Diagnostic investigation of bovine viral diarrhea infection in a Minnesota dairy herd

Transcription

1 J Vet Diagn Invest 1:57-61 (1989) Diagnostic investigation of bovine viral diarrhea infection in a Minnesota dairy herd Ronald E. Werdin, Trevor R. Ames, Sagar M. Goyal, Gordon P. DeVries Abstract. Bovine viral diarrhea (BVD) virus infection was diagnosed in neonatal calves with enteritis. Successful diagnostic procedures included direct immunofluorescence of frozen tissue sections, histopathology, and virus isolation. Virus isolation from buffy coats and serum was successful in detecting infected animals, whereas direct immunofluorescence of buffy coat samples was found to be less reliable. Virus was not isolated from any fecal samples. Booster vaccinations and the culling of animals shedding virus resulted in improved calf viability in this herd. It is suggested that procedures for the diagnosis of BVD virus infection should always be included in the diagnosis of neonatal calf enteritis. Bovine viral diarrhea (BVD) virus was first recognized in the United States in 1946 in association with disease outbreaks that were characterized by diarrhea and erosive lesions of the digestive tract. 17 The virus is recognized as having worldwide distribution, with serum antibody prevalence in cattle ranging from 50 to 90%. ll The wide spectrum of disease associated with BVD virus infection includes subclinical infections, diarrhea, immunosuppression, repeat breeding problems, abortion and mummification, congenital defects, immunotolerance and persistent infection, and acute and chronic mucosal disease. l,2 Recent experimental studies suggest new explanations for the pathogenesis of the various forms of clinical disease. 7,8 Cattle may become persistently infected with noncytopathic BVD virus while in utero? Persistently infected cattle may remain asymptomatic after birth 16 or may develop acute or chronic fatal mucosal disease. 3,7,8 Cattle not persistently infected may have subclinical infections or develop nonfatal acute bovine viral diarrhea. 5,10,12 Calves infected in utero may be born with congenital defects 1,2,5,12 or may appear normal? If infection occurs before 125 days of gestation, normal-appearing calves may be immunotolerant to BVD virus and as a result persistently infected with the virus. These calves may be subsequently infected with a cytopathic strain of BVD virus, which may result in signs similar to mucosal disease of adult cattle 6,8 Persistently infected From the Veterinary Diagnostic Laboratory, Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St. Paul, MN (Werdin, Goyal), the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minneosta, St. Paul, MN (Ames), and Veterinary Practitioner, Pierz, MN (DeVries). Presented at the 30th Annual Meeting of the AAVLD, Salt Lake City, UT, October 26-27, Received for publication October 27, calves have been reported to be unthrifty and immunocompromised, and as a result are prone to a variety of bacterial infections. 3 Calves that receive little colostrum but are not persistently infected may exhibit signs of bovine viral diarrhea and immunosuppression if infected with cytopathic BVD virus. l3 The form of disease that is expressed in calves is probably a function of the stage of gestation when infection occurred, how much virus is circulating on the farm during the neonatal period, and how much specific colostral protection was received by the calf. This report describes an investigation of a dairy herd with active BVD infection manifested, in part, by neonatal calf diarrhea. Materials and methods Herd history. A north-central Minnesota Holstein dairy herd with approximately 60 cows encountered severe calf mortality from July 1986 until June Information obtained from herd records disclosed that 29 of 55 calves born during that period died between the ages of 1 and 20 days. Some of the bull calves born during that period were sold at 1 week of age; mortality data on those calves were not available. Seven of 14 calves born in February 1987 died, most showing signs of diarrhea. All cows had been immunized with rota/coronavirus vaccine 3-4 weeks prior to calving and received 1 dose of a killed BVD, infectious bovine rhinotracheitis (IBR), and para-influenza type 3 (PI-3) vaccine approximately 1 year previous to the calf losses. Treatment of the diarrheic and weak calves did not prove effective. In early March 1987, a live 1-week-old and a dead 2-weekold calf were submitted to the Minnesota Veterinary Diagnostic Laboratory for necropsy. Bovine viral diarrhea virus infection was diagnosed in both. On March 25 a field trip was conducted to the premises of the infected herd to collect samples to determine the presence of BVD virus and its antibodies in this herd. A booster vaccination using a killed BVD, IBR, and PI-3 virus vaccine was given on May 28 and was repeated again 3 weeks later. Specimen collection. A total of 71 animals were included

2 58 Werdin et al. Figure 1. Photomicrograph of section of ileum. Crypts are dilated and contain mucus, neutrophils, and cellular debris (small arrow). Large arrow points to a Peyer s patch showing depletion of lymphocytes. HE stain. in the study. The animals were bled on March 25, 1987, and July 9, Paired samples were obtained from 54 animals. At each bleeding, a sample of blood was obtained in a plain tube a and a heparinized tube for the separation of serum and buffy coat cells, respectively. Fecal samples were obtained from 6 animals on initial visit and from 14 animals on the subsequent visit. Specimenpreparation. The blood samples from plain tubes were allowed to clot and the serum was separated by centrifugation and was used for virus isolation and serology. Buffy coat was obtained by mixing heparinized blood with an equal volume of Hanks balanced salt solution (HBSS) containing 5 units of heparin/ml. Ten milliliters of diluted blood was layered on 5 ml of Ficoll-Paque b and was centrifuged at 600 x g for 25 min. Buffy coat cells were washed twice with HBSS containing 0.5% sodium citrate. Direct immunofluorescence assay (FAT). A drop of buffy coat cell suspension was placed on a clean glass slide and fixed in acetone. Fixed cells were stained with fluorescein conjugate against BVD virus and other viruses as detailed in the Results section, counterstained with Evans blue, and examined by fluorescence microscopy. Tissue samples from the dead calves were flash frozen at -70 C in isopentane, mounted on a cryostat with OTC compound, d and sectioned at 6 μm. Mounted sections were fixed in acetone for 10 min and stained by routine methods using fluorescein conjugate and examined by fluorescence microscopy. Virus isolation. Attempts were made to isolate BVD virus from buffy coat cells, serum, fecal samples, and tissue samples (spleen and lung) of the dead calves. A 10% suspension of feces or tissues was made in HBSS, centrifuged to remove debris, and the supernatant was used for virus isolation. All 4 sample types were inoculated in duplicate in bovine turbinate (BT) cells in 24-well plates containing coverslips. Infected cells were incubated at 36 C in a 5% CO, incubator. Coverslips were harvested on the sixth day postinfection and were stained with fluorescein conjugate against BVD virus. Samples were blind passaged 3 times in BT cells before calling them negative. Viral serology. All sera were heat-inactivated and tested by virus neutralization (VN) test for the presence of antibodies against BVD virus by using a previously described procedure. 2O Pathological examination. Tissues for histologic examination were fixed in 10% neutral buffered formalin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE) by standard methods. 15 Bacteriological examination. Standard methods were used for bacterial isolation and identification. Results Gross lesions. Both calves necropsied in March 1987 revealed similar gross lesions and both were thin. There was serous atrophy of epicardial and perirenal fat depots. Umbilical abscesses and bronchopneumonia were observed in both. Intestinal tracts contained a pasty yellow-colored fetid material. No oral, esophageal, or gastric lesions were noted. Cryptosporidia were not found. Microscopic lesions. Sections of jejunum and ileum examined histologically showed moderate numbers of crypts containing mucus, sloughed necrotic epithelial cells, and neutrophils (Fig. 1). The lymphocytes in the Peyer s patches of the ileum were moderately to severely depleted (Fig. 1). Depletion of lymphocytes was observed in splenic sections (Fig. 2). Suppurative bronchopneumonia was observed in lung sections. No sig-

3 Diagnosis of BVD infection Figure 2. Photomicrograph of section of spleen with severe depletion of lymphocytes. HE stain. nificant lesions were observed in sections of other tissues collected. Bacteriologic studies. Pasteurella multocida was isolated from lung tissue. Pasteurella multocida, Actinomyces pyogenes, and Streptococcus sp. were cultured from the umbilical lesions. Intestinal cultures yielded nonhemolytic Escherichia coli (negative for K99 antigen by direct immunofluorescence). No Salmonella was isolated. Virologic studies. Direct immunofluorescence of frozen sections of colon and ileum from both calves was positive for BVD virus and negative for rota- and coronaviruses. Frozen sections of lung were positive for BVD by immunofluorescence, but were negative for IBR, PI-3, and bovine respiratory syncytial virus (BRSV). Noncytopathic BVD virus was isolated from tissue composites of both calves. Bovine viral diarrhea virus was isolated from buffy coats of 1 cow and 2 calves and from a serum sample of 1 cow bled on initial visit. No virus was isolated from fecal samples. Results of FAT of buffy coats were equivocal on multiple animals. From the samples collected on the second farm visit, 3 were positive for virus isolation (Table 1). Two of 3 calves positive for BVD virus in buffy coats also were positive for virus in serum (Table 1). Antibody titers of these calves on second bleeding were very low (1:2 and 1:4). Serology. Results of serologic studies are shown in Table 2. Serologic results of the first bleeding revealed that some of the animals had low serotiters to BVD virus. Most of the calves showed VN titers to BVD of 1:32. Seventeen of 54 animals had a 4-fold or more rise in antibody titers after booster immunization. In general the animals with low titers on initial bleeding Table 1. Isolation of bovine viral diarrhea (BVD) virus from cattle before and after booster vaccination. Animal Airian cow Tony cow Penger cow 0 calf Y-24 calf B-23 calf Virus isolation* Serology Buffy coat Serum BVD titer Before After Before After Before After Age (mo) booster booster booster booster booster booster :512 1: :2,048 1:1, :512 1:2, ND - ND 1:32 ND :32 1:2 2 ND + ND + ND 1:4 * Samples found negative (-) and positive (+) for BVD virus isolation. Sold after initial visit. ND = not done.

4 60 Werdin et al. Table 2. Antibody titers against bovine viral diarrhea (BVD) virus in cattle before and after booster vaccination. Virus neutralization titer <l:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 1:2,048 Total Number of animals showing serum neutralizing antibodies to BVD virus Before booster After booster ( 1:64) showed a 4-fold rise in titers after booster inoculations, whereas animals having high antibody titers on initial bleeding were less likely to show anamnestic response. Bovine viral diarrhea virus titers corresponding to the animals from which virus was isolated are shown in Table 1. Herd history following vaccination. One calf from which BVD virus was isolated on the second herd bleeding was brought to the Diagnostic Laboratory for necropsy. This calf had lesions similar to those of the original 2 calves that were necropsied. Noncytopathic BVD virus was isolated from blood and tissues (lung, spleen, mesenteric lymph node, and intestines). The owner reported that all calves born after the booster vaccination were alive and healthy to date. Discussion This case represents an example of a herd in which a BVD vaccination program was initiated but was discontinued. The case confirms the reports of others that BVD diagnostic procedures must be included in routine neonatal disease diagnosis, for both dairy and beef calves. 9 This herd was unusual in that the only form of the disease present was unthrifty, persistently infected calves that died from chronic viral or secondary bacterial infections. The lesions attributable to BVD virus observed in the affected calves in this case were restricted to the intestinal tract and lymphoid organs. No oral or esophageal lesions were observed in these calves, which is in agreement with some workers 9 but not with others. l3,l9 Secondary bacterial pneumonia observed in the 3 calves necropsied in this study has been observed by others. 9,14 Direct immunofluorescence techniques performed on tissue sections and virus isolation from tissue specimens appear to be successful diagnostic methods. Direct FAT performed on buffy coat smears was hampered by nonspecific fluorescence and proved to be unreliable compared to virus isolation in identifying animals harboring virus. This is in contrast to results obtained in calves experimentally infected with cytopathic strains of BVD. 4 Virus isolation from buffy coats, however, proved to be a successful technique. Buffy coat samples from cows were positive on first bleeding but were negative on the second bleeding. This may indicate an acute infection and not a persistent carrier state (S. R. Bolin, personal communication, 1987). Virus was isolated from buffy coats of several calves on 2 consecutive bleedings and was isolated from the sera of these calves. Serum is considered to be a preferred sample for virus isolation from persistent carrier animals (S. R. Bolin, personal communication, 1987). Bovine viral diarrhea virus has been shown to be stable in blood for up to 5 days postcollection stored at room temperature. 18 Bovine viral diarrhea virus antibody titers of these 2 calves at last bleeding were 1:2 and 1:4, suggesting that these 2 clinically poor-doing calves were probably immune-tolerant carriers. In this study blood appeared to be a better specimen in diagnosing carrier animals than feces because no virus was isolated from fecal samples. Despite relatively good management procedures, almost all of the newborn calves died in this herd. Boostering titers in the cows by repeated vaccinations with a killed BVD vaccine appears to have greatly improved calf viability. Infected animals in the herd were identified and removed. Removal of carrier cows may be helpful in decreasing future incidence of BVD. The results illustrate the benefits of serologic and virus isolation studies on a herd basis despite their relatively high costs. The results of this study demonstrate that BVD infection should always be considered as a possible cause of neonatal calf diarrhea. Diagnostic procedures for this pathogen should be included in all calf enteritis cases. Sources and manufacturers a. Vacutainer, Becton-Dickinson, Rutherford, NJ. b. Pharmacia, Piscataway, NJ. c. National Veterinary Services Laboratory, Ames, IA. d. LabTek Division, Miles Laboratories, Naperville, IL. References 1. Ames TR: 1986, The causative agent of BVD: its epidemiology and pathogenesis. Vet Med 81: Baker JC: 1987, Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 190: Barber DML, Nettleton JA, Herring JA: 1985, Disease in a dairy herd associated with the introduction and spread of bovine virus diarrhea virus. Vet Rec 117: Bezek DM, Baker JC, Kaneene JB: 1988, Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from

5 Diagnosis of BVD infection 61 experimentally infected immunocompetent calves. Can J Vet Res 52: Blood DC, Radostits OM, Henderson JA: 1983, Diseases caused by viruses and Chlamydia I. In: Veterinary medicine, 6th ed., pp Baillière-Tindall, London. 6. Bolin SR, McClurkin AW, Cutlip RC, et al.: 1985, Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus. Am J Vet Res 46: Bolin SR, McClurkin AW, Cutlip RC, et al.: 1985, Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and subsequent challenge exposure with cytopathic bovine viral diarrhea virus. Am J Vet Res 46: Brownlie J, Clark MC, Howard CJ: 1984, Experimental production of fatal mucosal disease in cattle. Vet Rec 114: Clark EG, Norman GR, Janzen ED: 1985, Pathologic and virology findings associated with bovine virus diarrhea-mucosal disease virus infection in neonatal beef calves in Saskatchewan. Proc Annu Meet Am Assoc Vet Lab Diagn 28: Duffel1 SJ, Harkness JW: 1985, Bovine virus diarrhea-mucosal disease infection in cattle. Vet Rec 117: Ernst PB, Baird JD, Butler DG: 1983, Bovine viral diarrhea: an update. Compend Contin Educ Pratt Vet 5:S581-S Kahrs RF: 1981, Bovine viral diarrhea. In: Viral diseases of cattle, pp. 89-l03. Iowa State University Press, Ames, IA. 13. Lambert G, McClurkin AW, Femelius AL: 1974, Bovine viral diarrhea in the neonatal calf. J Am Vet Med Assoc 164: Lloyd KC, Morris DD: 1985, Bovine viral diarrhea in a newborn calf. J Am Vet Med Assoc 186: Luna LG, ed.: 1968, Manual of histologic staining methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill Book Company, New York, NY. 16. McClurkin AW, Littledike ET, Cutlip RC: 1984, Production of cattle immunotolerant to bovine viral diarrhea virus. Can J Comp Med 48: Olafson P, MacCullum AD, Fox FH: 1946, An apparently new transmissible disease of cattle. Cornell Vet 36: Rae AG, Sinclair JA, Nettleton PF: 1987, Survival of bovine virus diarrhea virus in blood from persistently infected cattle. Vet Rec 120: Romvary W: 1985, Virus diarrhea in newborn calves. Acta Vet Hung 15: Rossi CR, Kiesel GK: 1971, Microtiter tests for detecting antibody in bovine serum to parainfluenza 3 virus, infectious bovine rhinotracheitis virus, and bovine virus diarrhea virus. Appl Microbial 22:32-36.