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.     

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.     

表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定

为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。     

犊牛呼吸道合胞体病毒和巴氏杆菌混合感染的调查研究

为了对疑似牛呼吸道合胞体病毒和巴氏杆菌混合感染的犊牛进行病原鉴定,本研究采用常规病毒经细菌分离鉴定和PCR方法分别进行分离与鉴定。结果表明,该病毒株能在BT细胞上增殖并产生特征性合胞体形态的细胞病变;无血凝性和血吸附特性;能被牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)标准阳性血清中和;分离的病毒经RT-PCR鉴定为牛呼吸道合胞体病毒;根据菌落形态、细菌染色特性及生化特性,鉴定分离的细菌为巴氏杆菌。提示,该牛场为牛呼吸道合胞体病毒和巴氏杆菌混合感染。     

Bovine respiratory syncytial virus infection: immunopathogenic mechanisms

Bovine respiratory syncytial virus (BRSV) causes severe respiratory disease in young cattle. Much like the human respiratory syncytial virus, BRSV induces immunomodulation in the infected host, favoring a Th2 response. Several groups have demonstrated IgE responses to BRSV proteins during infection and particularly in response to vaccination with formalin-inactivated vaccine in the field and experimentally. Newer vaccine modalities that favor a shift to Th1 cytokine production have provided promising results. Infection with BRSV is a major contributor to the multi-pathogen disease, bovine respiratory disease complex. This review stresses the unique immunomodulatory aspects of BRSV infection, vaccination and its interaction with the host's immune system.     

Severe respiratory disease in dairy cattle in New York State associated with bovine respiratory syncytial virus infection

Severe respiratory disease associated with bovine respiratory syncytial virus (BRSV) infection has been identified in dairy cattle in New York State. The cases identified occurred in dairy calves and heifers. The disease was characterized in 4 animals by pathologic changes including interstitial pneumonia, necrotizing bronchiolitis with multinucleated syncytial epithelial cells and interstitial emphysema. BRSV antigen was demonstrated in lung samples or was isolated in tissue culture in all 4 cases. A retrospective survey of 6279 bovine diagnostic accessions between 1977 and 1982 revealed 66 cases of interstitial pneumonia, often with concurrent bronchiolitis. In this 5 year period, only 1 case in 1981 had interstitial pneumonia and bronchiolitis with pathologic features consistent with BRSV infection. It is concluded that pathogenic BRSV has entered New York State and that it is contributing to clinical respiratory disease in dairy cattle.     

Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.     

Bovine respiratory syncytial virus-specific monoclonal antibodies

Five hybridomas were produced which secreted monoclonal antibodies to bovine respiratory syncytial virus (BRSV). Two antibodies (8G12, 15C7) neutralized the virus and inhibited syncytia formation in vitro. These monoclonal antibodies also stained, by indirect fluorescent assay, an external envelope protein of living virus-infected cells, and recognized the 48k subunit of the viral fusion protein by Western blot analysis of bovine respiratory syncytial virus-infected cell lysates. Three other monoclonals (6A12, 14D3, 14E3) stained, by indirect fluorescent assay, acetone-fixed virus-infected cells but not living cells. Three hybridomas (6A12, 8G12, 15C7) secreted monoclonal antibody of isotype IgG1, k; two hybridomas secreted monoclonal antibody of isotype IgG2a, k. This apparently is the first report of monoclonal antibodies specific for BRSV glycoproteins.     

Efficacy of a saponin-adjuvanted inactivated respiratory syncytial virus vaccine in calves

The objective of this study was to determine whether a commercially available, saponin-adjuvanted, inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from experimental infection with virulent BRSV. This was a randomized controlled trial comprising 14, 8- to 9-week-old calves seronegative for BRSV Group 1 calves (n = 8) were not vaccinated and group 2 calves (n = 6) were vaccinated on days 0 and 19 with an inactivated BRSV vaccine. All calves were challenged with virulent BRSV on day 46. Clinical signs, arterial PO2, and immune responses were monitored after challenge. Calves were euthanatized on day 54 (8 d after challenge) and lungs were examined for lesions. Vaccination elicited increases in BRSV-specific immunoglobulin (Ig) G and virus neutralizing antibody titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, but no signs of clinical disease and minimal or no pulmonary lesions in vaccinated calves. Arterial blood oxygen values on day 53 (7 d after challenge) in control calves were significantly lower than those in vaccinated calves, which remained within normal limits. Control calves shed BRSV for several days after challenge, whereas BRSV was not detected on deep nasal swabs from vaccinated calves. In summary, the results indicated that this inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus 27 d after vaccination and significantly decreased the prevalence and severity of pulmonary lesions. Efficacy was similar to that reported for other commercial inactivated and modified-live BRSV vaccines.     

Study on the etiologic role of bovine respiratory syncytial virus in pneumonia of dairy calves

Bovine respiratory syncytial virus (BRSV) was the viral agent most commonly identified in 14 epizootics of pneumonia in dairy calves. A microtiter serum-virus neutralization test proved to be the best means of identifying involvement of BRSV; seroconversion (fourfold or greater rise in titer) was demonstrated in 10 of the 14 epizootics. Only limited involvement of bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza 3 virus, and bovine adenovirus type 3 was recognized. Pasteurella multocida was isolated in 12 of 14 epizootics, and Pasteurella haemolytica in 4 of 14 epizootics. Mycoplasmal and ureaplasmal agents were isolated in all 14 epizootics.     

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.     

Investigation of causative agents of bovine respiratory tract disease in a beef cow-calf herd with an early weaning program.

Serum samples were collected from early weaned fall calves shortly after the onset of respiratory tract disease. Antibody titers to infectious bovine rhinotracheitis (IBR) virus, parainfluenza type 3 (PI-3) virus, bovine viral diarrhea (BVD) virus, bovine adenovirus type 3 (BAV-3), and bovine respiratory syncytial virus (BRSV) were determined on paired (acute and convalescent) serums. Seroconversion rate (a fourfold or greater rise in antibody titer) for IBR virus was 4.3%, PI-3 virus--16.3%, BVD virus--9.6%, and BAV-3--2.2%. Seroconversion for BRSV was 45.4%. An increased rate of seroconversion for IBR, PI-3, and BVD viruses and BAV-3 was observed in the presence of BRSV seroconversion. These results suggest that BRSV may facilitate infection by other viruses. Results of virus isolation procedures from these calves were negative.     

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.     

牛呼吸道合胞体病毒RT-PCR检测方法的建立

根据GenBankTM上发表的牛呼吸道合胞体病毒核衣壳蛋白N基因序列,设计合成了一对特异性引物,扩增大小为596bp的目的片段,通过特异性试验、敏感性试验和重复性试验建立了牛呼吸道合胞体病毒的RT-PCR检测方法。所建立的牛呼吸道合胞体病毒的RT-PCR方法与牛腺病毒、牛副流感病毒、牛传染性鼻气管炎病毒、牛无浆体均无交叉反应,该方法的敏感性可达1TCID50。结果表明,该方法具有快速、敏感、特异性强和重复性好等特点,可作为牛呼吸道合胞体病毒检测的一种方法。     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24–96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p<0.05).     

Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin (Ig) M antibodies to bovine respiratory syncytial virus (BRSV) in cattle was developed. Monoclonal antibody to bovine IgM was used as the catching antibody. The IgM-ELISA was used, as well as a BRSV-specific IgG ELISA to determine the kinetics of IgM and IgG antibody responses to BRSV infections in cattle. High IgM and IgG antibody titers developed after naturally occurring or induced BRSV infection of calves (6 to 7 months old). Induced infection resulted in an IgM response that was first detectable at postinoculation day (PID) 11 reached a maximum at PID 13, and became undetectable again about PID 28. An IgG response also was detected by PID 11. However, a maximum response was not reached before PID 23, and titers remained high (until PID 80). In naturally occurring infection, IgM and IgG responses in calves were observed in the acute phase of epizootics of respiratory tract disease. Patterns of IgM and IgG response curves were similar to those observed in experimentally infected calves. The involvement of BRSV in an epizootic of respiratory tract disease in 8 calves (2 to 3 weeks old) was demonstrated by the detection of BRSV in several lung lavage samples. All calves had existing IgG antibodies to BRSV which were interpreted to be maternally derived. None of the calves responded with an increase in IgG antibody titer. However, a weak but distinct BRSV IgM antibody response occurred in 6 calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological study of a population of alpine chamois (Rupicapra r rupicapra) affected by an outbreak of respiratory disease

A serological survey of respiratory virus infections was carried out from 1998 to 2001 in Lecco province, Italy, as part of a health monitoring programme in a population of alpine chamois, many of which died of pneumonia in autumn and winter 2000 to 2001; 194 carcases of all age classes were found over a short period and in a small area. Eighteen of them, which were examined postmortem, consistently showed signs of severe fibrinous lobar pneumonia or catarrhal bronchopneumonia. Samples of serum from 145 chamois collected from hunted animals and carcases were tested by a virus neutralisation test against bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus, bovine herpesvirus type 1 and parainfluenzavirus type 3. Positive results were detected only for BRSV. The area was divided into two subunits on the basis of the distribution of deaths; in the areas where fatalities were observed there was a significant increase of BRSV titres at the beginning of the outbreak. Furthermore, during the 2000 and 2001 hunting seasons antibody titres to BRSV were significantly higher in the areas where mortality occurred. The roe deer living in the same area were not affected by pneumonia and had a low prevalence of titres to BRSV which did not vary during the period of the study.     

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.     

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.