posthitis bij fokstien. Vlaams diergeneesk Tijdschr 29: 171-186. 1960. 6 DULBECCO R and VOGT M. Plaque formation and isolation of pure lines of poliomyelitis.
Volume 2
THE CANADIAN VETERINARY JOURNAL LA REVUE VETERINAIRE CANADIENNE June 1961
Number 6
AN OUTBREAK OF INFECTIOUS BOVINE RHINOTRACHEITIS IN ONTARIO M J Studdertt, 0 M Radostitst and M Savant (Received for publication Mar 14, 1961 ) INFECTIOUS BOVINE RHINOTRACHEITIS (IBR) is widespread in the USA but there have been no confirmed reports of its occurrence in Canada. This report contains a description of the clinical findings in a herd of feedlot cattle affected with an acute upper respiratory tract infection and the subsequent isolation of a virus from nasal discharges of the affected animals. Reciprocal cross neutralization tests in tissue culture and transmission studies have indicated that the agent which was isolated is indistinguishable from the virus known to cause IBR in the USA. REVIEW OF LITERATURE
Infectious bovine rhinotracheitis was apparently first observed in Colorado in 1950 (16), and since that time it has been reported in many western, midwestern and some eastern areas of the USA (14). A cytopathogenic agent has been isolated in fetal bovine kidney cell cultures from the nasal discharges of affected animals (10). This agent reproduced the disease when inoculated into susceptible animals (12). Infectious bovine rhinotracheitis has been defined as an acute, contagious, febrile infection of cattle, characterized by an intense inflammation of the upper respiratory passages and trachea and accompanied by dyspnea, depression, nasal discharge and loss of condition. Secondary bacterial complications may result in fatal pneumonia (14). Severe conjunctivitis, either as a single entity or accompanied by respiratory signs, has been reported to be characteristic of many outbreaks (1,13). Comparative studies have indicated that IBR virus and the virus which causes infectious pustular vulvovaginitis (IPV) are identical with the possible exception that IPV virus given intranasally induces a milder clinical syndrome than IBR given by the same route (7,15). IPV was first observed in Ontario in 1955 (4), and the viral agent associated with the outbreak was isolated in tissue culture (8). OBSERVATONS ON THE INFECTED HERD
The feedlot operation consisted of 200 head of cattle which had been purchased from various sale barns in the area. The herd was under weekly surveillance by a veterinarian as part of a disease prevention program. It had been noted that an increasing number of animals showed evidence of conjunctivitis; it was estimated that 50% of animals became infected. Bacteriologic fOntario Veterinary College, Guelph, Ont. 201 CAN VET JouR vol 2 no 6 June 1961
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TABLE I TOTAL AND DIFFERENTIAL WXHITE BLOOD CELL COUNTS OF 3 FIELD CASES OF INFECTIOUS BOVINE RHINOTRACHEITIS
Animal 29 30 31 'Cells per
Total 6,0001 4,000 4,800 cu mm
Neutro 600 760 384
Lympho 5,280 3,000 4,416
Eosino 120 240 -
examination of the ocular discharges of 6 animals revealed the presence of Moraxella bovis in each case and treatment with oxytetracycline eye ointment was instigated. Many animals showed bilateral involvement but no corneal opacities or ulcers were apparent, thus raising doubt as to whether the condition was contagious ophthalmitis as indicated by the presence of Moraxella bovis. After 5 days of treatment there was no improvement; the number of affected animals had if anything increased. The character of the lacrimal discharge had altered from a thin serous to a profuse mucopurulent discharge. A short explosive cough, drooling of saliva and reddened muzzles and conjunctivae were evident at this stage. Three of the more severely affected animals (#29, 30 and 31) were restrained for detailed clinical examination. They had temperatures of 1050, 1040 and 102°F; the heart and respiratory rates were slightly elevated and the respiratory movements were shallow but no marked dyspnea was evident. Ruminal movements were good and the rumens appeared well filled. The conjunctivae were intensely reddened and the periscleral vessels were injected. A profuse serous to mucopurulent lacrimal discharge tended to accumulate at the medial canthus but also ran down the face causing matting of the hair. The muzzles were pink and moist. The visible mucous membrane of the nasal passages was intensely reddened and much of it particularly on the septum and ventral portions, was covered with a pearly-white to pale green, irregularly contoured, diphtheritic membrane 2-4 mm thick. This membrane could be readily rubbed off revealing the underlying mucosa to be intensely reddened but intact. A serous to mucopurulent nasal discharge was present. Frothy rope-like strands of saliva drooled from the mouth but no lesions could be found on oral examination. The owner considered that many of the more severely affected animals were inappetent but not anorectic. The results of hematologic examination of the blood samples collected at the time of clinical examination are given in Table I. Bacteriologic cultures of nasal swabs did not reveal any signficant pathogens. The clinical signs progressively diminished and at the end of 10 days the cough was no longer audible and the nasal and ocular lesions had resolved. MATERIALS AND METHODS Nasal washings and serum samples were collected from 3 animals (#29, 30 and 31) in the affected herd. The washing material used was phosphate buffered saline (PBS) (6), to which had been added 200 units of penicillin, 20 mg of
203 streptomycin and 600 units of Nystatin1 per ml. Thirty ml of this fluid was poured into the nasal passages while the animal's head was held horizontally; the head was then quickly lowered and the fluid drained into a sterile basin. Flecks of pus that were adherent around the external nares were added to the washings. The washings were ground in a tissue grinder, centrifuged (1500 rpm for 10 minutes) and the supernatant fluid was dispensed into vials and stored at -60°C. A second serum sample was obtained from the same 3 animals 3 weeks later; both acute and convalescent phase sera were stored at -60°C. Primary cell cultures were prepared from fetal bovine kidneys (FBK) using the method outlined by Madin (11) with slight modifications. When monolayers were formed 0.1 ml of each nasal washing was inoculated into each of 5 tissue culture tubes; 5 tubes were left uninoculated as controls. Monolayers were grown on glass cover slips in a similar manner. The inoculum for these consisted of fluid harvested from the second tissue culture passage of the virus isolated from the nasal washings of animal #31. After 16 hour incubation, the cover glasses were removed, placed in Bouin's solution and the monolayers stained with hematoxylin and eosin. The second passage tissue culture fluid (from nasal washings of #31) was dispensed into glass ampoules which were sealed and stored at -60°C; this material formed the stock virus used in subsequent neutralization tests. Tenfold dilutions of this stock virus were made using PBS as diluent and 0.1 ml of each dilution (covering the range 10-1 to 10-7) was inoculated into each of 5 tissue culture tubes. The 50% end point was calculated by the Reed-Muench formula (18). Twofold dilutions of sera, to be tested for the presence of neutralizing antibodies, were prepared and to 0.5 ml of each serum dilution an equal volume of a virus dilution containing 200 tissue culture 50% infective doses (TCID50) per 0.1 ml was added. The serum virus mixtures were incubated at room temperature for 1 hour then 0.1 ml of each mixture was inoculated into each of 4 tissue culture tubes. Thus each tube received 100 TCID.50 plus the respective serum dilution. Inoculated tubes were examined daily for 7 days for evidence of cytopathogenic changes. Complete destruction of the monolayer was arbitrarily selected to indicate the presence of virus. Two virgin yearling bulls were experimentally infected with second passage tissue culture fluid diluted 1:10 with PBS. Both animals were inoculated intranasally and intrapreputially and one animal was inoculated per conjunctiva. The inoculum was rubbed onto the mucosal surfaces by means of a cotton gauze swab. Beginning 3 days before inoculation twice daily temperature recordings and once daily total and differential white blood cell counts were made. Serum samples were obtained before inoculation and 3 weeks postinfection, and tested for neutralizing antibodies. Nasal washings were collected for virus isolation on the 7th and 14th days postinfection. The sites of inoculation were examined daily for the presence of lesions. Anti-IBR rabbit serum2, prepared against a Colorado isolate of IBR virus, was tested for its neutralizing effect on the isolated virus. 1Mycostatin. E R Squibb & Sons, Montreal PQ. 2Kindly supplied by Dr T L Chow, Fort Collins, Colorado, USA. INFECTIOUS BOVINE RHINOTRACHEITIS
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RESULTS
Isolation of the Virus When examined at 24 hours all tubes inoculated with nasal washings showed evidence of cytopathogenic change. The cells appeared dark and round and had contracted into dense clumps resembling bunches of grapes. After 72 hours, the cells became dislodged from the glass. These changes, observed in more detail in the cover glass preparations, were indistinguishable from those we have observed with IPV virus (19). Large eosinophilic intranuclear bodies were present in many cells. Titration of the second passage tissue culture fluid showed a TCID50 10-5-68 per 0.1 ml. Experimental Transmission The 2 experimentally infected animals developed a marked febrile response which began 52 hours after inoculation and persisted for 5 and 9 days respectively; the temperatures varied between 1030 and 106.6°F. A few hours after the initial febrile response nasal lesions similar to those seen in field cases were observed in both animals. Small, discrete, white pustular lesions that developed on the conjunctiva of the animal infected via this route had not been observed in field cases. They were however similar to lesions produced by IPV virus when inoculated at the same site (19). Likewise the genital lesions observed on the penile and preputial surfaces were similar to those produced by IPV virus (19). The results of the blood counts for the 3 days preinfection and the 3rd, 4th and 5th days postinfection are shown in Table II. TABLE I I TOTAL
AND
DIFFERENTIAL WHITE BLOOD CELL COUNTS
OF 2 ANIMALS
EXPERIMENTALLY
INFECTED WITH IBR VIRUS. COUNTS SHOWN ARE THOSE OBTAINED ON THE 3 DAYS PREINFECTION AND THE 3RD, 4TH AND 5TH DAYS POSTINFECTION.
Day Total Neutro Lympho Mono Eosino
'Cells
Animal 744 Animal 743 Postiinfection Preinfection Postinfection Preinfection 4 5 1 2 3 3 4 5 3 2 1 3 6,5501 7,400 6,900 4,200 4,500 4,950 5,800 6,200 6,400 5,350 5,500 4,400 588 1,540 1,056 812 1,240 1,088 336 855 1,274 1,834 2,442 1,794 3,930 4,218 4,623 3,528 3,600 3,577 4,988 4,836 5,312 4,654 3,960 3,212 132 108 49 42 370 124 45 294 786 370 483 per cu mm
The virus was reisolated from the nasal passages of both animals 7 days after infection but it was not present in the nasal washings taken on the 14th day. Serum Virus Neutralization No virus neutralizing effect could be demonstrated in the acute phase sera obtained from the 3 field cases (#29, 30 and 31) although the convalescent phase samples did neutralize the virus. A similar result was obtained when these sera were tested against 100 TCID50 IPV virus.
205 Preinoculation sera from the experimentally infected animals did not neutralize the isolated agent but postinoculation samples exhibited a titre of 1:8. The specific IBR antiserum obtained from Colorado neutralized the virus at a titre of 1:16. INFECTIOUS BOVINE RHINOTRACHEITIS
DISCUSSION
It is possible that IBR has been present in Canada for a number of years, but that its presence has been masked for a number of reasons among which might be mentioned (a) the complex nature of bovine respiratory diseases, particularly with regard to the roll of viruses in their causation and (b) predisposing factors, analagous to those apparently responsible for the widespread occurrence of the disease in the western USA, have not existed. The IBR - IPV virus has been demonstrated to have a multiplicity of disease producing potentials viz vaginitis (IPV) (9); conjunctivitis (1, 13); a fatal disease in experimentally infected newborn calves (2); and, experimentally, mastitis (3). In a survey conducted in New York State IBR - IPV antibodies were demonstrated in 12% of 43 herds (7). Available evidence on the incidence of any of the above diseases together with IBR (which has never been reported) does not support the survey's findings. It is therefore apparent that subclinical infection is widespread in New York State and the same may be true of Ontario. There are obvious deficiencies in our knowledge of the epizootiology of these diseases. It has been suggested that IBR may be related to the bovine enteroviruses (17). If this claim is substantiated, then we have a reasonable basis for proposing not only the origin of the virus but also a means by which it may exist in the environment. It seems well established that in clinical cases of IBR and IPV, the virus cannot be isolated from the site of infection for periods longer than about 2 weeks after the initiation of the disease. However, McKercher (14) states that it is difficult to avoid the conclusion that cattle themselves maintain and serve as the chief means of spread of the virus. A diversity of opinion exists concerning the development of leukopenia resulting from infection with IBR - IPV virus. A systematic hematological study conducted in animals experimentally infected with IBR failed to reveal any qualitative or quantitative changes (13). However, neutropenia has been found to occur in cases of genital infection with IPV virus (5,9). Results obtained in this study suggest that neutropenia may occur in both field and experimental cases of IBR. The discrepancies may be related to the rapidity of secondary bacterial invasion. Nevertheless because of the variability of the finding, it is unlikely to be of great diagnostic value. SUMMARY Field observations on an outbreak of an acute upper respiratory tract infection in cattle and the subsequent isolation of a viral agent are reported. Experimental studies confirmed that the virus is indistinguishable from that known to cause infectious bovine rhinotracheitis (IBR) in the USA and infectious pustular vulvovaginitis (IPV) in Canada and the USA. The observations constitute the first confirmed report of the occurrence of the IBR syndrome in Canada. ACKNOWLEDGEMENTS The helpful criticism of Drs D C Blood and B J McSherry are greatly appreciated. Thanks are due to Mrs Florence Black for fine technical assistance and to Mr G E Fountain for the hematology.
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REFERENCES 1 ABINANTI F R and PLUMER G j. The isolation of infectious bovine rhinotracheitis virus from cattle affected with conjunctivitis-Observations on the experimental infection. Am J Vet Res 18: 932-941. 1961. 2 BAKER J A, MCENTEE K and GILLESPIE J H. Effects of infectious bovine rhinotracheitisinfectious pustular vulvovaginitis (IBR-IPV) virus on newborn calves. Cornell Vet 50: 156-170. 1960. 3 BAKER J A, GILLESPIE j H, MCENTEE K and LANGER P H. Production of mastitis in cattle by IBR-IPV virus when inoculated into the teat cystern. To be published. 4 BARKER c A V. Coital vesicular exanthema in cattle. Reported at the North American conference on infertility, International Fertility Association, Montreal, 1958. 5 BouTERs R, VANDEPLASSCHE M, FLORENT A, LEUNEN j and DEVOS A. De ulcereuse balanoposthitis bij fokstien. Vlaams diergeneesk Tijdschr 29: 171-186. 1960. 6 DULBECCO R and VOGT M. Plaque formation and isolation of pure lines of poliomyelitis virus. J Exptl Med 99: 167-182. 1954. 7 GILLESPIE j H, MCENTEE K, KENDRICK j w and WAGNER w c. Comparison of infectious pustular vulvovaginitis virus with infectious bovine rhinotracheitis virus. Cornell Vet 49: 288-297. 1959. 8 GRIEG A s, BANNISTER G L, MITCHELL D and BARKER c A V. Cultivation in tissue culture of an infectious agent from coital vesicular exanthema of cattle. A preliminary report. CJCM 22: 119-122. 1958. 9 KENDRICK j w, GILLESPIE j H and MCENTEE K. Infectious pustular vulvovaginitis of cattle. Comell Vet 48: 458-495. 1958. 10 MADIN S H, YoRKc J and MCKERCHER D G. Isolation of the infectious bovine rhinotracheitis virus. Science 124: 721-722. 1956. 11 MADIN s H, ANDRIESE P c and DARBY N B. The in vitro cultivation of tissues of domestic and laboratory animals. Am J Vet Res 18: 932-941. 1957. 12 McKERCHER D G, MOULTON j E, MADIN S H and KENDRICK j w. Infectious bovine rhinotracheitis-a newly recognised virus disease of cattle. Am J Vet Res 18: 246-256. 1957. 13 McKERCHER D G, SAITO j K, WADA E M and STAUB O. Current status of the newer virus diseases of cattle. Proc 62nd Ann Meeting US Livestock Sanit Assn. 136-156. 1958. 14 MlCKERCHER D G. Infectious bovine rhinotracheitis. Advances in Veterinary Science 5: 299-328. 1959. 15 MCKERCHER D G, STAUB O, SAITO j K and WADA E Nf. Comparative studies of the etiological agents of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis. CJCM 22: 119-122. 1958. 16 MILLER N J. Infectious necrotic rhinotracheitis of cattle. JAVMA 126: 463-467. 1955. 17 MOLL T and DAVIS A D. Serologic relationship between bovine enteroviruses and infectious bovine rhinotracheitis virus. Am J Vet Res 21: 1131-1132. 1960. 18 REED L T and MUENCH H. A simple method for estimating fifty percent end points. Am J Hyg 27: 493. 1938. 19 STUDDERT M j and BARKER c A V. Observation on the infectivity of infectious pustular vulvovaginitis (IPV) virus for bulls. Unpublished data.
ADDENDUM Since the preparation of the manuscript, a second isolation of IBR virus has been made from a separate herd of cattle affected with an acute upper respiratory tract infection. The isolation was made from a recently obtained nasal swab. The cotton pledget containing the nasal discharge was soaked in 4 ml PBS similar to that used to obtain nasal washings. This fluid was inoculated into tissue culture tubes and the virus thereby isolated. The reliability of this simple method of obtaining samples for isolation of IBR virus cannot at present be attested to. In addition Grieg (1) has published the results of a survey to detect the presence of IBR neutralizing antibodies in sera obtained from herds in southern Ont. It was found that 8.3% of serum samples representing 18.9% of herds tested were positive for IBR antibodies. REFERENCE 1 GRIEG A s. The detection of antibody to infectious bovine rhinotracheitis virus in Ontario cattle. CJCM 25: 31-34. 1961.
