弹性硬蛋白溶解试验鉴别反刍动物腐蹄病节瘤拟杆菌强毒株

根据腐蹄病节瘤拟杆菌毒力菌株具有溶解弹性硬蛋白的特性,利用弹性硬蛋白培养基建立了弹性硬蛋白溶解试验,以鉴别节瘤拟杆菌强毒株。试验表明,将不同毒力的菌株同时接种于弹性硬蛋白培养基,蛋白溶解带出现的时间和宽度都显著不同,强毒株大多在７ｄ内,少部分在７～１１ｄ内均出现一条明显的蛋白溶解带;而弱毒株在２１ｄ内基本上未见蛋白溶解带出现。     

PCR法诊断奶牛腐蹄病坏死梭杆菌的研究与应用

试验根据坏死梭杆菌白细胞毒素基因序列的特异性,设计1对引物FLP并建立了PCR检测方法.试验结果表明,引物FLP具有相对较高的特异性和敏感性,其敏感性达到了48.5pg.对现地采集奶牛蹄部病料进行PCR检测,发现引物FLP具有很好的特异性,能够准确诊断出坏死梭杆菌的存在.     

腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与表达载体的构建

用PCR技术从腐蹄病C型节瘤拟杆菌克隆出具有免疫保护性抗原0.85kb纤毛蛋白基因（pili基因），利用该基因构建了纤毛蛋白基因表达载体。提取C型节瘤拟杆菌染色体DNA；用所设计的专一性引物进行PCR，扩增出pili基因；将pili基因克隆于TE载体，TE－pili重组质粒pili基因序列测定结果正确，用EcoRI酶切，低熔点胶回收pili基因片段，经klenow补平后，用T4DNA连接酶将其与中间载体pPLλ连接，将pili基因克隆于pPLλ载体，经BamHI、BamHI HindⅢ、Dral酶切鉴定pili基因正向插入pPLλ载体；扩增pPLλ-pili重组质粒，用BamHI酶切出2.1kb大小片段，回收后，与PME290表达质粒连接，转化宿主细胞PAK／2pfs中，对获得的重组质粒用BamHI酶切，出现2.1kb大小的带,证明含有目的基因。     

腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与表达

用 PCR从腐蹄病 C型节瘤拟杆菌扩增出具有免疫保护性抗原 0 .85 kb纤毛蛋白基因 (pili基因 ) ,并构建了该基因的表达载体。转化宿主细胞 PAK/2 pfs,在营养肉汤中进行 pili基因的表达。培养 18～ 2 4h后 ,收集培养液上清 ,加入 0 .1mol/L Mg Cl2 ,离心提取重组纤毛蛋白。用羊抗兔 C型节瘤拟杆菌抗血清与提取的纤毛蛋白进行对流免疫电泳试验 ,结果表达的重组纤毛蛋白有特异性 ;用 SDS- PAGE和 Western- blot证明表达的蛋白是 C型节瘤拟杆菌纤毛蛋白。     

腐蹄病C型节瘤拟杆菌纤毛蛋白基因T程疫苗对家兔的免疫试验

将转化C型节瘤拟杆菌纤毛蛋白基因表达子粒的工程菌PAK/2pfsC在含羧苄青霉素营养肉汤培养基中培养,用MgCl2法在培养物上清中提取表达产物(纤毛蛋白).将表达的纤毛蛋白产物用弗氏完全佐剂乳化,制成疫苗.用疫苗免疫3只健康家兔,21 d后再次接种;定期采血,用对流免疫电泳和K凝集实验检测试验家兔的体液免疫应答及抗体水平.结果发现,免疫7 d即可产生相应抗体,21 d后抗体效价达到8 000×以上,而且高滴度抗体可维持6个月以上.试验表明,腐蹄病C型节瘤拟杆菌纤毛蛋白基因工程疫苗具有较好的免疫应答和免疫原性.     

腐蹄病节瘤拟杆菌的分子致病机制的研究进展

腐蹄病是反刍动物绵羊、山羊、鹿和奶牛等常见的一种高度接触性传染病。节瘤拟杆菌是致病作用的主要病菌之一,它是通过IV型纤毛和细胞外蛋白酶而产生作用。但从腐蹄病发病症状看,节瘤拟杆菌所致疾病的严重程度不是相同的,据此节瘤拟杆菌被分为毒性、弱毒性和良性菌,这种分类通过对该菌基因组毒性关联蛋白Vap(Virulence-associated protein)区域和毒性相关位点vrl(Virulence Related Locus)区域的研究,发现其致病特点与基因的顺序有很大联系。     

节瘤拟杆菌K-凝集试验方法的建立

根据节瘤拟杆菌(D.nodosus)特异的表面抗原———K抗原具有凝集反应的特性,建立了检测抗节瘤拟杆菌抗体的方法———K-凝集试验方法。节瘤拟杆菌液体培养物接种兔,制备节瘤拟杆菌阳性血清;福尔马林灭活肉汤培养物制备抗原,结果表明,该方法使用方便、特异性强。     

奶牛腐蹄病的诊治

奶牛腐蹄病又称坏死性蹄皮炎,多由坏死厌气丝杆菌、节瘤拟杆菌引起,是牛的一种急性或亚急性坏死性传染病,以蹄真皮或角质层腐败、蹄间皮肤及其深层组织腐败化脓为主要特征。脓性棒状杆     

国内奶牛腐蹄病综合防治研究进展

文章通过对国内奶牛腐蹄病的产生原因、临床症状、预防措施等方面的研究的综述，为奶牛腐蹄病综合防治提供参考。     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites

Objective To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment.
Design and procedure Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus a t each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season.
Results The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted.
Conclusion Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.     

Protection of sheep against experimental footrot by vaccination with pili purified from Bacteroides nodosus

Merino sheep vaccinated with either whole Bacteroides nodosus organisms, a crude surface antigen preparation or highly purified pili (>99% homogeneity) in oil adjuvant, developed significant resistance to artificial footrot infection when compared with unvaccinated control sheep inoculated with saline-in-oil emulsion (Freund;s incomplete adjuvant) alone. The pili-vaccinated sheep generally had higher K-agglutinating antibody titres than sheep vaccinated with whole B. nodosus. These results confirmed the role of B. nodosus pilus protein both as a protective antigen and the K-agglutinogen. Vaccines prepared with Freund;s incomplete adjuvant containing either purified pili, crude pili or B. nodosus whole cells did not produce significantly different injection-site reactions.     

The virulence of Dichelobacter nodosus,measured by the elastase test,is an important predictor for virulent footrot diagnosis in New South Wales sheep flocks

Ovine footrot is a contagious bacterial disease that causes foot lesions, and depending on the virulence of the causative strains, may lead to severe underrunning of the hoof and lameness. Virulent footrot can be identified, treated and controlled more effectively than less virulent benign forms. The in vitro elastase test for virulence of the causative bacteria, Dichelobacter nodosus, has been used to support clinical diagnosis. However, not all laboratory-designated virulent D. nodosus strains cause clinical signs of virulent footrot. This study evaluated retrospectively how well the elastase test supported clinical footrot diagnosis in 150 sheep flocks examined for suspect footrot in New South Wales between August 2020 and December 2021. Flocks were included if measures of clinical disease, environmental conditions and the virulence of D. nodosus isolates were available. Variation in the elastase activity result between D. nodosus isolated from the same flock made bacterial virulence hard to interpret, but calculating the mean elastase rate for all isolates from the same flock made correlations between bacterial virulence and flock footrot diagnosis possible. Simplifying bacterial virulence into whether there were any elastase-positive D. nodosus isolates before 12 days increased the predictive value of elastase results for virulent diagnosis, compared with using the first day that any isolate was elastase positive or the percentage of elastase-positive isolates by 12 days, but not all clinically virulent flocks had isolates with elastase activity before 12 days. Logistic regression models were fitted to identify the minimum number of predictors for virulent footrot diagnosis, with models suggesting that virulent footrot diagnosis was best predicted by adding the elastase test result and environmental conditions to the prevalence of severe foot lesions (score 4 and 5). However, performing the same analysis with different breeds, ages of sheep and seasons might highlight other factors important in the diagnosis of virulent footrot.     

The effect of dissociation of Bacteroides nodosus pili on their efficacy as a protective antigen against ovine footrot.

Previous studies have shown that pili from homologous Bacteroides nodosus provide protective immunity in sheep against footrot, whereas denatured pilin subunits are ineffective. The aim of the present study was to examine whether pili that were dissociated into pilin subunits under less vigorous, non-denaturing treatment conditions, would provide an effective level of protective immunity. Using the techniques of gel permeation chromatography, light scattering and susceptibility to proteolysis as measures of disruption, it was shown that pili were dissociated either by the neutral detergents n-octyl-beta-D-glucopyranoside (NOG) and Tween 80 or by lowering the pH with 1 M phosphoric acid to pH 2.2. Circular dichroic spectra indicated, however that the samples were not denatured by these treatments. Electron microscopic monitoring of detergent dissociated material following exhaustive dialysis showed the presence of protein-detergent micelles and "in-line" aggregates which gave the appearance of short fibres. Within these monitored preparations, there was no evidence of native undissociated pili. Pili dissociated by NOG or acid were tested in protection trials and shown to provide protective immunity, although agglutination titres of serum taken from the vaccinated sheep were significantly lower than those of animals inoculated with intact pili.     

家蚕微粒子病PCR快速检测技术研究及诊断试剂盒的研制

根据比对大量微孢子虫ssu rRNA序列的基础上自行设计了检测家蚕微粒子病的通用型引物。本试验确定了PCR反应体系和反应条件,并进行了相关病原检测试验,且对家蚕的不同材料进行了PCR检测,结果表明,本试验建立了有效、特异、敏感的检测家蚕微粒子病的PCR检测方法,为家蚕微粒子病在口岸快速检出,提供了有效的手段。     

应用PCR方法对奶牛早期体外受精胚的性别鉴定

试验以牛雄性特异序列BOV97M和牛属1.715卫星DNA序列设计引物进行PCR扩增,以进行牛的性别鉴定.共检测76枚胚胎,37枚为雌性,39枚为雄性,把雌性胚胎进行移植,受胎11头,受胎率29.73%,产出母犊10头,准确率为90.91%.