Laboratory diagnosis methods for bovine respiratory syncytial virus

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.

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Kurzfassung Die Erkrankungen der Atemwege die durch den Sinzizialatmungsvirus der Rinder hervorgerufen werden, können zur Zeit mit Leichtigkeit diagnostiziert werden. Die klinische Diagnostik, die auf die Anzeichen eines Lungenemphysems beruhen und im Herbst auftreten, muss durch eine Labordiagnose bestätigt werden. Die Sichtbarmachung der viralen Antigenen mittels Färbung ultradünner Schnitte mit einem durch Fluoreszeinisothiozianat markierten Serum erweist sich wirksam und zuverlässig. Die Isolierung des Virus in den Zellkulturen ist oft sehr schwierig. Bei der Aufstellung der Diagnose ist die Suche nach Antikörpern in den gekoppelten Seren, mit der Komplementbindungsmethode und besonders mit der indirekten Immuno-Fluoreszenz, von grosser Wichtigkeit.

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Resume En cas de troubles respiratoires dus au virus Respiratoire Syncytial Bovin (RSB) le diagnostic peut être posé actuellement sans grande difficulté. Le diagnostic clinique, basé sur les signes d'emphysème pulmonaire, apparaissant en automne, doit être confirmé par un diagnostic de laboratoire. L'isolement de l'agent viral sur culture cellulaire est souvent difficile. La mise en évidence des antigènes viraux par coloration de coupes ultra-fines à l'aide d'un sérum marqué à l'isothiocyanate de fluorescéine est efficace et fiable. La recherche d'anticorps dans des sérums couplés, par les méthodes de fixation du complément et principalement d'immunofluorescence indirecte, est de grande utilité pour l'établissement du diagnostic.

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Riassunto Attualmente la diagnosi sulle turbe respiratorie causate dal virus respiratorio sinciziale bovino (RSB) non presenta difficoltà di rilievo. La diagnosi clinica, basata sui sintomi di enfisema polmonare, che si manifestano in autunno, deve essere confermata mediante una diagnosi di laboratorio. L'isolamento dell'agente virale su coltura cellulare risulta spesso difficile. La messa in evidenza degli antigeni virali mediante colorazione di tagli ultrafini con un siero marcato all'isotiocianato di fluorescina è efficace e ed affidabile. Per stabilire la diagnosi è di grande utilità la ricerca di anticorpi nei sieri combinati, con i metodi del la fissazione del complemento e in particolare con l'immunofluorescenza indiretta.

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This article was originally written in French. Copies of the French version may be obtained free of charge by writing to: Mr J. Rodesch, Commission of the European Communities, DG XIII, Bâtiment Jean Monnet, Rue Alcide de Gasperi, Kirchberg, Luxembourg.     

Enzootic bovine leukosis virus in Brazil

Summary A sero-epidemiological survey for antibodies to the glycoprotein of enzootic bovine leukosis virus showed that the infection is widely disseminated in the State of Rio de Janeiro, Brazil. Sera from 1,290 females and 154 males from 12 dairy herds were tested by the agar gel precipitin test. Seven hundred and one females (54.3%) and 68 males (44.2%) were found to have specific antibodies. These antibodies were demonstrated in all 7 age groups tested. The older age groups contained the highest percentage of reactors. The results are briefly discussed in relation to management practices and environmental conditions.

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El Virus De La Leucosis Bovina Enzootica En Brasil

Resumen Una encuesta suero-epidemiológica por anticuerpos contra la glicoproteina del virus de la leucosis bovina enzootica mostró que la infección está ampliamente diseminada en el Estado de Rio de Janeiro, Brasil. Sueros de 1290 hembras y 154 machos pertenecientes a 12 rebaños lecheros fueron examinados en la prueba de precipitación en agar. Se encontró que 701 hembras (54.3 por ciento) y 68 machos (44.2 por ciento) poseían anticuerpos específicos. Estos anticuerpos fueron demonstrados en todos los siete grupos de edad examinados. Los grupos de mas edad contenian un porcentaje mayor de reactores. Los resultados son discutidos brevemente en relación a las prácticas de manejo y a las condiciones ambientales locales.

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Virus De La Leucose Bovine Enzootique Au Bresil

Résumé Une enquête séro-épidémiologique pour la détection de la présence de la glycoproteine du virus de la leucose enzootique bovine a montré que cette affection est largement disséminée dans l'Etat de Rio de Janeiro, au Brésil. Des échantillons de sérum provenant de 1290 femelles et 154 mâles appartenant à 12 troupeaux laitiers ont été étudiés par précipitation en gélose—701 femelles (54,3 p. 100) et 68 mâles (44,2 p. 100) ont été reconnus comme possédant des anticorps spécifiques. Ces anticorps ont été mis en évidence dans la totalité des 7 groupes d'âges étudiés. Les groupes d'âges plus élevés contenaient le plus haut pourcentage d'animaux positifs. Les résultats sont brièvement discutés, dans leur relation avec les pratiques de l'élevage et les conditions de l'environnement.

     

Enzootic bovine leukosis virus in Brazil

A sero-epidemiological survey for antibodies to the glycoprotein of enzootic bovine leukosis virus showed that the infection is widely disseminated in the State of Rio de Janeiro, Brazil. Sero from 1,290 females and 154 males from 12 dairy herds were tested by the agar gel precipitin test. Seven hundred and one females (54.3%) and 68 males (44.2%) were found to have specific antibodies. These antibodies were demonstrated in all 7 age groups tested. The older age groups contained the highest percentage of reactors. The results are briefly discussed in relation to management practices and environmental conditions.     

The diagnosis of enzootic bovine leukosis

This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficient to do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera).     

Serology for diagnosis and epizootiological studies of bovine respiratory syncytial virus infections

The complement fixation test (CFT) and the virus neutralisation test (VNT), performed as a plaque reduction test, were employed to measure antibodies to bovine respiratory syncytial virus. The CFT with bovine sera was performed with supplementation of the complement factors in fresh guinea pig serum by an adequate amount of Clq-factor of the bovine species. Kinetics of maternally derived antibodies and the antibody response after spontaneous and experimental infections and after intramuscular vaccination were studied by both tests. Patterns of development of complement fixing and virus neutralising antibodies were generally similar and titres equalled each other in the test systems that are described. However a VNT detected antibodies a few days earlier after an infection than a CFT and peak-levels reached after a naturally acquired infection decreased faster in a CFT than in a VNT: a mean decrease of 3.1 and 1.4 log2 units was found in 13 weeks respectively. Mean half-life of passive antibodies was 25 days in a VNT. An infection with bovine respiratory syncytial virus could be diagnosed by serology, using a CFT on acute and convalescent serum samples of a number of animals in a group. Serology is preferable to virus isolation for routine diagnosis of bovine respiratory syncytial virus infections. Paired sera, collected at 14-day intervals and examined by CFT, are recommended for the diagnosis of the cause of respiratory disease. A VNT is preferable if low antibody levels are to be detected because non-specific reactions occur in a CFT at low serum dilutions.     

Protection against bovine leukosis virus infection in sheep with the BL 20 bovine lymphoblastoid cell line

The bovine lymphoblastoid BL 20 cell line derived from a case of sporadic bovine leukosis when inoculated into sheep did not induce an antibody response directed against bovine leukosis virus (BLV) structural proteins. Sheep were inoculated twice with the BL 20 cell line and then challenged with BLV infected lymphocytes. Three out of four sheep challenged four weeks after BL 20 inoculation did not develop BLV antibodies. Of the 12 sheep challenged later, three sheep did not develop BLV antibodies. BLV was isolated from all the seropositive animals and from none of the seronegative animals.     

Induction of neutralizing antibodies against bovine leukosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leukosis virus envelope glycoprotein

Three kinds of recombinant vaccinia virus (RVV)--mO-HA/ATI, LO1-HA/ATI and mO-HA/7.5kD--expressing bovine leukosis virus (BLV) envelope glycoprotein (gp60) were constructed. The BLV envelope gene of RVV mO-HA/ATI and LO1-HA/ATI or of RVV mO-HA/7.5kD was expressed under control of the promoter of A-type inclusion body (ATI) protein gene of cow-pox virus or vaccinia virus 7.5-kD protein gene, respectively. The vaccinia virus strain, LC16mO, was used as vector for RVV mO-HA/ATI and mO-HA/7.5kD, and strain LO-1 was used for RVV LO1-HA/ATI. Strains LC16mO and LO-1 are attenuated vaccine virus strains originating from the Lister original vaccinia virus. All 3 kinds of constructed RVV expressed gp60 in cultured rabbit kidney cells after infection; mO-HA/ATI expressed more antigen than did mO-HA/7.5kD. Rabbits vaccinated with RVV produced considerable antibody capable of inhibiting syncytium formation, as well as antibody with virion-binding ability. The RVV that used ATI promoter induced higher antibody titer than did the RVV that used 7.5-kD promoter. Results indicate that BLV gp60 is responsible for induction of neutralizing antibodies that suppress in vitro formation of syncytia among BLV-infected cells. Applicability of RVV, especially those using ATI promoter, was evaluated in a vaccine against bovine leukosis.     

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.     

Association between the strength of serologic recognition of bovine leukosis virus and lymphocyte count in bovine leukosis virus-infected cows

OBJECTIVE: To determine whether strength of serologic recognition of bovine leukosis virus (BLV) by use of ELISA is associated with blood lymphocyte counts. DESIGN: Prospective study. ANIMALS: 161 cows with positive results of ELISA for BLV. PROCEDURE: Sample-to-positive ratio (S:P), which is the ratio between the test sample and a positive control sample, was compared among lymphocytotic and nonlymphocytotic cows. A regression model was constructed to evaluate the association between blood lymphocyte concentration and S:P, age, and the interaction of these terms. RESULTS: Mean S:P differed significantly between lymphocytotic (2.58 +/- 0.36) and nonlymphocytotic (2.38 +/- 0.39) cows. Age and S:P were significantly associated with lymphocyte count. CONCLUSIONS AND CLINICAL RELEVANCE: Sample-to-positive ratio and lymphocyte count were related; however, cows with high S:P were not always lymphocytotic. Culling cows on the basis of S:P will reduce the herd load of infectious virus faster than random culling of ELISA-positive cows; however, culling on the basis of lymphocyte count will eliminate a greater proportion of the reservoir of infection.