A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Detection of IgE antibodies to bovine respiratory syncytial virus

The role of IgE antibodies against respiratory syncytial virus has attracted attention for both human and bovine disease. To detect such antibodies, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for bovine respiratory syncytial virus (BRSV). Firstly, anti-serum strongly positive for BRSV-specific IgE was produced by immunizing a levamisole-treated calf with BRSV. The presence and specificity of BRSV-specific IgE in this animal was confirmed with the Praunitz-Kustner (PK) technique. Potential interference in an ELISA by other BRSV-specific immunoglobulin isotypes was eliminated by preferential precipitation of serum samples with 27.5% saturated ammonium sulfate. The correlation between the PK and the ELISA assay was greater than 93% and the ELISA was found to be more specific than the PK. Indeed, in a pilot experimental infection study, the serum levels of BRSV-specific IgE were found to correlate with the symptom expression following repetitive live virus aerosolization. This may prove to be a useful rapid test to study both herd immunity and the potential pathogenic influence of IgE.     

A comparison of two ELISAs for the detection of antibodies to bovine leucosis virus in bulk-milk

OBJECTIVE: To estimate the sensitivity, specificity and detection limits for two bulk-milk enzyme-linked immunosorbent assays, the Svanovir BLV-gp51-Ab and the Lactelisa BLV Ab Bi indirect tank 250, for the detection of antibody to bovine leucosis virus in milk. PROCEDURE: Milk samples from 27 cows known to have enzootic bovine leucosis (EBL) were serially diluted with milk from a herd known to be free from the disease. The dilution at which antibodies could no longer be detected by each test was determined. A total of 1959 bulk-milk samples submitted to a laboratory for the Victorian (EBL) eradication program were tested with both the Svanovir and the Lactelisa assays. A Bayesian approach was used to calculate maximum-likelihood estimates of test sensitivity and specificity. An additional 660 bulk-milk samples were tested with both the Svanovir and the Lactelisa assays. Herds that had positive results on either or both of the assays were subjected to blood or milk testing of individual cattle. RESULTS: The dilution of milk at which the Svanovir assay failed to detect enzootic bovine leucosis antibody in half of the samples was 1 in 40, whereas the comparable value for the Lactelisa was 1 in 200. Computer modeling of the operating characteristics of the Svanovir assay indicated that the sensitivity of that assay would be considerably lower than that for the Lactelisa, and the specificity was estimated to be higher. Evaluation of the assays using 660 bulk-milk samples showed that the Lactelisa assay detected four infected herds that were not detected by the Svanovir test. No false positive results were recorded for either assay. CONCLUSION: Use of the Lactelisa assay in the Victorian EBL eradication program will enhance disease detection and eradication, but may also result in an increased frequency of false positive bulk-milk test results.     

牛呼吸道合胞体病毒RT-PCR检测方法的建立及初步应用

本研究旨在评估日粮补充以枯草芽孢杆菌、乳酸菌和酵母菌组成的复合益生菌对育肥猪生长性能、小肠绒毛形态及肝脏脂代谢相关指标的影响。试验将208头体重接近的18周龄三元育肥猪随机分为2组,每组4个重复,每个重复26头。对照组育肥猪饲喂基础日粮,处理组育肥猪饲喂基础日粮+1.5 g/kg由乳酸菌、枯草芽孢杆菌和酵母菌组成的复合益生菌,饲养试验时间为8周。结果：处理组试验28 d育肥猪体重、1～28 d和1～56 d平均日增重较对照组显著提高2.28%、10.33%和4.56%（P＜0.05）,但1～28 d和1～56 d料重比显著降低11.15%和5.72%（P＜0.05）。处理组空肠绒毛高度、绒毛高度与隐窝深度比值较对照组分别显著提高5.45%和17.52%（P＜0.05）,而隐窝深度显著降低10.27%（P＜0.05）,对照组与处理组育肥猪血清低密度脂蛋白和高密度脂蛋白浓度及肝脏胆固醇、高密度脂蛋白和低密度脂蛋白浓度无显著差异（P＞0.05）,但处理组育肥猪血清甘油三酯和胆固醇浓度及肝脏甘油三酯浓度显著低于对照组（P＜0.05）。结论：在本研究条件下,日粮添加1.5 g/kg以枯草芽孢杆菌、乳酸菌和酵母菌组成的复合益生菌可通过改善空肠绒毛形态,提高育肥猪生长前期和全期的平均日增重和饲料效率,降低肝脏和血清胆固醇浓度。 [关键词]益生菌|育肥猪|生长性能|绒毛形态|脂代谢     

Indirect haemagglutination test for the detection and assay of antibody to bovine respiratory syncytial virus

An indirect haemagglutination (IHA) test was used for the rapid assay of antibody to bovine respiratory syncytial virus. Antigens for the sensitisation of formalised tanned erythrocytes were prepared by treatment of virus infected cells with non-ionic detergent. A close serological relationship was shown by the IHA test between the strain of bovine respiratory syncytial virus used and the A2 strain of human respiratory syncytial virus. The IHA test was sensitive and reproducible. A linear correlation was demonstrated between antibody titres obtained by the IHA test and the serum neutralisation test. Titres obtained by the IHA test were approximately 60 times greater than serum neutralisation titres. Serum samples from 803 two-year-old heifers in 48 herds in England were examined by the IHA test. Ninety-four per cent of the animals had antibody to respiratory syncytial virus. Examination of paired serum samples from outbreaks of respiratory disease by the IHA test showed that respiratory syncytial virus was associated with seven out of 15 outbreaks.     

Comparison of four RT-PCR assays for detection of bovine respiratory syncytial virus

RT-PCR assays for detection of BRSV, based on four different sets of primers were optimized and evaluated for their sensitivity and specificity. Primers used in this study were specific for genes encoding three BRSV proteins, nucleoprotein N and glycoproteins F and G. Our results indicated that RT-PCR with primers B7:B8 for G protein was the most efficient in detecting BRSV. Starters B7:B8 reacted specifically only with BRSV strains, no cross-reaction with other closely related viruses to BRSV was observed. RT-PCR sensitivity was also high and amounted to 10(1.66) TCID50. Starters for F and N genes of BRSV were not sufficiently specific and cross-reacted with RNA of HRSV. RT-PCR with primers for the genes F and N of BRSV was characterized by a lower sensitivity than RT-PCR with primers B7:B8. In conclusion, RT-PCR specific to a sequence of glycoprotein G gene, seemed to be the most useful for BRSV detection.     

Use of the indirect fluorescent antibody test in the detection of bovine respiratory syncytial virus antibodies in bovine serum.

The indirect fluorescent antibody test was adapted for identifying bovine respiratory syncytial virus and its specific antibody, using goat turbinate (GTU) cells. The virus caused maximal cytopathic effects in GTU cells 4 to 8 days postinfection, but fluorescence was not readily detected during this period. Fluorescence was maximal in infected GTU cells at 24 to 36 hours postinfection, but could be detected 48 hours postinfection. Bovine serums (331) which had been submitted to the Oklahoma Animal Disease Diagnostic Laboratory were tested for antibodies to this virus, and 73.6% were found to be positive.     

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.     

Dot-enzyme linked immunosorbent assay as an alternative technique for the detection of bovine respiratory syncytial virus (BRSV) antibodies

A dot-ELISA test for the detection of anti-BRSV antibodies is described. The objective of this study was the standardisation of a test as a fast, inexpensive and effective alternative to detect anti-BRSV antibodies. Its sensitivity, specificity and usefulness were compared to a commercial ELISA-kit and to the standard serum neutralisation (SN) test. The standardisation of the technique was done using nitrocellulose disks soaked with a viral sample isolated in Brazil, BRSV-25-BR. The best results were obtained when the disks were sensitised with a purified antigen at a concentration of 0.7 microg/disk and the bovine serum was diluted 1: 200. The experiment used 423 samples of bovine serum collected in the main cattle breeding centres in Brazil. The standard SN, dot-ELISA technique and commercial ELISA kits scored 67.8%, 71.8% and 72.3% of the samples as positive, respectively. When compared to the SN test, the standardised dot-ELISA and the commercial ELISA tests presented relative sensitivities of 92.3% and 91.6% and relative specificities of 71.3% and 68.4% respectively. The results demonstrated that the dot-ELISA test is adequate for the objectives proposed by this study, being easy to use and economically viable. Thus, this test represents an alternative for BRSV serological diagnosis in the substitution of SN and commercial ELISA tests, recommendable for utilisation in laboratories with few resources.     

Application of a modified indirect fluorescent antibody test to the detection of antibodies to bovine respiratory syncytial virus in Ontario cattle.

A modified indirect fluorescent antibody test for the detection of serum antibodies to bovine respiratory syncytial virus was developed. The test made use of Terasaki plastic microtiter plates in which bovine respiratory syncytial virus (Saskatchewan strain) infected Georgia bovine kidney cells were grown and fixed in situ by a modified acetone fixation procedure. Evans blue dye was used as a counterstain to reduce nonspecific fluorescence. In a study of 986 field sera from a geographically broad cross-section of mature Ontario cattle, 95% of the samples were found to be positive at or above a 1:2 dilution. No seronegative regions, counties or herds were identified. When representative samples covering a range of indirect fluorescent antibody titers were further examined by a microtiter virus neutralization assay, a significant agreement was found between the two tests. Up to a fourfold decrease in titer was observed when antigen coated plates were stored at -70 degrees C for four months. The modified indirect fluorescent antibody test for bovine respiratory syncytial virus antibody detection proved to be a rapid, practical procedure for use in the diagnostic laboratory. This study confirms that bovine respiratory syncytial virus is widespread in the Ontario cattle population and that most mature cattle can be assumed to have been exposed to this virus.     

Comparative evaluation of indirect and sandwich ELISA for the detection of antibodies to bovine respiratory syncytial virus (BRSV) in dairy cattle

Seroprevalence of bovine respiratory syncytial virus (BRSV) infection in both exotic and crossbred cattle were described. A baculovirus expressed recombinant purified nucleocapsid (N) protein was used in indirect and sandwich ELISA for screening of 499 bovine sera samples from all over the state for the presence of BRSV antibodies. The seroprevalence rate of BRSV was found to be 46.09% through indirect ELISA while it would found to be 65.33% by sandwich ELISA. The result also indicated that exotic breeds were more susceptible to BRSV infection compared to crossbred cattle. A comprehensive analysis on susceptibility to BRSV as regards to various factors like age and sex was also summarized.     

Cell-mediated immune response in cattle to bovine respiratory syncytial virus

Cattle inoculated with bovine respiratory syncytial virus (BRSV) were evaluated for the development of a cell-mediated immune response. Results of the leukocyte migration-inhibition test under agarose and the delayed hypersensitivity test indicated that a cell-mediated immune response was elicited after intranasal inoculation of calves with BRSV. Migration inhibition in the leukocyte migration-inhibition test was detected by postinoculation day (PID) 5 and reached maximum inhibition on PID 21. Inhibition of leukocyte migration was still evident by PID 42 when values were still appreciably greater than preinoculation values. All of the calves inoculated with BRSV developed a delayed hypersensitivity skin response when challenge exposed intradermally with BRSV antigen.     

Prevalence and specificity of antibodies to bovine respiratory syncytial virus in sera from feedlot and range cattle

The specificity of serum antibodies for the polypeptides of bovine respiratory syncytial virus (BRSV) was examined, using sera obtained from feedlot and range cattle. Test results in sera from feedlot cattle indicated a 60% rate of seroconversion and 95% seropositivity to BRSV, associated with lack of clinical signs indicative of respiratory tract disease. Exposure to other common respiratory tract viruses also was high (greater than or equal to 92% to bovine herpesvirus type 1, bovine viral diarrhea virus, and para-influenza virus type 3). Test results in sera from range cattle indicated BRSV seropositive rates of 28% in calves, 49% in yearling cattle, and 70% in mature cows; clinical signs of respiratory tract disease were not observed in these cattle. Antibodies to BRSV in sera from cattle in both environments reacted predominantly with polypeptides of molecular weight 80,000 through 85,000, 40,000, and 28,000. Reactivity to a glycoprotein of molecular weight between 43,000 and 44,000 and to several glycopolypeptides of smaller molecular weight increased in serum specimens obtained from feedlot cattle between time of entry into the feedlot and slaughter.     

Failure to detect antibody to bovine respiratory syncytial virus in bovine fetal serum.

Sera obtained from 147 bovine fetuses estimated to be between 120 and 270 days of gestation at an abattoir were tested for antibody to bovine respiratory syncytial virus. Antibody to bovine respiratory syncytial virus was not detected in any of the sera examined. Based on the results of this study and a review of the literature, it appears that transplacental infection by bovine respiratory syncytial virus does not occur, or is uncommon.     

The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska stained BRSV antigen in infected bovine lung with acceptable background staining of uninfected tissues. This method offers advantages over other techniques for BRSV diagnosis in that fresh tissue is not required and all reagents may be purchased commercially.     

Prevalence of antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine herpesvirus-1, and bovine parainfluenza-3 virus in sheep and goats in Quebec

Serum samples were collected from 1,075 clinically normal sheep and goats from 77 flocks in 7 agricultural regions of Quebec from June to August 1982. Sheep and goats were tested for antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes-virus-1 by the indirect fluorescent antibody technique and for parainfluenza-3 virus by the hemagglutination inhibition test. The prevalence of antibodies in animals to respiratory syncytial virus was 31%; to bovine viral diarrhea virus, 22.2%; to bovine herpesvirus-1, 10.8%; and to parainfluenza-3 virus, 23.2%. Antibodies prevailed in similar proportions in young (less than 1 year) and adult (greater than 1 year) animals.     

Experimental reproduction of respiratory tract disease with bovine respiratory syncytial virus

An experiment was conducted to reproduce respiratory tract disease with bovine respiratory syncytial virus (BRSV) in one-month-old, colostrum-fed calves. The hypothesized role of viral hypersensitivity and persistent infection in the pathogenesis of BRSV pneumonia was also investigated. For BRSV inoculation a field isolate of BRSV, at the fifth passage level in cell culture, was administered by a combined respiratory tract route (intranasal and intratracheal) for four consecutive days. Four groups of calves were utilized as follows: Group I, 6 calves sham inoculated with uninfected tissue culture fluid and necropsied 21 days after the last inoculation; Group II, 6 calves inoculated with BRSV and necropsied at the time of maximal clinical response (4-6 days after the last inoculation); Group III, 6 calves inoculated with BRSV and necropsied at 21 days after the last inoculation; Group IV, 6 calves inoculated with BRSV, rechallenged with BRSV 10 days after initial exposure, and necropsied at 21 days after the initial inoculation. Clinical response was evaluated by daily monitoring of body temperature, heart rate, respiratory rate, arterial blood gas tensions, hematocrit, total protein, white blood cell count, and fibrinogen. Calves were necropsied and pulmonary surface lesions were quantitated by computer digitization. Viral pneumonia was reporduced in each principal group. Lesions were most extensive in Group II. Disease was not apparent in Group I (controls). Significant differences (p less than 0.05) in body temperature, heart rate, respiratory rate, arterial oxygen tension, and pneumonic surface area were demonstrated between control and infected calves. Results indicate that severe disease and lesions can be induced by BRSV in one-month-old calves that were colostrum-fed and seropositive to BRSV. BRSV rechallenge had minimal effect on disease progression. Based on clinical and pathological response, results did not support viral hypersensitivity or persistent infection as pathogenetic mechanisms of BRSV pneumonia.     

Immunology of bovine respiratory syncytial virus infection of cattle

Bovine respiratory syncytial virus (BRSV) is a respiratory pathogen of cattle that causes severe disease in calves alone and as one of several viruses and bacteria that cause bovine respiratory disease complex. Like human RSV this virus modulates the immune response to avoid stimulation of a vibrant CD8+ T cytotoxic cell response and instead promotes a Th2 response. The Th2 skew sometimes results in the production of IgE antibodies and depresses production of the Th1 cytokine interferon γ. Innate immune cells have a pivotal role in guiding the adaptive response to BRSV, with selective secretion of cytokines by pulmonary dendritic cells. Here we review some of the pertinent observations on immune responses to BRSV infection and vaccination and illustrate how experimental infection models have been used to elucidate the immunopathogenesis of BRSV infection. Recent experiments using intranasal vaccination and/or immune modulation with DNA based adjuvants show promise for effective vaccination by the stimulation of Th1 T cell responses.     

Reinfection of lambs with bovine respiratory syncytial virus.

Eight lambs which were experimentally infected with bovine respiratory syncytial virus (RSV) when they were six to eight weeks old were challenged with the same virus seven months later. After reinfection, lambs developed mild clinical disease and the virus was isolated from nasal swabs from three lambs and peripheral blood from two lambs. Reinfection resulted in changes in peripheral blood cell populations. There was an early increase in the number of CD8+ T lymphocytes and B (LCA p220+) lymphocytes but the proportions of CD4+ and CD4-CD8- T lymphocytes were significantly reduced. Peripheral blood mononuclear cells obtained from lambs reinfected with bovine RSV showed significantly higher responses to bovine RSV antigen in vitro than those obtained from control lambs but their responses to the mitogen phytohaemagglutinin were significantly lower than in control lambs. RSV-specific IgG, IgM and IgA levels of serum samples obtained 10 days after challenge were significantly higher than those of serum samples obtained before challenge.