Prevalence of antibodies to bovine virus diarrhoea-mucosal disease virus in Tanzanian cattle

A serological survey to determine the prevalence of antibodies to bovine virus diarrhoea-mucosal disease (BVD-MD) virus was conducted on 419 bovine serum samples originating from 18 of 20 regions (except Mwanza and Shinyanga) of the Tanzania mainland. The sera were a small proportion of samples collected for the appraisal of immune response to rinderpest vaccination. The survey indicated that the virus is prevalent in cattle populations and approximately 12% of sera tested contained demonstrable neutralising antibodies against BVD-MD virus.     

Pathogenesis of bovine viral diarrhoea-mucosal disease (BVD-MD) virus infection in tracheal organ cultures

Bovine foetal tracheal organ cultures were infected with two strains of BVD-MD virus and observed for 35 days. The effect of the virus was assessed by observing ciliary activity in gross tissue explants and histological changes in haematoxylin and eosin stained sections. Decrease in ciliary activity and mild epithelial degeneration were first observed at 4 days post infection (p.i.), the epithelial degeneration progressing to complete destruction by day 35 p.i. Viral titres in extracellular fluids rose sharply from day 4 p.i., reached peak levels (10⁵ TCID₅₀/ml) between 15 and 18 days p.i., and eventually declined but persisted at lower titres up to day 35 p.i., when observations were terminated.     

The effects of bovine viral diarrhoea-mucosal disease (BVD) virus on the ovine foetus

Bovine viral diarrhoea-mucosal disease (BVD) virus has been incriminated as a cause of abortion, hairy birth coat and unthriftiness in sheep. Intravenous inoculation of 40 ewes 34 to 45 days pregnant with the V/TOB strain of virus produced death in two of four foetuses 9 days after inoculation and in all but one of 31 foetuses between 11 and 56 days. The highest levels of virus in placentomes and foetal tissues occurred between 9 and 15 days after inoculation and in foetal fluids between 11 and 18 days. Virus was not detected in any foetus later than 21 days after inoculation. Groups of 10 ewes infected between 59 and 62 days (Group B) and 70 and 76 days (Group C) of gestation had 73% and 62%, respectively, of abortions or perinatal foetal deaths. Birth weights of lambs born to infected ewes in groups B and C were significantly lower than those born to uninfected control ewes. Virus was recovered consistently from the cotyledons of the foetal membranes of live lambs, and irregularly from the tissues of full term foetuses that were dead at birth but on no occasion from mummified foetuses. There were no specific gross or microscopic lesions in tissues selected from aborted foetuses and the results highlight the difficulties associated with the diagnosis of BVD abortions and perinatal death of foetuses under field conditions.     

Cell titration assay for measuring blastogenesis of bovine lymphocytes

The blastogenic response of bovine peripheral blood lymphocytes to phytohemagglutinin (PHA) and to microbial antigens was measured using a lymphocyte titration assay. Culture conditions, including lymphocyte concentrations, incubation periods and medium formulation, were established which produced linear or nearly linear responses over a range of cell concentrations. These conditions were established by testing lymphocytes from unimmunized cattle and from heifers infected with Brucella abortus with PHA and a B. abortus extract. Four cell concentrations in 2-fold increments were selected for measuring responses to PHA (3.125 X 10(3) to 2.5 X 10(4) cells/well) and to antigens (5.0 X 10(4) to 4.0 X 10(5) cells/well). The strength of response varied among animals and also over time for individual animals, but the titration assay allowed exponential proliferation to be distinguished from decline, which may have been due to overcrowding of microtiter wells, exhaustion of nutrients or induction of regulatory events. This assay provided a more reliable and discriminating method of evaluating lymphocyte proliferation responses than that achieved by single point assays. The displacement of the titration curves could be used to estimate the relative frequency of lymphocytes responding to antigens or mitogens.     

A microculture syncytia assay for bovine leukemia virus

A microculture syncytia assay for the detection of bovine leukemia virus (BLV) has been described and compared with the conventional macroculture assay. The microculture assay required fewer indicator cells, was as sensitive as the macroculture assay and provided a reproducible test for the detection and titration of BLV.     

Bovine virus diarrhoea-mucosal disease virus: pathogenicity for the fetal calf following maternal infection

Fifteen pregnant, bovine virus diarrhoea-mucosal disease (BVD-MD) antibody-free Jersey heifers were infected experimentally with a mixture of 10 cytopathic strains of BVD-MD virus isolated from cattle in Britain. Each cow was inoculated intramuscularly on gestation day 100 with a high or a low dose of virus grown in primary calf testis tissue cultures. None of the cows showed clinical signs of illness following exposure, but all had seroconverted within six weeks. Six fetuses, including one set of twins, died in utero following infection. Of these five were aborted between days 136 and 154; the sixth one was mummified and still retained at day 300. The remaining 10 fetuses survived to term, but all showed evidence of intrauterine growth retardation with or without gross malformation and/or dysmyelination of the central nervous system. Three were clinically affected with congenital nervous disease. Of the 10 liveborn fetuses, two had specific serum antibodies to BVD-MD. Non-cytopathic BVD-MD virus was recovered from all of the remaining eight. When non-immune cows become infected with BVD-MD virus in mid gestation: transplacental infection of the fetus will probably result; apart from the risk of fetal death, with or without abortion, there is a high probability of fetal mal-development which may not always be clinically obvious; the immunological competence of the fetus may be impaired; congenital infection is likely in a substantial proportion of liveborn calves. About one in 16 bovine fetuses in British herds are estimated to be at risk from BVD-MD virus infection.     

A rapid virus neutralization assay for Newcastle disease virus with the swine testicular continuous cell line.

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.     

牦牛病毒性腹泻/粘膜病的防制研究

20世纪80年代以来，我国牦牛群中陆续发现牛病毒性腹泻／粘膜病(BVD／MD)，血清阳性率在30％～42．4％之间，病死率在30％左右，本研究先后从四川、西藏等地牦牛中分离出病毒，并对其进行各种生物学特征鉴定后，表明该病毒与标准毒属同一种，所不同的是四川牦牛病毒株属非致细胞病变型，即属NCP型。但回归本动物能复制出典型病例。目前尚无国产牛粘膜病疫苗用于生产。本研究依据猪瘟病毒与牛病毒性腹泻／粘膜病病毒具有交叉免疫性的原理，用猪瘟弱毒苗对牦牛病毒性腹泻／粘膜病进行预防，试验证明用猪瘟弱毒苗可以预防牛病毒性腹泻／粘膜病，且安全可靠，具有实用价值。     

A rapid enzyme-linked immunosorbent assay for the detection of foot-and-mouth disease virus in epithelial tissues

A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.     

A simple, rapid syncytial-inhibition test for antibodies to bovine leukemia virus.

Cocultivation of equal numbers of cells from a fetal lamb kidney line infected with bovine leukemia virus and African green monkey (Vero) cells results in the rapid production of syncytia. The effect was blocked or inhibited by serum containing antibodies to bovine leukemia virus. A serological test based on syncytial inhibition was compared to the agar gel immunodiffusion test and the modified direct complement fixation test for the detection of bovine leukemia virus antibodies in sera from leukosis-free cattle, cases of adult enzootic bovine lymphosarcoma and cattle from herds in contact with enzootic lymphosarcoma. The results showed the syncytial inhibition test to react positively with sera from all cases of adult enzootic lymphosarcoma, but to be much less sensitive than the other tests in detecting bovine leukemia virus antibodies in sera of exposed animals.     

牛病毒性腹泻病毒E2基因在毕赤酵母GS115中的表达与鉴定

将牛病毒性腹泻病毒(Bovine viral diarrhoea-mucosal disease virus,BVDV)河北分离株HB-bd毒株E2基因去除跨膜区获得sE2基因,将sE2基因克隆入巴斯德毕赤酵母(P.pastoris)分泌型表达载体pPIC9K中,筛选培养后提取阳性重组质粒,经酶切和PCR鉴定,命名为pPIC9K-sE2。重组质粒pPIC9K-sE2经SalⅠ酶切后,电穿孔导入P.pastorisGS115基因进行整合,使外源基因sE2稳定地整合到P.pastoris染色体中,G418筛选得到阳性高拷贝转化子GS115-pPIC9K-sE2。经甲醇诱导表达后,sE2融合蛋白获得了表达,表达产物经SDS-PAGE、Western-blot和Dot-ELISA分析,确定其表达的sE2融合蛋白相对分子质量大小为37 000,且具有天然蛋白的抗原特异性。免疫活性研究证明,P.pastoris表达的sE2蛋白能刺激动物产生抗体。     

牛病毒性腹泻病毒E2基因在毕赤酵母GS115中的表达与鉴定

将牛病毒性腹泻病毒(BVDV)河北分离株HB-bd毒株E2基因去除跨膜区获得sE2基因,将sE2基因克隆入巴斯德毕赤酵母(P.Pastoris)分泌型表达载体pPIC9K中,筛选培养后提取阳性重组质粒,经酶切和PCR鉴定,命名为pPIC9K-sE2.重组质粒pPIC9K-sE2经Sal Ⅰ酶切线性化,电穿孔导入P.Pastoris GS115基因进行整合,使外源基因sE2稳定地整合到P.Pastoris染色体中,G418筛选得到阳性高拷贝转化子GS115-pPIC9K-sE2.经甲醇诱导表达后,sE2融合蛋白获得了表达,表达产物经SDS-PAGE、Western-blot和Dot-ELISA分析,确定其表达的sE2融合蛋白相对分子质量大小为37 000,且具有天然蛋白的抗原特异性.免疫原性研究证明P.Pastoris表达的sE2蛋白能刺激动物产生特异抗体.