key: cord-0033630-5g6dn95u authors: Wellemans, G. title: Laboratory diagnosis methods for bovine respiratory syncytial virus date: 1977 journal: Vet Sci Commun DOI: 10.1007/bf02267648 sha: 0494819d15e98cb28ca72a02d9b4e95c20d3ab0e doc_id: 33630 cord_uid: 5g6dn95u Laboratory diagnosis of bovine respiratory syncytial (BRS) virus no longer poses a problem. Clinical diagnosis, based on signs of pulmonary emphysema manifest in autumn, should be confirmed by laboratory techniques. Direct isolation of the BRS virus from field samples in cell cultures is often unsuccessful, whereas detection of the viral antigens by staining ultra-thin tissue sections with fluorescein isothiocyanate antibody conjugates is highly effective. Complement fixation and especially indirect immunofluorescence tests are still very useful for the detection of BRS specific antibodies in serum and nasal mucus. Care should be taken to determine the aetiology of diseases before embarking on their treatment. Symptomatic treatments usually have little effect against bovine respiratory diseases. The symptoms observed in these disorders are the result of synergism in which various agents are predisposing conditions, followed by infection by one or more viruses and bacteria, and sometimes further complicated by the immune reaction of the body. The bacterial infection can be overcome by the use of antibiotics and sulphonamides, but predisposing conditions are more difficult to counteract. Two important predisposing factors are due to the demands of 1S0 over-intensification of modern beef production and the over-crowding of yarded animals, which for economic reasons are unavoidable. Neasures to combat the viral component in the aetiology of these diseases depend on first isolating and identifying the agent or agents and then producing specific vaccines. Respiratory disorders are causing considerable economic losses in cattle rearing, predominantly in intensive beef production. The aetiological agents in these disorders were formerly thought to be organisms of the genus Pasteurella. It has since been discovered on isolatin~ them from the lungs that these bacteria are not the main factor in the disease. In the USA a virus was isolated from the respiratory system by T,~din and coworkers (1956) . However, this virus, I~R (infectious bovine rhinotracheitis virus) is not involved in the general respiratory syndrome but causes a separate set of symptoms. Again in the USA, Reisinger and coworkers (1959) isolated the p~rainfluenza-3 virus (PI-3) and the role of this organism in respiratory disorders was soon confirmed by numerous European investigators. In 1969, researchers of this Institute (Wellemans and Leunen, 1969) demonstrated that BVD (bovine viral diarrhoea virus) played a part in respiratory disease. The Hungarian scientists Bartha and Aldasy (1964) isolated adenoviruses of type B, which came to be regarded as important factors in some cases of respiratory disorder. Other investigators (Rosen and Abinanti, 1960; Bogel and Bohm, 1962) have isolated reoviruses, rhinoviruses and ECBO viruses with less clearly established pathogenic action. Finally, Paccaud and Jacquier (1970) , Inaba and coworkers (1972) and Wellemans and Lsunen (1975) About two or three days later, when everything seems to be satisfactory again, the animals suddenly present difficulty in breathing, accompanied by bouts of dry coughing. The body temperature when these symptoms are first manifested is close to normal. The breathing of the sick calves becomes increasingly rapid and shallow and the condition is aggravated by bouts of coughing. There is little or no discharge from the nostrils. Frequently there is frothing at the commissure of the lips. Constipation is a commonly occurring symptom and there is complete loss of appetite. The animals can neither lie down nor eat and they make desperate efforts to breathe through an open mouth. On auscultation some harshnes~ in the breathing can be detected but rales are not often heard and towards the end of the disease state the classic signs of emphysema begin to appear. There can be up to 30% mortality in the herd and farms specialising in late calves tend to have the highest losses. Opening up of the thoracic cavity in ~utopsy reveals widespread pulmonary emphysema and distended air spaces. In some cases the walls are broken, so that pneumothorax results. The presence of tracheitis or of rhinitis is comparatively rare. Even where characteristic symptoms occur, the only means of determining the aeticlogical diagnosis with certainty is laboratory investigation. Constant improvement in diagnostic techniques has led to easier identification of the virus. Infection by BRS virus can be ascertained by: I. isolation of the viral agent, in cell cultures; 2. detection of the viral antigens in ultra-thin sections stained by a serum labelled with fluorescein isothiocyanate; 3. antibody detection in conjugated sere; 4. antibody dctection in nasal mucus. Diagnosis from isolation of the BRS virus is the most laborious technique. Severe or fatal damage is not always directly due to the effects of the virus, but is often caused by complications such as bacterial infection or hypersensitive reaction of the body of the host animal. We have discovered that infection by BRS virus often produces very rapid immunological response in the animal, so that the maximum titre is attained within a week. Ground lung tissue from a dead animal may contain not only the virus and its antigens but also neutralising antibodies to the virus, which impede its isolation. Attempts are also made to isolate the virus from animals which still seem to be healthy. But even in favourable conditions there is no certainty that isolation will be achieved, because the BRS virus produces a very slow cytopathogenic effect in the initial passages. It sometimes takes more than a month to obtain discernible cell lesions. Paccaud and Jacquier, in Switzerland, identified their virus after 32 days (1970) and Inaba e% el., in Japan, had to wait 17 days ( phages, sometimes in great abundance (Fig. 1) . The investigation of antibodies in sera by means of complement fixation (CF) or indirect immunofluorescence (IIF) is the most reliable and straightforward diagnostic technique at present available 9 However, it usually ~ecessitates the use of conjugated sera, which deters many veterinary ~ractitioners from performing such tests at a time when they are more concerned with restoring the surviving animals to health than obtaining a belated diagnosis of the aetiology of the disease 9 Complement fixation (C.F.). -If specific antigen is present, some antibodies form a stable complex into which complement is taken up. The CF iest, which is simple to perform, demonstrates the existence of the anti-~n-ccmplement-antibody complex, by fixing the complement which causes lysis of sensitised red blood cells. The antigen is contained in the supernatant fluid of a cell culture lysed by the action of a strain of BRS virus adapted to cell cultures. This showing BRS antigens in the mucous membranes. supernatant fluid is used either without modification or after concentration by dialysis with a PEG 20,000 solution. The complement is guinea pig serum to which 5% of fresh calf serum has been added. The procedure for the antigen-complement-antibody combination is carried out at 4~ overnight and then for one hour at 37~ Titres of 1:8 to 1:64 are obtained with the second set of sera. I/here the blood specimens have not been collected until a few days after the onset of the disease it is already possible to detect high titres in the first set of sera. We have found that the antibodies to BRS virus develop very rapidly (Fig. 2a) . Indirect immunofluorescence. -Some antibodies can attach to the specific antigen and complex with it. A labelled serum which reacts with the immunoglobulins belonging to the species of animal under study reveals the complex. In serum tests the titre corresponds to the highest dilution with which fluorescence is obtained. The indirect immunofluorescence (IIF) reaction enables diagnosis to be made very rapidly. In a successful test the results can be read within 24 hours. We have not found any cross-reaction with the viruses used in differential diagnosis (BVD, parainfluenza-3, IBR, rhinovirus, papular stomatitis, adenovirus). The antigen is produced on a PK 15 cell line. Its use obviates non-specific reactions due to the presence in the ser~n of antibodies which react with the cells of bovine origin. The titres obtained are as high as 1:405 to 1:3,735 and such titres are often obtained in the first set of sera (l,-~ig. 2b). We have used this technique for monitoring various farms where the cattle were affected with respiratory disorders caused by the BRS virus. The accompartu diagram illustrates the test results for 6 animals from which we had taken blood specimens by chance a few days before the disease was manifested. We found average titres of h32 in CF and l:810 in IIF in the first few days of the disease. The antibody level was elevated for a month and then the titre steadily declined. After two months the average CF ti• was ~nly 1:4 and the IIF titre was 1:135. Lo~ring of the CF titre continued ~id after the end of the third month the antibodies could no longer be detected. There is some degree of excretion of antibodies in the nasal mucus, for brotection of the respiratory mucosa. The availability of highly absorbent Ein Rhinovirus des Rindes Bovine respiratory syncytial virus Isolation of a respiratory syncytial virus from cattle in Britain Isolation of the infectious bovine rhinotracheitis virus A respiratory syncytial virus of bovine origin A myxovirus (SF4W) as~sociated with shipping fever of cattle Natural and experimental infection of cattle with human types of reoviruses Isolation, characterization and pathogenicity studies of a bovine respiratory syncytial virus Examen s@rologique de quelques troupeaux de bovins atteints de troubles respiratolres Le virus Respiratoire Syncytial et les Troubles Respiratoires des Bovins Diagnostische Nethoden des Sinzizialatmungsvirus beim Rind bet Erkr,nk-amgen der Atemwege Die klinische Diagnostik, die auf die Anzeichen eines Lungenemphysems beruhen und im Herbst auflreten, muss durch eine Labordiagnose hest~tigt werden. Die Sichtbarmachung der viralen Antigenen mittels F~bung ultrad'ttnner Schnitte mit einem dutch Fluoreszeinisothiozianat markierten Serum erweist sich wirksam und zuverl~ssig. Die Isolierung des Virus in den Zellkulturen ist oft sehr schwierig Les m~thodes de diagnostic du virus respiratoire syncytial bovin (RSB) lots d'affeetiens respiratoires Le diagnostic clinique, bas@ sur les signes d'emphys~me pulmonaire, apparaissant en automne, doit ~tre confirm~ par un diagnostic de laboratoire. L'isolement de l'agent viral sur culture cellulaire est souvent difficile. La mise en @vidence des antig~nes viraux par coloration de coupes ultra-fines l'aids d'un s@rum marqu@ ~ l'isothiocyanate de fluoresc~ine est efficace et fiable. La recherche d'anticorps dans des s@rums couples, par les m~thodes de fixation du compl@ment et principalement d'immunofluorescence indirecte I metodi di diagnostica del virus respiratorio simciziale del bovimo (RSB) melle affeziomi respiratorie La diagnosi clinica, basata sui sintomi di enfisema polmonare, che si man• festano in autunno, deve essere confermata mediante una diaguosi di laboratorio. L'isolamento dell'agente virale su coltura cellulare risulta spesso difficile. La messa in evidenza degli antigeni viral• medi~ite colorazione d• tagli ultrafini oon ~i siero marcato all'isotioci~uato di fluorescina ~ efficace e ed affidabile. Per stabilire la dia~nosi & di gTande utilitA la ricerca d• anticorpi he• sieri combh~ati, con i metod• della