ISOLATION AND CHARACTERISATION OF BOVINE HERPESVIRUS MAMMILLITIS VIRUS AND ITS PATHOGENICITY FOR CATTLE
A. J. TURNER,M.V.Sc., Ph.D., L. KOVESDY, Dip. Med. Lab. Tech., and I. R. MORGAN,B.V.Sc., M.P.V.M. Attwood Veterinary Research Laboratory, Division of Animal Health, Department of Agriculture, Westmeadows, Victoria, 3047 IntaoducHoa
A virus isolated from a teat scab of a cow was demonstrated to be serologically and morphologically similar to bovine herpes mammillitis (BHM) virus by Turner et a1 (1974). In a subsequent communication Turner et a1 (1975) described the clinical and epidemiological features of an outbreak of BHM in two districts of north-central Victoria. This paper records the isolation and characterisation of BHM viruses from cattle in these districts and the experimental production of mammillitis with the virus in two cattle. Materials a d Me(h0ds Preparation of Cell Cultures Primary cell cultures of BK, BT, ovine embryonic kidney (OEK) and pig embryonic kidney ( P F ) were grown in Roux flasks at 37°C. Growth medium was Hank‘s lactalbumin solution* (BK and OEK) or Medium 199* (PEK and BT) .with 7.5% foetal calf serum (FCS), 200 units penicillin and 200 gm streptomycin per ml. Established monolayers of these cell cultures were dispersed and seeded into glass tubes with growth medium as above and incubated at 37°C until monolayer cultures were established. When grown, .the cell cultures were maintained in the respective medium with FCS reduced to 1.5%. Virus Isolation Procedures Samples of teat lesions were obtained from cattle on 15 dairy properties between mid-November and midDecember 1973. Solid samples were homogenised to an approximate 10% suspension in phosphate buffered saline (PBS; Dulbecco and Vogt, 1952) containjng 0.5% bovine serum albumint (BSA). The suspenslon was centrifuked at 750g for 10 minutes and passed through a Millipore filter of average pore diameter 0.45 p. Cotton wool tipped swabs of lesions were transported in glass containers containing 3 ml Hank’s lactalbumin solution. The fluid was centrifuged and filtered as above Vesicular fluid was inoculated directly into tubes containing cell cultures. Aliquots of the filtered samples (0.1 to 0.2 ml) were inoculated into each of 4 tubes of bovine kidney (BK) and bovine testicular (BT) cell cultures. The cultures were incubated stationary at 37°C and were examined daily for cytopathic effect. Cell line cultures of baby hamster kidney (BHK21) were grown in Hank’s lactalbumin and African green monkey kidney (VERO) were grown in Eagle’s minimum essential medium* using similar concentrations of *GIBCO. Grand. Jslqd, New York. United States of America tsigma, St. Louis, Missouri, Umted States of America.
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FCS and antibiotics as described above. BHK21 cell cultures in tubes were maintained in Eagle’s basal medium* containing 2% FCS and antibiotics. VERO cell cultures in tubes were maintained in Eagle’s minimum essential medium containing 2% FCS and antibiotics. Viral Assay Virus titrations were performed in tube cultures using 4 replicate cultures per tenfold dilution; 0.2 ml was inoculated into each tube. Titres were expressed as the 50% tissue culture infective dose (TCID,) per ml as determined by the method of Karber (1931). Cytopathology Cytopathic effects were studied in BK and BT cell cultures in coverslip preparations both unstained and following fixation in Bouin’s fluid for 10 minutes and staining with haematoxylin and eosin. BHM-1 virus grown in BK cell cultures was titrated in BK. BT, PEK, OEK, BHK 21 and VERO cell cultures as described previously.
Physico-chemical Properties Ether sensitivity was determined by the method of Andrewes and Horstmann ( 1949) and chloroform sensitivity by the method of Feldman and Wang (1961). Infectious bovine rhinotracheitis (IBR) and bovine entero (BE) viruses were included in the tests as control ether sensitive and ether resistant viruses respectively. The nucleic acid type of BHM-1 virus was determined using 5-bromodeoxyuridine$ (BUDR). BK cell culture monolayers in tubes were maintained with 0, 20, 50 and 100 pgm BUDR per ml for 24 hours when 1,000 TCIDm of virus was inoculated into 5 tubes of each treatment. IBR and BE viruses were utilised as control viruses. When 90% cytopathic effect was observed in the tubes with no BUDR treatment, all treatment tubes for the particular virus were frozen to -70°C and thawed 3 times. The fluids from the tubes were centrifuged at 750 g for 10 min and the supernatant fluid of each treatment was titrated in BK cell cultures as described previously. Antiserwn Produciion BHM virus suspensions were prepared from supernatant cell culture fluids by centrifugation as described above and mixed with equal volumes of Freund‘s complete adjuvant. Blood samples were obtained from 2 adult rabbits which were inoculated intramuscularly with 0.5 ml at 4 sites and subcutaneously with 0.5 ml at 2 sites. This procedure was repeated twice at weekly intervals with Freund’s incomplete adjuvant being used instead of Freund’s complete adjuvant. Blood samples were obtained from the rabbits 2 weeks after completion of the iniections. Serums were stored at -20°C. SCalbiochem, San Diego, California, United States of America.
Australian Veterinary Journal, Vol. 52, April, 1976
Serological Identification of Viruses Serums for neutralisation tests were treated at 56°C for 30 min then serially diluted twofold in PBS containing 0.5% BSA. Equal volumes of serum or serum dilution and virus dilution estimated to contain 200 TClDm per 0.2 ml were allowed to stand at room temperature for 60 min. Two tubes of BK cell cultures were inoculated with 0.2 ml of serum-virus mixture. Serology Acute and convalescent blood samples were collected from 68 animals on 19 farms and stored at -20°C until tested. Animals were considered to have been infected with BHM virus if the titre of serum neutralising antibody in the convalescent serum was at least 4 times higher than in the acute serum. Reference Viruses The BHM virus isolated by Turner el a1 (1974) was designated BHM-1 virus. Third passage BHM-1 virus was passaged 3 times in BK cell cultures at limiting dilution and stock preparations of the virus were stored at -80°C. This stock preparation, a 1CF dilution of the virus from the original teat material, was used for all serological procedures, antiserum production in rabbits and inoculation of cattle. IBR and BE viruses isolated from nasal and mouth swabs of cattle in Victoria were used for physiochemical comparisons with BHM viruses. Inocularion of Cattle One 16 month old non pregnant Hereford heifer and one aged lactating Jersey cow which had been recently joined were used for inoculation. The cow was not milked. The animals were housed in isolation and blood samples were taken to determine that there was no serum neutralising antibody. The cattle were injected intramuscularly with xylazine* to produce analgesia and 0.1 ml of BHM-1 virus so1u:ion was inoculated intradermally along two 5 mm tracts on the lateral aspects of the right teats. Rectal temperatures were recorded twice daily for 5 days before inoculation and for 10 days after inoculation. Samples for virus isolation were obtained on days 7 and 10 after inoculation. Blood samples were obtained before inoculation and days 7, 10, 13, 16, 26 and 39 after inoculation. On day 39 after inoculation the cattle were inoculated daily for 6 days with 20 mg dexamethasonel by the intravenous route The teats were sampled for virus on days 39, 43, 44 and 45. On day 46 the cattle were slaughtered and the teats collected. Approximately 10% homogenates of the skin and dermis where infection had occurred were prepared and Millipore filtrates were inoculated into BK cell cultures as described pre viously. Swabs of the nasal and vaginal mucosa were taken on days 39. 44 and 46 and cultured for virus in BK cell cultures. Results
Virus isolation was attempted from 54 specimens from 15 properties. Ten viruses were isolated in BK cell cultures from 38 scab and 1 vesicular fluid samples received from 4 properties. Virus was not isolated from 15 swabs received from the other 1 1 urouerties. Only 8 * Rom-un. Bayer Australia Ltd, Sydney. ~~
8 Dexadreson, Organon Lab. Ltd, Moreden. United Kingdom.
Australian Veterinary Journal, Vol. 52, April, 1976
of the viruses were isolated in BT cell cultures. Isolations were made from samples received during November but not from any samples received during December. Cytopathic effect was observed as soon as one day and as late as five days after inoculation. In only 2 instances was passaging into further tissue cultures required before cytopathic effect was observed. In unstained preparations of BK and BT cell cultures cytopathic effects were observed initially as refractile areas in the monolayer where cell outlines were indistinct. The cytopathic effects developed rapidly with similar areas developing throughout the monolayer. Affected areas contracted from adjacent cells and the areas without distinct cell boundaries became larger and sloughed from the glass surface until the whole monolayer became affected. In stained preparations the refractile areas were identified as syncytia which, when fully developed, frequently contained more than 200 nuclei. The nuclei in small syncytia contained eosinophilic masses similar to Cowdry type A inclusion bodies. In large syncytia some inclusion bodies stained eosinophilic where others stained basophilic. BHM-1 virus that had been passaged 7 times in BK cultures produced cytopathic effects in the in cell cultures tested and gave titres of in BHK-21 and PEK, 103.43in OEK, in VERO cells. Physico-Chemical Properties The cell culture infectivity of one virus isolated from each property was destroyed by ether and chloroform treatment. The results for BHM-1 virus are summarised in Table 1 . The growth of BHM-1 and IBR viruses was depressed by BUDR treatment but the growth of BE virus was not affected (Table 1). TABLE 1 The Effect of Treatment o f Bovine Herpes Mammillitis, Infectious Bovine Rhinotracheitis and Bovine EnteroViruses with Ether, Chloroform and 5- Brom-deoxyuridine
Virus Treatment Ether Chloroform Control BUDR 0 ue/ml 20 ug/ml 50 u g h 1 100 u g h 1
* log10 infective
BHM-1 d 1.00.* 1.00 4.95
A. J. TURNER,M.V.Sc., Ph.D., L. KOVESDY, Dip. Med. Lab. Tech., and I. R. MORGAN,B.V.Sc., M.P.V.M. Attwood Veterinary Research Laboratory, Division of Animal Health, Department of Agriculture, Westmeadows, Victoria, 3047 IntaoducHoa
A virus isolated from a teat scab of a cow was demonstrated to be serologically and morphologically similar to bovine herpes mammillitis (BHM) virus by Turner et a1 (1974). In a subsequent communication Turner et a1 (1975) described the clinical and epidemiological features of an outbreak of BHM in two districts of north-central Victoria. This paper records the isolation and characterisation of BHM viruses from cattle in these districts and the experimental production of mammillitis with the virus in two cattle. Materials a d Me(h0ds Preparation of Cell Cultures Primary cell cultures of BK, BT, ovine embryonic kidney (OEK) and pig embryonic kidney ( P F ) were grown in Roux flasks at 37°C. Growth medium was Hank‘s lactalbumin solution* (BK and OEK) or Medium 199* (PEK and BT) .with 7.5% foetal calf serum (FCS), 200 units penicillin and 200 gm streptomycin per ml. Established monolayers of these cell cultures were dispersed and seeded into glass tubes with growth medium as above and incubated at 37°C until monolayer cultures were established. When grown, .the cell cultures were maintained in the respective medium with FCS reduced to 1.5%. Virus Isolation Procedures Samples of teat lesions were obtained from cattle on 15 dairy properties between mid-November and midDecember 1973. Solid samples were homogenised to an approximate 10% suspension in phosphate buffered saline (PBS; Dulbecco and Vogt, 1952) containjng 0.5% bovine serum albumint (BSA). The suspenslon was centrifuked at 750g for 10 minutes and passed through a Millipore filter of average pore diameter 0.45 p. Cotton wool tipped swabs of lesions were transported in glass containers containing 3 ml Hank’s lactalbumin solution. The fluid was centrifuged and filtered as above Vesicular fluid was inoculated directly into tubes containing cell cultures. Aliquots of the filtered samples (0.1 to 0.2 ml) were inoculated into each of 4 tubes of bovine kidney (BK) and bovine testicular (BT) cell cultures. The cultures were incubated stationary at 37°C and were examined daily for cytopathic effect. Cell line cultures of baby hamster kidney (BHK21) were grown in Hank’s lactalbumin and African green monkey kidney (VERO) were grown in Eagle’s minimum essential medium* using similar concentrations of *GIBCO. Grand. Jslqd, New York. United States of America tsigma, St. Louis, Missouri, Umted States of America.
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FCS and antibiotics as described above. BHK21 cell cultures in tubes were maintained in Eagle’s basal medium* containing 2% FCS and antibiotics. VERO cell cultures in tubes were maintained in Eagle’s minimum essential medium containing 2% FCS and antibiotics. Viral Assay Virus titrations were performed in tube cultures using 4 replicate cultures per tenfold dilution; 0.2 ml was inoculated into each tube. Titres were expressed as the 50% tissue culture infective dose (TCID,) per ml as determined by the method of Karber (1931). Cytopathology Cytopathic effects were studied in BK and BT cell cultures in coverslip preparations both unstained and following fixation in Bouin’s fluid for 10 minutes and staining with haematoxylin and eosin. BHM-1 virus grown in BK cell cultures was titrated in BK. BT, PEK, OEK, BHK 21 and VERO cell cultures as described previously.
Physico-chemical Properties Ether sensitivity was determined by the method of Andrewes and Horstmann ( 1949) and chloroform sensitivity by the method of Feldman and Wang (1961). Infectious bovine rhinotracheitis (IBR) and bovine entero (BE) viruses were included in the tests as control ether sensitive and ether resistant viruses respectively. The nucleic acid type of BHM-1 virus was determined using 5-bromodeoxyuridine$ (BUDR). BK cell culture monolayers in tubes were maintained with 0, 20, 50 and 100 pgm BUDR per ml for 24 hours when 1,000 TCIDm of virus was inoculated into 5 tubes of each treatment. IBR and BE viruses were utilised as control viruses. When 90% cytopathic effect was observed in the tubes with no BUDR treatment, all treatment tubes for the particular virus were frozen to -70°C and thawed 3 times. The fluids from the tubes were centrifuged at 750 g for 10 min and the supernatant fluid of each treatment was titrated in BK cell cultures as described previously. Antiserwn Produciion BHM virus suspensions were prepared from supernatant cell culture fluids by centrifugation as described above and mixed with equal volumes of Freund‘s complete adjuvant. Blood samples were obtained from 2 adult rabbits which were inoculated intramuscularly with 0.5 ml at 4 sites and subcutaneously with 0.5 ml at 2 sites. This procedure was repeated twice at weekly intervals with Freund’s incomplete adjuvant being used instead of Freund’s complete adjuvant. Blood samples were obtained from the rabbits 2 weeks after completion of the iniections. Serums were stored at -20°C. SCalbiochem, San Diego, California, United States of America.
Australian Veterinary Journal, Vol. 52, April, 1976
Serological Identification of Viruses Serums for neutralisation tests were treated at 56°C for 30 min then serially diluted twofold in PBS containing 0.5% BSA. Equal volumes of serum or serum dilution and virus dilution estimated to contain 200 TClDm per 0.2 ml were allowed to stand at room temperature for 60 min. Two tubes of BK cell cultures were inoculated with 0.2 ml of serum-virus mixture. Serology Acute and convalescent blood samples were collected from 68 animals on 19 farms and stored at -20°C until tested. Animals were considered to have been infected with BHM virus if the titre of serum neutralising antibody in the convalescent serum was at least 4 times higher than in the acute serum. Reference Viruses The BHM virus isolated by Turner el a1 (1974) was designated BHM-1 virus. Third passage BHM-1 virus was passaged 3 times in BK cell cultures at limiting dilution and stock preparations of the virus were stored at -80°C. This stock preparation, a 1CF dilution of the virus from the original teat material, was used for all serological procedures, antiserum production in rabbits and inoculation of cattle. IBR and BE viruses isolated from nasal and mouth swabs of cattle in Victoria were used for physiochemical comparisons with BHM viruses. Inocularion of Cattle One 16 month old non pregnant Hereford heifer and one aged lactating Jersey cow which had been recently joined were used for inoculation. The cow was not milked. The animals were housed in isolation and blood samples were taken to determine that there was no serum neutralising antibody. The cattle were injected intramuscularly with xylazine* to produce analgesia and 0.1 ml of BHM-1 virus so1u:ion was inoculated intradermally along two 5 mm tracts on the lateral aspects of the right teats. Rectal temperatures were recorded twice daily for 5 days before inoculation and for 10 days after inoculation. Samples for virus isolation were obtained on days 7 and 10 after inoculation. Blood samples were obtained before inoculation and days 7, 10, 13, 16, 26 and 39 after inoculation. On day 39 after inoculation the cattle were inoculated daily for 6 days with 20 mg dexamethasonel by the intravenous route The teats were sampled for virus on days 39, 43, 44 and 45. On day 46 the cattle were slaughtered and the teats collected. Approximately 10% homogenates of the skin and dermis where infection had occurred were prepared and Millipore filtrates were inoculated into BK cell cultures as described pre viously. Swabs of the nasal and vaginal mucosa were taken on days 39. 44 and 46 and cultured for virus in BK cell cultures. Results
Virus isolation was attempted from 54 specimens from 15 properties. Ten viruses were isolated in BK cell cultures from 38 scab and 1 vesicular fluid samples received from 4 properties. Virus was not isolated from 15 swabs received from the other 1 1 urouerties. Only 8 * Rom-un. Bayer Australia Ltd, Sydney. ~~
8 Dexadreson, Organon Lab. Ltd, Moreden. United Kingdom.
Australian Veterinary Journal, Vol. 52, April, 1976
of the viruses were isolated in BT cell cultures. Isolations were made from samples received during November but not from any samples received during December. Cytopathic effect was observed as soon as one day and as late as five days after inoculation. In only 2 instances was passaging into further tissue cultures required before cytopathic effect was observed. In unstained preparations of BK and BT cell cultures cytopathic effects were observed initially as refractile areas in the monolayer where cell outlines were indistinct. The cytopathic effects developed rapidly with similar areas developing throughout the monolayer. Affected areas contracted from adjacent cells and the areas without distinct cell boundaries became larger and sloughed from the glass surface until the whole monolayer became affected. In stained preparations the refractile areas were identified as syncytia which, when fully developed, frequently contained more than 200 nuclei. The nuclei in small syncytia contained eosinophilic masses similar to Cowdry type A inclusion bodies. In large syncytia some inclusion bodies stained eosinophilic where others stained basophilic. BHM-1 virus that had been passaged 7 times in BK cultures produced cytopathic effects in the in cell cultures tested and gave titres of in BHK-21 and PEK, 103.43in OEK, in VERO cells. Physico-Chemical Properties The cell culture infectivity of one virus isolated from each property was destroyed by ether and chloroform treatment. The results for BHM-1 virus are summarised in Table 1 . The growth of BHM-1 and IBR viruses was depressed by BUDR treatment but the growth of BE virus was not affected (Table 1). TABLE 1 The Effect of Treatment o f Bovine Herpes Mammillitis, Infectious Bovine Rhinotracheitis and Bovine EnteroViruses with Ether, Chloroform and 5- Brom-deoxyuridine
Virus Treatment Ether Chloroform Control BUDR 0 ue/ml 20 ug/ml 50 u g h 1 100 u g h 1
* log10 infective
BHM-1 d 1.00.* 1.00 4.95
