ISRAEL JOURNAL OF VETERINARY MEDICINE BOVINE HERPESVIRUS 4 IN ISRAELI DAIRY CATTLE ISOLATION, PCR AND SEROLOGY Fridgut O. and Stram Y. Division of Virology, Kimron Veterinary Institute, Beit Dagan POB 12 Introduction In the winters of 2000 and 2001, outbreaks of postparturient necrotic vulvovaginitis reported in three dairy herds in northeastern Israel. In two of the affected herds the outbreaks began several months after introduction of pregnant animals, in the context of merging dairy herds into large production units. In the first season cows of all ages were affected following parturition, with primiparous and introduced stock being most severely affected. Clinical signs included vaginitis and metritis that did not respond to antiseptic and antibiotic treatments, decreased milk production, prolonged non-conception, peritonitis and death. In 2000 and 2001 cases emerged over a period of about four months. During the first season, vaginal swabs were sent to Kimron Veterinary Institute (KVI). Some of these swabs provided isolations of bovine herpesvirus 1 (BHV1) and mixed bacteria. When the disease reappeared in 2001, a multi-disciplinary team from KVI visited two herds. Samples taken during the visit yielded a novel, unidentified virus and an unusual black anaerobic bacterium, identified as Porphyromonas levii. The viral isolate was identified as a herpesvirus by negative-stain electron microscopy, following which we set up PCR and immunofluorescent assays for bovine herpesviruses. The virus was identified as bovine herpesvirus 4 (BHV4). BHV4 is present in cattle worldwide and has been isolated from a variety of clinical states such as mastitis, pneumonia, metritis, vaginitis, conjunctivitis, interdigital dermatitis, abortions,, and from healthy cattle (5). BHV4, like all herpesviruses, can cause a chronic latent infection, which is activated when the immune system is stressed. Contrary to other bovine herpesviruses, there is no proof that BHV4 is a pathogen causing a well-defined disease. Controlled infection studies have rarely reproduced disease. In one study, pregnant heifers were infected with the virus and developed metritis after parturition (14). Calves infected with the virus developed a mild respiratory disease (6, 13). Other studies suggest that BHV4 is a risk factor for abortion, metritis and reproductive deficiencies (2,8,9,10,13). There are studies examining molecular variation and varying pathogenic potential of different isolates (11,12). Results of serological surveys differ between countries, regions and herds, ranging from very low to over eighty percent seroprevalence. There is one report from 1989 describing the isolation of BHV4 from vaginal swabs from cows in Israel which had aborted or suffered from metritis (1) Once assays for BHV were established, BHV4 was identified several times from aborted fetuses in 2001 and 2002. The objectives of this study were to examine the relationship between the presence of BHV4 and development of post-parturient necrotic vulvovaginitis, to determine the seroprevalence of BHV4 in dairy herds and to test for BHV4 in aborted fetuses by PCR and by isolation in cell culture Materials and Methods Animals We conducted a clinical and laboratory investigation at one of the affected farms during 6 visits in January to the end of April 2002, and at each visit we took vaginal swabs for bacteriological and virological tests from 32 first calvers that developed post-parturient necrotic vulvovaginitis, and from 10 asymptomatic animals - 5 pregnant heifers, and 5 post-parturient cows. In April 2002, 28 of the first calvers were bled for antibody testing, from 127 heifers, ages 1 to 16 months for BHV4 antibodies, and for BHV1 in an attempt to determine the time of infection. A total of 819 sera from 146 herds were submitted to KVI during 2003, mostly because of abortions, and were tested for BHV4 antibodies. A total of 93 aborted fetuses were tested for BHV4 by PCR and by 2 passages in cell culture. Vaginal swabs were placed in physiological medium supplemented with antibiotics and fetal bovine serum. The samples were kept at -80oC until testing. Virus isolation was made on Madin Darby bovine kidney (MDBK) cell line over three weeklong passages for vaginal swabs, and two passages for abortions. Samples that caused a cytopathic effect were tested by immunofluorescense for BHV4 and BHV1 with antisera provided by USDA NVSL, Ames Iowa, and an anti-bovine IgG FITCconjugate (Sigma Israel). Uninfected MDBK and positive virus controls (NVSL, Ames Iowa) were included in all immunofluorescent and PCR assays. BHV4 serology was made with Cypress Diagnostics ELISA (Langdorp, Belgium), and for BHV1 using SVANOVA ELISA (Uppsala, Sweden). Cut-off values were calculated according to the manufacturers' instructions. PCR We analyzed the sequences of bovine herpesviruses 1, 2 and 4 stored in Genbank to select unique sequences for selecting primers that could differentiate between the 3 viruses. The gB gene showed the greatest differentiation, and Oligo program (Molecular Biology Insight, USA) was used to design 3 sets of primers. These primers were analyzed by Fasta 3 or Blast programs to assure specificity for the designated viruses (results not shown). DNA was extracted using DNeasy tissue kit (Qiagen Germany) according to the manufacturer's instructions, and was denatured by boiling for 5 minutes. PCR conditions were 98oC for 1 minute, 33 cycles of 66 oC for 45 seconds, 72 oC for 50 seconds and 98oC for 25 seconds. PCR products were separated by electrophoresis in an agarose gel stained with 2% ethidium bromide. Results Clinical and diagnostic investigation of the outbreak of necrotic vulvovaginitis (NVV) All 32 heifers that calved during the period of the study developed NVV, and of these BHV4 was isolated or identified by PCR in 30 of them. In several heifers, BHV4 was isolated repeatedly over a period of more than a month, and in some cases BHV1 was isolated at the same time. Only BHV1 was isolated following clinical resolution of NVV. In addition to virus recovery, Porphyromonas levii was a consistent finding in all 32 first-calvers showing clinical signs of NVV. A description of the bacteriological findings has been published elsewhere(7). Table 1 with some modification, originates from this publication. Five asymptomatic postparturient cows and 5 pregnants heifers were also sampled, BHV4 was isolated from 2 heifers but not from the cows, while 4 cows were positive by PCR. No further cases of NVV were reported in the investigated herd between April 2002 and January 2003. The correlation between virus isolation and PCR made directly on the swabs was poor at first. However, performing 3 passages in cell culture and changing the PCR reaction parameters resulted in an improved in the correlation. Serology Following resolution of all clinical cases, blood samples were taken from 29 animals from the study: 19 first-calvers, 5 postparturient cows and 5 pregnant heifers. Table 2 presents the antibody results and virus recoveries. Female calves from the outbreak herd were transferred after birth to separate premises to be raised until pregnant, whereupon they were moved back to the adult herd. All the heifers were tested for antibodies to BHV4 and BHV1. The results are given in Table 3. Four single cases of NVV in first-calvers were examined. BHV4 and P. levii were isolated from vaginal swabs from all 4 cases. Samples taken from first-calvers not showing NVV on the same farms were all negative. Other BHV4 findings BHV4 was isolated from single cases in 4 other herds: the brain of a cow with encephalitis, the lungs of a cow with pneumonia, a case of chronic conjunctivitis, and in addition to BHV1 in an outbreak of rhinotracheitis-keratoconjuntivitis. Abortions. By PCR, we retroactively identified 4 isolations from aborted bovine fetuses from 2001 and 2002. In 2003, in addition to 2 passages in MDBK cell culture, 96 bovine abortions were tested by PCR. BHV4 was not isolated, but 6 samples were PCR positive. One of the fetuses was from a farm with multiple abortions, whereas 4 other samples from this farm were negative. Serology 819 serum samples from 146 herds submitted to the laboratory, mostly because of abortions were examined; overall, the seroprevalence was 40 percent. Ninety nine herds (68 percent of the herds tested) had seropositive cows. 106 sera were from herds from which the virus had been isolated. In this subgroup, seroprevalence was 59 percent (62 positive sera). PCR Each set of primers was tested with each of 3 reference viruses (NVSL, Ames Iowa) and reacted only with their designated herpesvirus (Figure1). Figure 1. PCR for identification of bovine herpesviruses A. Primers BHVB1f and BHV1r to identify BHV1. Lanel 1 BHV1, lane 2 BHV2, lane 3 BHV4 B11-42, lane 4 BHV4 86-068, lane 5 Israeli isolate. B. Primers BHVB2f and BHV2r to identify BHV2. Lanes as for A. C. Primers BHVB4f and BHV4r to identify BHV4. Lanes as for A. Discussion The outbreaks of necrotic vulvovaginitis raised several questions: was BHV4 an exotic incursion into the affected herds, or were the initial findings incidental? Should BHV4 tests be mandatory for herds that are to be merged? And considering reports from world literature and BHV4 isolations from abortions in Israel, is it a major abortigenic pathogen here? Our study indicated that BHV4 is widespread in Israeli dairy herds and was not an exotic incursion into the 3 affected herds in the Golan Heights. Serological testing of cows from 146 herds showed reactors in 99 herds. BHV4 was isolated or identified by PBR in 21 herds from a variety of clinical samples received from 2001 to 2003. The virus was found most often in aborted fetuses and vaginal swabs from cattle with NVV, where the anaerobic bacterium, Porphyromonas levii was found consistently. Our findings both in the outbreaks and sporadic cases of NVV seem to indicate a close relationship between the virus, the bacterium and the clinical syndrome. Yet despite the prevalence of BHV4 in dairy herds and sporadic cases of NVV in some of them, the 3 outbreaks occured in winter, on the Golan Heights, and 2 of them appeared within months of herd merging. These findings thus indicate a multifactorial background for outbreaks of the disease. The seroprevalence in the follow-up herd was low compared to the general prevalence of 40 percent and the prevalence in other herds with sporadic isolations (60 percent). 17 first-calvers from which BHV4 had been isolated in January and February 2002 were not seropositive in April. Other groups have reported that in herds suffering endometritis with involvement of BHV4, serological tests showed low antibody levels that disappeared with time (9). The same result occurred in controlled infectivity experiments in pregnant cows (14). Perhaps the ELISA we used was not sufficiently sensitive, and perhaps the lack of a serological response indicated a true deficiency of the immune response that might have contributed to the clinical outcome. A more structured serological study is required to answer these concerns. In any case, at the present time, antibody testing is an insensitive method to monitor BHV4 presence. Although PCR was more sensitive for detecting BHV4 in aborted fetuses than 2 passages in MDBK, BHV4 remained a sporadic finding and was not implicated as a cause of abortion storms. Acknowledgments This study was supported by an ISRAEL DAIRY BOARD grant 848-0302-03. Thanks to Shoshi Raz and Yekutiel Shreiver from the investigated farm, to Dr Daniel Elad, Director of the Bacteriology division KVI, to clinical veterinarians Doron Tiomkin and Nir Alpert, and to Raya Blumenkrantz, Rita Paz, Larissa Kuznetzov, Dr. Ditza Rotenberg, Virology division KVI. References 1. Elad D, Friedgut 0, Alpert N, Stram Y, Lahav D, Tiomkin D, Avramson M, Grinberg K, Bernstein M (2004). Bovine necrotic vulvovaginitis associated with Porphyromonas levii. EED vol 10, 2004. 2. Egyed L Bovine herpesvirus type 4: A special herpesvirus (Review article). Acta Veterinaria Hungarica 48 (4), 501-513, 2000. 3. 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