Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies

Monoclonal antibodies against hog cholera and bovine viral diarrhoea viruses were assayed on organ tissue sections of experimentally infected animals. The animals had been infected simultaneously with both viruses. The antibodies were tested using an indirect immunofluorescence test and an indirect enzyme immunoassay with a biotin/streptavidin/peroxidase detection system. A polyclonal hyperimmune serum was used as a control in direct immunofluorescence tests. Both techniques based on monoclonal antibodies were more sensitive and more specific than the conventional test, the enzyme immunoassay being more sensitive than the immunofluorescence test. Small amounts of BVD viral antigen were demonstrable with monoclonal antibodies in most organ tissues.     

Serological comparison of cytopathogenic and non-cytopathogenic bovine viral diarrhea-mucosal disease viruses isolated from cattle with mucosal disease

Ten cattle that died with mucosal disease were examined for bovine viral diarrhea-mucosal disease (BVD-MD) viruses. Both cytopathogenic (CP) and non-cytopathogenic (NCP) BVD-MD viruses were isolated concomitantly from 9 of them, and only a CP virus was recovered from the other. Then each pair of CP and NCP viruses was compared serologically by a serum neutralization test. Each pair of CP and NCP viruses from the same cattle was found to be serologically indistinguishable, although a minor antigenic difference was observed among the groups of the paired viruses. These results seem to support the hypotheses that mucosal disease occurs in persistently infected cattle which were induced by in utero infection with NCP virus when they are superinfected with CP virus, and the antigenic homology in CP and NCP BVD-MD viruses may be an important factor in the pathogenesis of the disease.     

Variation in immunogenicity of ruminant pestiviruses as determined by the neutralisation assay

Immunogenicity in naive three-month-old Friesian bull calves of nine ruminant pestiviruses, three each of type 1 bovine virus diarrhoea virus (BVDV), type 2 BVDV and border disease virus (BDV) was directly compared in reciprocal cross-neutralisation tests using sera obtained eight weeks after intranasal and intravenous inoculation with live virus. Cytopathic (CP) type 1 BVDV strain C86, non-cytopathic (NCP) type 2 BVDV strain 890 and NCP BDV strain V2536/2 were found to elicit significantly broad cross-neutralising antibodies against viruses in other species whereas other virus strains in all three species produced a much more pronounced homologous and/or species specific response. Results are clearly relevant in the selection of strains for vaccines against diseases caused by these successful, economically important ubiquitous viruses.     

牦牛病毒性腹泻病研究进展

牦牛感染牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)后常发生胃肠炎等消化系统疾患和呼吸系统疾患、繁殖障碍,造成机体损伤,给牦牛产业发展造成一定影响。基于5"-UTR的差异将BVDV分为BVDV-I和BVDV-II两种基因型,牦牛源BVDV的主要基因型为BVDV-1型;根据BVDV对宿主细胞的致病性,分为致细胞病变型(Cy-topathogenic,CP)和非致细胞病变型(Non-cytopathogenic,NCP)两个生物型,牦牛源BVDV既有CP型又有NCP型,且这两种生物型能相互转化。牦牛生活的高原地区风、鸟类的迁徙等因素可能都会导致牦牛BVD传播范围广泛,牛群中大量的BVDV携带者和牦牛特殊而又复杂的生态系统导致了BVDV种间传播的巨大潜力,影响着牦牛产业的健康发展和养殖经济收益。针对本病在牦牛群中的感染流行等情况未发现相关详细的研究报道,笔者就牦牛BVD病原学、流行病学调查、实验室诊断、综合防治等方面研究进展情况进行整理,供同仁在研究和防控中参考借鉴。     

Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies

Five monoclonal antibodies against the bovine viral diarrhoea (BVD) viral strain NADL were isolated and characterized by an indirect immunofluorescence assay. Extensive cross-reactions were detected when the antibodies were tested with 12 heterologous BVD and four hog cholera (HC) viral strains. One antibody reacted with all strains tested. Two antibodies were specific for cytopathogenic BVD viruses, but failed to react with HC virus. The other antibodies reacted to varying degrees with BVD and HC viral strains.     

Demonstration of the agent of bovine viral diarrhea-mucosal disease (BVD/MD) in comparison between organ fluoresence freezing method and the direct cultivation in tissue culture]

Organs of 100 calves whose clinical symptoms indicated a BVD/MD viral infection have been examined in the laboratory by different methods on BVD/MD virus in order to compare the effectivity of the single test-systems. By inoculation of organ material into tissue culture from foetal calf kidneys and additional staining with swine-fever conjugate in the CCSC-system 59 calves were detected as BVD/MD virus carriers. Taking this result for comparative purposes equal to 100% there could be stated an effectivity of 73% by inoculation of tissue cultures and judging cpe instead of staining with fluorescent antibody as above. Only 34% reactions could be demonstrated by means of heterotypic immunofluorescence in organ tissues. For diagnostic purposes a combination of the easy and quick method of heterotypic immunofluorescence in organ tissues and a cultural virus isolation from organ material with additional immunofluorescence is recommended.     

Complement-Fixing And Neutralizing Antibody Response To Bovine Viral Diarrhea And Hog Cholera Antigens

In calves inoculated with bovine viral diarrhea (BVD) viruses and soluble antigen, the complement-fixing (CF) antibodies appeared before serum-neutralizing (SN) antibodies and remained at high levels throughout the test period. A rapid rise in SN antibodies occurred after challenge with homologous virus with no apparent effect on CF antibody levels.

The CF antibody responses in calves infected with cytopathogenic NADL-MD and noncytopathogenic CG-1220 viruses were similar whereas SN antibody responses indicated strain specificity by reciprocal cross-neutralization tests.

The CF antibody levels in 5 hog cholera (HC) antisera were assayed using the soluble antigen of NADL-MD BVD virus. No demonstrable SN antibodies were present in four HC antisera tested against NADL-MD virus, but a significant titer was present in the commercially prepared antiserum.

Virus was reisolated from animals infected with BVD viruses by buffy coat culture technique during 3 weeks postinoculation, even when significant levels of CF and SN antibodies were present.

     

Serologic detection and practical consequences of antigenic diversity among bovine viral diarrhea viruses in a vaccinated herd.

Samples of sera were obtained from 5,725 cows in a semiclosed herd. In each of the preceding 7 years, the herd was vaccinated against bovine viral diarrhea (BVD) with killed virus. Neutralizing antibody tests were done on all samples of sera, using cytopathic virus, BVD-TGAC virus, that was antigenically distinct from the vaccine virus. Most samples of sera had high titers of neutralizing antibodies against BVD-TGAC virus. In 48 samples of sera, neutralizing antibodies were not detected against BVD-TGAC virus, but were detected against the vaccine virus. Neutralizing antibodies against selected noncytopathic BVD viruses were not detected in several samples of serum that had neutralizing antibodies against the vaccine virus and BVD-TGAC virus. Noncytopathic BVD virus was isolated from sera obtained from 3 cows less than 4 years old. Two cows were available for further testing, and persistent infection with BVD virus was confirmed in both cows. The BVD viruses isolated from those cows were not neutralized by several samples of sera. Immunoprecipitation of polypeptides induced by the vaccine virus was done with selected samples of serum. Two patterns of immuno-precipitated viral-induced polypeptides were identified. One pattern was consistent with exposure of cows with live virus. The other pattern was consistent with exposure of cows with only the killed virus vaccine.     

Bovine viral diarrhoea virus (BVDV) subgenotypes in diagnostic laboratory accessions: distribution of BVDV1a, 1b, and 2a subgenotypes

The prevalence of bovine viral diarrhoea virus (BVDV) biotypes and subgenotypes was determined from 131 BVDV positive samples from a diagnostic laboratory. The majority of the isolates were from Oklahoma; however, other states including Kansas, Texas, and Arkansas were represented. These BVDV samples were from submissions of 76 live animals and 55 necropsy samples. There were 131 BVDV samples represented by 117 noncytopathic (NCP), 11 cytopathic (CP) and 3 cases with mixed NCP and CP biotypes. The NCP isolates were more common (P < 0.05) than the CP and NCP/CP combination. The BVDV samples were segregated into three subgenotypes by differential PCR and sequencing of a viral genomic region, 5'-untranslated region (5'-UTR). There were more BVDV1b subgenotypes 60/131 (45.8%) than BVDV1a, 37/131 (28.2%) or BVDV2a, 34/131 (26.0%) (P < 0.05). The organ system involvement included the major categories such as respiratory, digestive, mixed/multiple organs, abortions, and persistent infections (PI). All three BVDV subgenotypes were found in persistently infected (PI) cattle and respiratory diseases, both major requests for BVDV diagnosis. Only one of the 131 viruses was genetically similar to the strains present in U.S. vaccines.     

Hog cholera diagnostic techniques.

Clinical signs and lesions can sometimes provide the basis for a presumptive diagnosis of hog cholera (HC). However, an accurate diagnosis requires laboratory testing. The usual procedure for the detection of viral antigen is the examination of cryostat sections stained with fluorescein-conjugated HC antiserum. A more definitive technique is isolation of the virus in PK-15 cell cultures and identification of the viral antigen in cells using an HC fluorescent antibody conjugate. As bovine viral diarrhea (BVD) virus will cross-react with HC virus, isolation must be confirmed by the comparison of BVD and HC staining or, preferably, by the use of monoclonal antibodies that can differentiate between HC and BVD viruses. Hog cholera surveillance must rely on serology. The fluorescent antibody virus neutralization (FAVN) test is the classical technique, and HC and BVD antibody can usually be differentiated if HC-positive serum samples are tested against both viruses. Recently the enzyme-linked immunosorbent assay (ELISA) and peroxidase-labeled antibody tests have become the commonly used techniques.     

Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro

Isolates of non-cytopathogenic bovine viral diarrhoea (BVD) virus from 18 persistently infected calves from one herd were compared by using monoclonal antibodies directed against the major viral glycoprotein gp53. All the isolates displayed an almost identical reaction pattern. Based on this antigenic analysis three cytopathogenic BVD and three non-cytopathogenic BVD viruses closely related to the non-cytopathogenic BVD herd isolate were selected. Six of the persistently infected calves were inoculated with a pool of the three closely related cytopathogenic BVD viruses and two with a pool of the three non-cytopathogenic BVD viruses. In addition three animals were infected with one closely related cytopathogenic BVD strain (Indiana) and two animals with the antigenetically different cytopathogenic BVD viral strain A1138/69. Regardless of the inoculation route all the animals superinfected with closely related cytopathogenic BVD viruses developed the characteristic lesions of mucosal disease within 14 days of infection. Animals which were inoculated with non-cytopathogenic BVD viruses which closely resembled the herd isolate, or with cytopathogenic BVD viruses which did not resemble the herd isolate did not develop any signs of disease. However, the latter group produced antibodies to the superinfecting virus.     

牛病毒性腹泻病毒间接ELISA方法的建立

持续性感染和免疫耐受是牛病毒性腹泻病的重要特征,也是该病的防控难点.本试验将2种生物型的牛病毒性腹泻病毒(BVDV):致细胞病变(CP)型(BVDV OregonC24株)和非致细胞病变(NCP)型(BVDV Yak株),灭活、浓缩作为抗原,分别免疫家兔制备高免血清.同时,使用BVDV全病毒蛋白作为包被原,建立了检测BVDV的间接ELISA检测方法.本试验利用该方法对高免血清进行检测,探讨CP型BVDV与NCP型BVDV抗原免疫原性差异.结果显示,CP型BVDV的高免血清效价均比NCP型BVDV高免血清的效价高,平均高35%.结果表明,CP型BVDV的免疫原性优于NCP型BVDV,且CP型BVDV与NCP型BVDV的血清效价具有明显差异.这为在实践中疫苗毒株筛选及生产提供了理论指导.     

吉林某牛场牛病毒性腹泻流行病学调查及分析

为全面了解吉林省某规模化牛场牛病毒性腹泻(BVD)的流行情况,试验采集临床血清样品157份,粪便样品18份,肝脏、精液等组织样品17份,应用BVDV抗体检测试剂盒进行血清抗体检测,利用BVDV1型引物,采用纳米PCR方法对血清及临床样品进行BVDV抗原检测,对抗原阳性样品进行测序分析;将抗原阳性样品经研磨、稀释后用0.22μm滤膜过滤除菌,接入牛肾细胞(MDBK)进行病毒分离培养,盲传3代后再次进行抗原检测;采用免疫荧光技术检测病毒对MDBK细胞的侵染作用;利用Mega软件绘制系统进化树并进行同源性比对分析。结果显示,该牛场临床血清BVDV抗体阳性率为77.1%,血清抗原阳性率为12.1%,临床粪便等样品抗原阳性率为74.3%;病料接入细胞未观察到细胞病变,分离毒株PCR产物测序分析结果与样品抗原检测结果一致;免疫荧光检测结果显示,分离株毒液正常吸附于MDBK细胞中,有明显荧光反应;抗原测序分析显示,该牛场BVD主要流行毒株与BVDV JL-1株同源性高达99.0%,且均为BVDV 1型毒株。本研究对该牛场BVDV流行情况进行了全面调查,为开展净化工作奠定了基础。     

A Controlled Field Study Using Live Virus Vaccines and an Antiserum in a Preconditioning Program

Thirty-five vaccinates and 29 control beef calves from five farms were studied. Vaccinates in group 1 received a modified live virus vaccine against infectious bovine rhinotracheitis (IBR) and bovine virus diarrhea (BVD) 30 days after shipment; vaccinates in groups 2, 3 and 4 received live virus vaccines agains IBR and bovine parainfluenza 3 (PI3) seven to 17 days before shipment. Half of group 5 were given bovine origin antiserum containing antibodies against IBR, BVD and PI3. Three weeks later, the animals that had received serum were given a live modified vaccine containing IBR, BVD and PI3. In group 1, WBC counts were lower in the vaccinates than in the controls for two weeks after vaccination. WBC counts in groups 3 and 4 were higher in vaccinates than in controls after addition to the feedlot. Seroconversions to BVD virus occured in all groups. Clinical disease apparently due to BVD affected one vaccinated calf in group 2 and eight calves in group 5. Combined weight gains were significantly higher in three groups of calves vaccinated before shipment compared to unvaccinated control animals after addition to the feedlot. Vaccination with IBR and PI3 live virus vaccines should be given at least 17 days before shipment to feedlots containing infected cattle. Antiserum containing antibodies against the three viruses showed no apparent advantage in preventing clinical respiratory disease over control calves not receiving the serum.     

Demonstration and quantitation of immunoglobulins in bovine serum, follicular fluid, and uterine and vaginal secretions with reference to bovine viral diarrhea and infectious bovine rhinotracheitis.

Immunoglobulin concentrations (IgG, IgM, and IgA) in bovine serum, follicular fluid, and uterine and vaginal secretions were determined. The specificities of IgG, IgM, and IgA for virus-neutralizing antibody against bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses were also examined. High concentrations of IgG were present in both serum and follicular fluid. The IgG, IgM, and IgA concentrations were low in uterine and vaginal secretions. There was more IgG in the uterus during estrus than at any other time. Virus-neutralizing antibodies against BVD and IBR in serum of cows were mainly the IgG class. There was positive correlation between serum and follicular fluid virus-neutralizing antibody titers fro BVD and IBR. These antibodies may provide some protection for recently ovulated ova.     

Virus Neutralizing Antibodies Against a Panel of 18 BVDV Isolates in Calves Vaccinated with Rispoval™ RS‐BVD

Seven of nine colostrum‐deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval^? RS‐BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non‐cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine‐induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1 : log₂), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum‐deprived BVDV seronegative calves, Rispoval^? RS‐BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Studies on Bovine Virus Diarrhea: Serum Neutralization, Complement-fixation and Immunofluorescence

The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle.

At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections.

The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests.

     

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.     

Frequency of persistent bovine viral diarrhea virus infection in selected cattle herds

Sera and blood buffy coat samples were obtained from 3,157 cattle in 66 selected herds. Antibodies to bovine viral diarrhea (BVD) virus were detected in 89% of the serum samples by immunoprecipitation or virus-neutralization tests. Cytopathic or noncytopathic BVD viruses were isolated from blood buffy coat samples from 60 cattle in 6 herds. A second blood buffy coat sample was obtained from 54 of the 60 cattle 2 months after the initial sampling, and BVD virus was isolated again from each cow. The 54 cattle were considered persistently infected with BVD virus. The frequency of persistent infection was 1.7%.