One-step RT-PCR for the Detection of Infectious Bursal Disease Virus in Clinical Samples

A single-tube, non-interrupted, one-step RT-PCR has been standardized to amplify the hypervariable region of the VP2 gene sequence of infectious bursal disease virus (IBDV). The technique standardized on purified viral RNA was successfully applied to the detection of the virus directly in clinical samples. The amplified products were confirmed to be IBDV specific by their size in ethidium bromide-stained agarose gel, nested PCR and restriction enzyme digestion. Digestion of the amplicons with StyI restriction enzyme also differentiated classical virus from six very virulent field isolates. The sensitivity of the one-step RT-PCR was found to be 0.2 pg of viral RNA.     

Demonstration of Heterogeneous Genotypes of Taylorella equigenitalis Isolated from Horses in Six European Countries by Pulsed-Field Gel Electrophoresis

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.     

牛羊4种寄生虫病联合诊断技术的研究与应用

疫区牛羊同时寄生有捻转血矛线虫、日本血吸虫、伊氏锥虫和肝片吸虫等多种寄生虫。目前血清学诊断方法对这 4种寄生虫病的检测需 2～ 4次操作。将捻转血矛线虫、日本血吸虫、伊氏锥虫和肝片吸虫抗原分别以 0 1 5 ,0 0 4 ,0 0 6和 0 1 3μg/ μL的最佳浓度依次定点在同一硝酸纤维素膜条上 ,制成 4种寄生虫病联合诊断膜。用黄牛IgG ,水牛IgG和山羊IgG联合免疫兔 ,获得了高效价兔抗黄牛、水牛和山羊 (简称兔抗牛羊 )IgG抗血清。应用兔抗牛羊IgG抗血清连接酶标SPA ,建立了 1份血纸能同时用于检测牛、羊捻转血矛线虫、日本血吸虫、伊氏锥虫和肝片吸虫病抗体技术。经浙江、湖北、四川、安徽等省应用 ,该项技术具有省工、省时、经济、实用等优点。适宜于牛、羊捻转血矛线虫、日本血吸虫、伊氏锥虫和肝片吸虫病流行区的普查或监测     

Detection of the German grapevine yellows (Vergilbungskrankheit) MLO in grapevine,alternative hosts and a vector by a specific PCR procedure

A polymerase chain reaction procedure was developed which enables specific amplification of a ribosomal sequence from the mycoplasmalike organism (MLO) associated with German grapevine yellows (Vergilbungskrankheit, VK) and stolbur-related diseases of solanaceous plants. Successful amplification from all samples prepared from various cultivars collected in different viticultural areas indicates that the causal agent is a relatively homogeneous organism. Amplification was also achieved with template DNA prepared from naturally infected weeds in vineyards such asConvolvolus arvensis andSolanum nigrum, and from the planthopperHyalesthes obsoletus that was collected in the vineyards. Feeding of insects of this species on grapevine seedlings resulted in the development of typical yellows symptoms by the grapes.H. obsoletus could therefore be identified as a vector of Vergilbungskrankheit.Abbreviations FD Flavescence dorée - GY Grapevine yellows - MLO Mycoplasmalike organism - PCR Polymerase chain reaction - RFLP restriction fragment length polymorphism - VK Vergilbungskrankheit (German grapevine yellows)     

奶牛发情周期中毛 唾液和乳汁孕酮水平的变化

本实验采用放射免疫分析法(RIA)测定了11头奶牛一个发情周期里毛、唾液、乳汁中孕酮(P_4)含量。经产牛(n=8)的毛和唾液中P_4水平变化与乳汁中P_4水平变化一致。青年牛(n=3)的毛和睡液中的P_4水平变化一致,并分别与经产牛毛和唾液中P_4水平变化一致。7头配种后的经产牛和5头配种后的青年牛分别取20和23天的样品进行妊娠诊断,与配种后60天的直检结果相对照,依据经产牛毛、乳P_4水平进行妊娠诊断的阳性准确率分别是85.7％(6/7)和100％(7/7),依据青年牛毛P_4水平进行妊娠诊断的阳性准确率为100％(5/5)。     

Studies on the Epidemiology of Tropical Theileriosis (Theileria annulata Infection) in Cattle in Central Anatolia,Turkey

An epidemiological survey for Theileria annulata infection was conducted in 12 selected villages around Ankara in Central Anatolia, Turkey, during the period April 1990 to January 1993. During the survey, 198 cattle of 30 local breeds, 84 Holstein-Friesian×local breeds and 84 Holstein-Friesian breed were examined for antibodies to T. annulata and the presence of the vector ticks. Four species of Hyalomma ticks were identified: Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavtum, Hyalomma detritum and Hyalomma marginatum marginatum. Salivary gland staining indicated that infected adult ticks of all four species were present and, therefore, were implicated in the transmission of tropical theileriosis in the field. Generally, the Hyalomma infestation rate was low, with the heaviest infestations occurring on the older animals. Young adults and calves had very low infestation rates. Most ticks seen on cattle were adults, very few nymphs were found. The blood smear and serological examination of the 198 cattle conducted in March, before the start of the first disease season, showed that the prevalence of piroplasmosis was 11.1% (22 out of 198) and the seroprevalence of T. annulata was 10.6% (21 out of 198). Forty-three animals were then excluded from the study because they were seropositive and/or harboured piroplasms. Ninety-two seronegative animals showed piroplasmosis (92 out of 155) and 34 seronegative animals became seropositive for T. annulata (34 out of 155) during the three disease seasons. One animal became clinically ill with tropical theileriosis and required treatment. The incidence of cattle showing piroplasmosis and disease in the total study sample was 50.7% and 0.5% per disease season, respectively. The seroconversion rate of new infection with T. annulata in the total study was 14.3% per animal season. The number of cattle showing piroplasmosis was much greater than the number of seropositive cattle, which may indicate the presence of another species of Theileria. The two different management systems encountered in the study were considered to have influenced the tick infestation levels.     

Critical Observations on BSE Control in Italy

Eleven native sheep, 1–2 years old, of both sexes were randomly divided into two groups, 6 sheep being allocated to the experimental group and 5 serving as controls. The sheep in the experimental group were fed 80% Tribulus terrestris and 20% alfalfa hay and wheat straw, while the control sheep were given a mixture of 40% alfalfa hay and 60% wheat straw. Clinical signs of hepatogenous photosensitivity were observed from day 11, including reddening and crust formation on the muzzle, nose, ears and eyelids, depression, weight loss, icterus, conjunctivitis, and yellow discoloration of the urine. Laboratory findings on weekly samples indicated significant differences (p<0.05) in white blood cell count, total plasma protein and fibrinogen, total and direct bilirubin, blood urea nitrogen and creatinine concentrations, and aspartate aminotransferase and alkaline phosphatase activities. There were no significant differences in the packed cell volume, in the neutrophil, lymphocyte or eosinophil counts, or in the serum calcium, phosphorus, potassium, sodium or chloride concentrations. At necropsy of the experimental animals, there were various degrees of generalized icterus and the livers were swollen and discolored by bile pigment. Histopathological examination revealed varying amounts of crystalloid material in the bile ducts and renal tubules, hepatocellular degeneration, biliary fibrosis and proliferation, renal tubular necrosis and focal necrosis of cardiac muscle.     

The Value of Tissue Imprint Hybridization for Rapid Detection of Infectious Bursal Disease Virus from Field Outbreaks

The use of a peroxidase labelled PCR generated probe followed by enhanced chemiluminescence hybridization assay detected infectious bursal disease virus directly from bursal imprints on a nylon membrane. Tissue imprint hybridization proved to be a simple, rapid and safe means of detecting IBD virus for screening large numbers of field samples. The PCR generated probe was highly specific for IBD virus and did not hybridize with cellular nucleic acids in control imprints. Tissue imprint hybridization was found to be a more sensitive method than conventional antigen detection assays.     

Detection of Antibodies Against Peste des Petits Ruminants Virus in Sera of Cattle,Camels, Sheep and Goats in Sudan

Detection of antibodies against peste des petits ruminants virus in sera of cattle, camels, sheep and goats in Sudan.     

Evaluation of the Abbott LCx Mycobacterium Tuberculosis Assay for Direct Detection of Mycobacterium Bovis in Bovine Tissue Samples

The commercial LCx amplification assay, usually employed to detect the Myocobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Löwenstein-Jensen solid medium and pathological findings on 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.