DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

A simulated surveillance program for bovine paratuberculosis in dairy herds in Norway

Monte Carlo simulation models were used to evaluate the feasibility and potential results of a proposed national survey of the prevalence of bovine paratuberculosis (PTB) in dairy herds in Norway. The expected herd prevalence was assumed to be 0.2% in the simulations. The low sensitivity of the ELISA test, the assumed low herd prevalence, the typical low within-herd prevalence of PTB and the small herd sizes all present problems in detection of the disease. Simulations with 500, 1000, 2500 and 6000 herds tested were done. Our results suggest that a national survey would not be feasible at present, due to the low probability of detecting infected herds and because of the high number of false-positive reactions that would be expected to occur.     

七星瓢虫对两种麦蚜控制作用的模拟研究

应用二次回归旋转组合设计方法研究了七星瓢虫成虫、幼虫与两种麦蚜共存系统中瓢虫对麦长管蚜和禾谷缢管蚜的捕食量模型。结果表明七星瓢虫对两种麦蚜的捕食量随着瓢虫密度的增加而减少．随着该种麦蚜密度的增加而增加．且七星瓢虫无选择性。七星瓢虫不同个体间的干扰作用对其捕食麦长管蚜数量有显著影响,两种麦蚜数交互作用对七星瓢虫捕食禾谷缢管蚜数量影响显著。该模型可用来预测田间蚜虫的变化．指导麦田蚜虫防治。     

牛分枝杆菌抗原MPB70、MPB83和ESAT-6的融合表达及重组蛋白的初步应用

应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术(splicing by overlap extension,SOE)获得融合基因mpb70-mpb83后,将mpb70-mpb83和esat-6串连于同一表达载体pET32a( )中得到重组质粒pET70-83-E6。转化BL21(DE3)大肠杆菌感受态后,经IPTG诱导以可溶的形式表达融合蛋白。用Ni2 亲合层析的方法纯化该融合蛋白。Western blot分析显示:该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应,而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法,并检测了117份临床血清样本(其中67份为PPD阳性牛血清),阳性率为39.32%(46/117)份,与PPD皮试诊断的符合率为82.05%(96/117)。     

牛分支杆菌MPT83基因的表达及重组蛋白的纯化

从牛分支杆菌培养物中抽提总DNA，扩增了MPT83基因，并克隆入pMD-18-T Simple Vector，将测序正确的目的基因插入表达载体pET-32a（＋）中，转化BL21（DE3）宿主菌，IPTG诱导表达，用亲和层析方法对表达蛋白进行了纯化。经测序，构建的克隆质粒pMD-MPT83与Gen-Bank中的序列一致；构建的表达质粒pET-MPT83经PCR和BamHI＋HindⅢ双酶切鉴定构建正确；经SDS-PAGE分析，在约42ku处出现新的蛋白条带，4h后表达量即达到高峰；Westernblotting分析表明，融合蛋白能够被牛分支杆菌阳性血清所识别；纯化的表达蛋白经SDS-PAGE电泳，出现清晰的单一条带。表明MPT83基因原核表达载体构建成功。     

复合天然添加剂对鹌鹑蛋品质影响的研究

用300只10月龄产蛋鹌鹑随机分成3组,对照组A饲喂全价基础日粮,试验组B添加0.5%的含有红椒粉和油菜花粉的天然添加剂,试验组C添加0.5%的含有红椒粉和甜茶粉的天然添加剂,试验期15天。测定第0、5、10、15天的蛋重、蛋形指数、蛋壳厚度、蛋黄指数及蛋黄色泽度(RCF)。结果表明:B和C蛋黄色泽度明显高于A(P<0.01),同时B高于C(P<0.05);B和C蛋黄指数显著高于A(P<0.05,P<0.01),且第15天时B显著高于C(P<0.05);蛋重、蛋形指数和蛋壳厚度组间差异不显著(P>0.05)。试验说明:该两种复合天然添加剂能够明显提高鹌鹑蛋黄色泽度和蛋黄指数。     

日本血吸虫候选疫苗免疫保护力研究进展

血吸虫病是危害严重的寄生虫病之一,其疫苗的研究是血吸虫病防治的一项工作重点也是一大难题。本文综述了日本血吸虫疫苗侯选分子筛选、模拟表位疫苗和多价疫苗的研究现状,并对血吸虫疫苗的深入研究进行了分析。     

抑制鹿源多药耐药结核菌中药的筛选

为了筛选对鹿源多药耐药结核菌有效的中药耐药抑制剂,通过MABA法检测24种中药的水和乙醇提取物对多药耐药结核菌及标准菌H37Rv的体外抗菌效果,将筛选出的抗菌效果好的黄连醇提物和黄连水提物采用紫外分光光度法进行活性成分含量测定,同时以MABA法与标准品比较体外抗结核菌活性。结果显示,黄连、独活和侧柏叶有明显的抗结核活性,其中黄连水粗提物和乙醇粗提物效果最佳,测得它们活性成分即总生物碱含量分别为22.74%和35.23%,并显示二者与小檗碱的体外抗结核作用相同。     

高铜日粮对肉鸡肝细胞线粒体呼吸链复合物活性的影响

在对照组（Cu 11mg／kg，A组）基础上设高铜组3个（110mg／kg、220mg／kg和330mg／kg，分别对应为试验组B、C、D），试验期为60d，在12、24、36、48、60日龄采集肝组织，提取线粒体，测定线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ的活性变化。结果表明，11mg／kg和110mg／kg铜日粮添加组线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ的活性呈增加趋势。220mg／kg和330mg／kg铜日粮添加组肉鸡肝细胞线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ活性减弱，但主要在试验后期，尤其是48日龄之后，比对照组和各组内的试验前期下降明显（P〈0．05）。说明高铜日粮在一定程度上对线粒体复合物活性是有利的，但过高的铜添加到日粮中（≥220mg／kg）可降低肉鸡肝细胞线粒体呼吸链复合物的功能活性。据此可以推测，线粒体是机体铜过量状态下侵害的靶部位之一，而且能作用于线粒体各个复合物，造成线粒体呼吸功能减弱，呼吸链中电子传递发生故障，从而影响线粒体功能的正常发挥。