in production categorized. A knowledge base was established based on literature citations, sources of potential contamination, experience, and examination of cell lines without sufficient knowledge of BVDV permissiveness. Based on this information, products were identified that had the potential to support BVD replication. The BVD extraneous agent test code (906-PU0) was added to all such identified MLV products. From March 2, 2001, through March 15, 2001, and from January 22, 2002, through February 4, 2002, every serial with assigned test code 906-PU0 was selected by the Biological Materials Processing Section, National Veterinary Services Laboratories, for testing by the MVS. The results of this testing will be discussed.

20 TESTING OF BVDV-CONTAINING INACTIVATED VIRUS VACCINES FOR LIVE BVDV P. Foley*, D. Toms, and L. Wilbur Center for Veterinary Biologics-Laboratory, USDA, APHIS, VS, Ames, IA USA An in vitro test method was developed using differential centrifugation, cell culture, and fluorescent antibody techniques for detecting live BVDV in killed virus (KV) vaccines containing BVDV. The procedures were recorded in the draft Supplemental Assay Method for the Detection of Live Bovine Viral Diarrhea Virus in Inactivated Vaccines containing BVDV (SAM 136). Vials of final product for nine US-licensed KV vaccines were inoculated directly with one of four CVB-L reference viruses. The reference viruses included Singer, Oregon C-24V, 890, and New York-1 strains. These vials were stored at 4 o C for a year, and periodically tested for live BVDV. The amounts of added virus per vial resulted in 4, 20, 100, or 500 TCID 50 per inoculum into a 75 cm 2 flask containing a BVDV-susceptible cell monolayer. In addition to fluorescent antibody testing on cell culture slides, PCR was performed using cell lysates from the third pass harvest of inoculated monolayers. Results of this testing will be presented.

21 THE SWEDISH BVDV SCHEME IN THE FINAL PHASE B MEASURES NEEDED TO CONCLUDE THE ERADICATION AND TO MAINTAIN FUTURE FREEDOM A. Lindberg Swedish Dairy Association, Uppsala, Sweden A nationally available scheme to eradicate BVDV was started in Sweden on 1 September Farmers have affiliated on a voluntary basis and have to conform to a sampling and biosecurity protocol in order to attain and maintain certification as being free from the infection. In 1993, 52 % of the dairy herds had signs of recent or current infection as judged from results from bulk milk screenings. In January 2002, 93 and 88% of the dairy and beef herds, respectively, were declared free from BVDV. The incidence of new infections has been reduced to below 1 case per 200 herd-years-at-risk. Today, less than 4% of all herds have an ongoing infection. To avoid a low prevalence steady state situation, work has now been initiated to minimise the time left before the infection is eradicated. Focus is on finding means to shorten the time from infection to detection, from detection to action and from action to the conclusion of virus clearance in infected herds. In addition, measures are iteratively being taken to (re- )establish a high awareness about biosecurity issues among farmers and professionals providing ambulatory services within the agri-business. Also, the regulations concerning the importation of semen, embryos and livestock are being revised and updated to be in concordance with the needs of a BVDV free region, in cooperation with the other Scandinavian countries.

22 THE ERADICATION OF BVD IN DENMARK Viggo Bitsch 1*, Karen-Else L. Hansen 1, Kurt B. Larsen 1, Ole Andersen 1 & Leif Rønsholt 2 1 Danish Milk and Livestock Federation, Frederiks Alle 22, DK-8000 Aarhus C. 2 Danish Veterinary Institute, Lindholm, DK-4771 Kalvehave The BVD eradication programme in Denmark - with a very high prevalence of BVD - was initiated January Before that, several pilot studies had been undertaken in order to reveal the possibilities of eradication. (1) The Samsoe infection control study was initiated early in New ELISA tests for demonstration of BVD virus and BVD antibody had just been developed at the Danish Veterinary Virus Research Institute, and the study showed that the ELISAs were reliable and that spread of the infection ceased after removal of the persistently infected animals found. (2) The BVD vaccination study using a new Danish BVD vaccine did not demonstrate sufficient protection of foetuses of vaccinated heifers after exposure to contact infection, because of which the possibility to use a vaccine for infection control was given up. Finally, (3) the bulk tank milk study demonstrated the value of a bulk milk antibody test in monitoring the infection status of dairy herds. As from early 1996 legislative measures were introduced to support the programme. The most important lines of the BVD order were (1) obligation for all farmers to determine the infection status of their herds, (2) individual serological control of all animals moved to other herds, auction sales, exhibitions, and common pastures, and (3) no admission of animals from non-bvd-free herds to pastures unless after control to secure that they were not persistently infected. At present less than 1 % of dairy herds and 2 % of non-dairy herds are registered as BVDinfected, but all these can be considered to be under eradication. An important feature in the last few years has been the relatively high but steadily decreasing number of new infected herds. The poster will present details of the basic lines and the course of the eradication programme. The importance of legislation and the causes of new infections will be elucidated.

23 THE PREVELANCE OF BOVINE VIRAL DIARRHEA VIRUS (BVDV) INFECTIONS IN TRAKYA DISTRICT OF TURKEY AND THE DISTRIBUTION OF VIRAL ANTIGENS IN THE GENITAL SYSTEM TISSUES OF CATTLE Ibrahim FýRAT 1 *, HHakan BOZKURT 2, Seyyal AK 3, Veli GULYAZ 4, Kemal AK 5 1 Istanbul University, Veterinary Faculty, Department of Pathology, Ýstanbul, Turkey 2 Istanbul University, Veterinary Faculty, Department of Histology and Embriyology, Ýstanbul, Turkey 3 Istanbul University, Veterinary Faculty, Department of Microbiology, Ýstanbul, Turkey 4 Veterinary Research Institute, Pendik, Istanbul-Turkey 5 Istanbul University, Veterinary Faculty, Department of Reproduction and Artificial Insemination, Ýstanbul, Turkey The aim of this study was to detect prevalance of BVDV infection and existence of PI cattle in Trakya District. In total 260 samples of leucocytes were isolated from dairy cows (65 samples) and breeding bulls (65 samples) also from cows and bulls in the slaughterhouses (65 samples from each). It was found that in total of 35 (13.46 %) cattle, 16 alive, and 19 (15 female, 4 male) slaughtered were positive for BVDV antigen. BVDV antigens were also detected on 4 of the second leucocyte samples, which were taken from 16 BVDV positive animals. Also, the cellular localisation and distrubition of BVD viral antigens were investigated in genital system tissues of those 65 non-pregnant dairy cows and in 65 bulls from slaughterhouses. For this, genital system tissue samples were marked using the immunperoxidase method on their paraffin block. BVD viral antigens were determined in 15 of the 65 non-pregnant cows (consistent with the cell culture results) using the indirect immunoperoxidase method. BVD viral antigens which were presents in macrophage-like cells in the stroma of the ovaries and uterus were detected in all these tissues. No BVD viral antigens were seen in the samples of testicles, epididymis, vesicula seminalis and prostate in the male animals. In all animals, including the BVDV positive ones, no pathologic lesion was observed except periodically non-specific subepithelial or stromal mononuclear cell infiltrations. This project was supported by the Research Fund of Istanbul University. Project number:1126/010598

24 BOVINE VIRAL DIARRHEA VIRUS IMPAIRS MACROPHAGES FUNCTION AND MARKER EXPRESSION IN VITRO G Elmowalid, BL Ransom, LJ Braun, A Young and CCL Chase Dept. Vet. Sci., South Dakota State University, Brookings, SD U.S.A. Mononuclear phagocytes (monocytes and macrophages) are known to play a significant role in the cellular defense against infection with bovine viral diarrhea virus (BVDV). Infection with BVDV has several different outcomes and results in alteration of important mononuclear cell inflammatory functions. A bovine monocyte-derived macrophages (MDM) experimental system was established for studying the interaction with BVDV. Both genotypes (BVDV-1 and BVDV-2) and biotypes: cytopathic (cp) and non-cytopathic (ncp) were studied in this system. Experiments were carried out to determine the viral antigen expression and replication in MDM and the effects of BVDV infection on macrophages functional properties and their surface marker expression. BVDV binds, penetrates and expresses its protein at h post infection (PI). Indirect immunofluorescence indicated that MDM are susceptible to both cp and ncp BVDV-1 and BVDV-2. Although the cells were infected, no infectious cp or ncp BVDV type 1 and/or BVDV type 2 virus was produced at h PI in MDM. Phagocytic and microbicidal activities were significantly reduced in MDM infected with cp and ncp BVDV-1 and BVDV-2 whereas the control displayed a significant increase in these functions. Cp, but not ncp BVDV induced changes in MDM morphology and viability. Some of the infected MDM showed cell fusion, cell rounding and cell size variation. MDM surface marker expression level was following infection with BVDV. MHC-I, MHC-II and CD14 were significantly down regulated at h PI. CD11b expression level was slightly down regulated while CD11c expression level did not change at h post infection. These results demonstrated that BVDV infection can reduce the mononuclear phagocytes inflammatory functions and alter the virus pathogenesis and can predispose animals to other microbial infections.

25 INHIBITION OF BOVINE VIRAL DIARRHEA VIRUS REPLICATION BY INTERFERON-ALPHA THROUGH INHIBITION OF VIRAL RNA SYNTHESIS. AA Elsheikh, D Benfield, LJ Braun and CCL Chase Dept. Vet. Sci., South Dakota State University, Brookings, SD USA Viral and cellular protein synthesis stop abruptly in interferon (IFN) treated infected cells. This lethal defense is carried out by at least two cellular proteins, dsrna-activated protein kinase (PKR) and ribonuclease L (Rnase L). PKR function is mainly rendering the cells incapable of new protein synthesis, while Rnase L function is degradation of most cellular and viral RNA species. Prolonged activity of IFN-induced proteins leads to cell death by apoptosis. Here, we demonstrated that INF-alpha inhibit both BVDV biotypes growth in vitro, however, its effect on CP strain vastly exceed the effect on NCP strains. The maximum effect of IFN-alpha was at the early stages of infection (up to 10 h post infection). At the late stages of infection (around 30h post infection), treatment with IFN showed insignificant effect. The addition of 2-aminopurine (2-AP), an inhibitor of PKR, did not rescue the virus growth, suggesting little role for PKR in viral apoptosis. In contrast, IFN treatment greatly diminished the RNA level especially of CP biotype. In summary, this study demonstrated IFN-alpha inhibits BVDV growth and its effect is dose, time and biotype dependent. It also suggests that IFN inhibition of virus replication and induction of apoptosis is carried out through Rnase-L rather than through PKR.

26 COMPARISON OF NEUTRALIZING ANTIBODIES TO TYPE 1A, 1B OR 2 BOVINE VIRAL DIARRHEA VIRUS FROM EXPERIMENTALLY INFECTED AND VACCINATED CATTLE. *Hana Van Campen 1, Lisa Jones 2, Zhi-Chang Xu 3 and John A. Schnackel 3 1 Department of Microbiology, Colorado State University, Fort Collins, CO 2 College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 2 Fort Dodge Animal Health, Fort Dodge, IA Serum neutralizing (SN) antibody titers to cytopathic (cp) type 1a, type 1b and type 2 bovine viral diarrhea viruses (BVDV) were determined for yearling beef heifers immunized with a modified live cytopathic type 1a BVDV vaccine, or inoculated with a noncytopathic (ncp) type 1b or a ncp type 2 BVDV. Type-specific and cross-reactive SN titers were found to all three test viruses in an individual animal, with the highest SN titers to the same genotype of BVDV as the challenge or vaccine virus. Exceptions occurred in individual animals emphasizing the importance of comparing the SN titers for several animals in a herd. Comparing SN titers to different BVDV genotypes during field investigations can be used to distinguish BVDV infection from vaccination.