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。     

藏羊腐蹄病主要病原的分子生物学鉴定

利用分子生物学技术扩增藏羊腐蹄病病料中坏死杆菌和节瘤拟杆菌的特异性基因片段。结果显示:坏死杆菌lkt A基因和节瘤拟杆菌fim A基因序列与Genebank中JX678850.1和X52407的序列同源性分别为99%和95%,表明青海省藏羊腐蹄病病原中有坏死杆菌和节瘤拟杆菌存在,为今后防治提供参考依据。     

奶牛蹄病病因分析及防治

<正>蹄病是奶牛饲养过程中常见的3大疾病之一,治疗不及时或治疗方法不当,会降低奶牛生产性能,增加奶牛淘汰率,给奶牛养殖户带来损失,严重危害着奶牛业的发展。1病因分析1.1病原微生物侵袭奶牛腐蹄病的主要病原体是坏死杆菌和节瘤拟杆菌。坏死梭杆菌能分泌透明质酸酶,节瘤拟杆菌能分泌水解蛋白酶,生成酶     

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。     

羊场环境中绵羊腐蹄病病原的检测

以近年来国内外最新研究资料为基础,采用PCR方法对采集于绵羊腐蹄病发病地区水样和土样环境中坏死杆菌和节瘤拟杆菌的存在情况进行检测。结果显示:34份样品中有1份水样为坏死杆菌阳性,其余为阴性,节瘤拟杆菌检测全为阴性。将扩增所得基因KY130428通过BLAST序列比对,发现该基因片段与FJ230831序列同源性为99%,表明样品中存在坏死杆菌。从羊圈周围动物饮水中检测到坏死杆菌的存在,国内尚未见相关报道,本研究旨在摸清羊场环境中腐蹄病病原的分布情况,为该病的防制提供参考依据。     

牛腐蹄病病因及综合防治

正牛腐蹄病一般发生于反刍动物,牛和绵羊最易感。该病主要由节瘤拟杆菌引发,具体表现为牛蹄部皮肤出现显著炎症,伤口出现腐坏化脓症状,牛蹄出现红色的粘稠分泌物,伴有恶臭味,严重的呈现全身性败血症状。1病原特点牛腐蹄病的病原有很多例如坏死梭杆菌、结节状类杆菌、球菌、以及产黑色素类杆菌和酵母菌等。引发牛腐蹄病的病原主要是坏死梭杆菌和节瘤拟杆菌。病原菌通过分泌含有毒素的物质,或高活性的蛋白酶,     

不同培养工艺制备的禽多杀性巴氏杆菌菌苗抗原的免疫原性比较试验

为比较不同培养工艺制备的禽多杀性巴氏杆菌抗原的免疫原性,对禽多杀性巴氏杆菌标准强毒株C48-1株采用固体表面培养法和液体高密度发酵法制备疫苗并进行了免疫对比试验.结果表明,固体表面培养法制备的抗原菌体形态结构较均一,抗原成分稳定一致,并且含有较丰富的荚膜;而液体高密度发酵法制备的抗原形态结构大小不一,抗原成分不够稳定一致,含荚膜也较少.将两种方法制备的抗原按相同工艺制备成禽霍乱蜂胶灭活疫苗,效力检验结果显示二者差异极显著,固体表面培养法制备的疫苗免疫后14 d和90 d时保护率均为80%～100%,平均93%;液体高密度发酵法制备的疫苗免疫后14 d和90 d时保护率分别为40%～60%、60%～80%,平均为53%和67%.     

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。     

一起牛腐蹄病的诊治

腐蹄病是由坏死梭杆菌和节瘤拟杆菌协同感染引起的各种哺乳动物和禽类的创伤性传染病。牛、羊、鹿等反刍动物常见且发病率较高,以侵害反刍动物趾间皮肤及深层软组织为主。该病的特征为动物蹄部组织化脓、坏死、腐败以及角质层破坏。目前,在我国牛、羊饲养场中,腐蹄病的发生率可达到50%,是牛、羊,尤其是奶牛养殖业中危害性最大的传染病性疾病之一。患腐蹄病的奶牛,通常表现为食欲减退,泌乳量下降,繁殖能力降低,严重者被迫淘汰,从而严重地影响到奶牛的生产性能和产奶质量,给奶牛业造成了巨大的经济损失。因此,我国牛、羊,尤其是奶牛腐蹄病的防治工作显得十分重要,一般为慢性经过,多位散发,有时表现为地方流行性。坏死梭杆菌主要以白细胞毒素、内毒素、溶血素、血凝素和各种蛋白酶起致病作用。节瘤拟杆菌主要通过细菌纤毛和蛋白酶起致病作用,其他杂菌在致病中起辅助作用,所以腐蹄病的主要判定依据是检测到病料中是否有节瘤拟杆菌和坏死梭杆菌的存在。     

Pilus ELISA and an anamnestic test for the diagnosis of virulent ovine footrot and its application in a disease control program in Nepal

The immunological memory (anamnestic) responses in sheep recovered from virulent footrot (VFR) can be aroused by subcutaneous injection of outer membrane protein (OMP) antigens of Dichelobacter nodosus. The magnitude of this response is directly correlated to the highest antibody response attained during infection and memory lasts at least a year after recovery from VFR. However, some older animals show non-specific responses to OMP antigens. In this study an evaluation of D. nodosus pilus antigen for the anamnestic diagnosis of footrot in sheep was undertaken. The results indicated that the primary and anamnestic responses to pilus were similar in character to OMP antigen but were highly specific. The sensitivity of the procedure for detection of sheep with a history of VFR was approximately 80%. A low proportion of sheep with mild lesions due to virulent strains of D. nodosus reacted to anamnestic challenge. Anamnestic challenge with 10 microg pilus was used in a VFR surveillance program in migratory sheep flocks in Nepal. Conventional diagnostic methods could not be applied during the disease transmission periods in these flocks because of their migration to alpine pastures far away from human habitation. The results supported clinical and bacteriological findings suggesting that virulent strains of D. nodosus have apparently been eliminated from these flocks in Nepal.     

Current understanding of the aetiology and laboratory diagnosis of footrot

Footrot is a highly contagious disease of the feet of ruminants caused by the synergistic action of certain bacterial species of which Dichelobacter nodosus (D. nodosus) is the main transmitting agent. The infection is specific to sheep and goats, although it has also been reported in cattle, horses, pigs, deer and mouflon. The antigenic diversity of D. nodosus is due to variations in the DNA sequence of its fimbrial subunit gene (fimA) and provides the basis for classification of the organism into at least 10 major serogroups (A-I and M), the distribution of which varies with different geographical locations. Host immune response to vaccination is serogroup specific. There are three different clinical forms of disease caused by virulent, intermediate and benign strains of D. nodosus, respectively. In order to facilitate rapid and reliable clinical diagnosis, virulence determination, strain differentiation and serogroup identification for effective control measures, immunological tests, DNA probes and PCR based techniques have been introduced. This review summarises the current understanding of the mechanisms of antigenic diversity of D. nodosus as well as advances made in its strain differentiation and diagnosis.     

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.     

Update on ovine footrot in New Zealand: isolation, identification, and characterization of Dichelobacter nodosus strains [corrected

Dichelobacter nodosus, a Gram-negative strict anaerobe, is the essential causative agent of ovine footrot. Despite its worldwide presence, the disease has significant economic impact in those sheep-farming countries with a temperate climate and moderate to high rainfall, such as New Zealand (NZ) and Australia. In this study, we aimed to isolate, identify, and characterize as many D. nodosus strains as possible from NZ farms by using polymerase chain reaction (PCR)-based technology. Understanding the virulence of this bacterium and showing extensive genomic variation in the fimbrial subunit gene (fimA) in different D. nodosus strains was very important to produce serogroup specific and effective vaccine for NZ. More than 100 footrot samples were collected from four different farming regions in NZ. Thousands of primary plates were cultured anaerobically and examined with Gram-staining in order to detect single colonies of D. nodosus. Approximately 500 plates that had potential D. nodosus colonies were subcultured several times to eliminate contaminating colonies until single colonies were obtained. Variable and a part of the conserved regions of the fimbrial subunit gene (fimA) were amplified directly from bacterial DNA extracted from footrot lesions and also from cultured NZ D. nodosus isolates, using the polymerase chain reaction. Different fimA amplimers were analyzed by DNA sequencing. On the basis of DNA sequence analysis, 16 new D. nodosus isolates belonging to eight different serogroups were identified from NZ. These new D. nodosus fimA sequences from NZ were different to previously reported strains and strains used in a commercial vaccine.     

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping).The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.     

Diagnosis of footrot in goats: application of ELISA tests for response to antigens of Dichelobacter nodosus

Goats are an important natural host for footrot and are infected with Dichelobacter nodosus that have virulence characteristics similar to those of sheep strains. However, the humoral response of goats to D. nodosus antigens and the possibility of a serological diagnosis of footrot in goats have not been studied. With the aim of evaluating a diagnostic ELISA test, we investigated the primary immune response of goats to experimental and natural infection, the memory response in recovered animals, and the transfer and persistence of colostral antibodies in kids. Footrot stimulated the goat's immune system and, as in sheep, under-running lesions were the primary stimulus for production of anti-D. nodosus antibodies. The immune response could be detected in ELISA using either fimbrial or outer membrane protein (KSCN) antigens of D. nodosus. Antibody titres resulting from infection declined quickly after recovery and reached pre-infection levels within 3-4 months. Previously affected animals, however, mounted a memory response when injected with purified D. nodosus antigens. Antibody levels attained after anamnestic challenge were correlated with the maximum levels attained during infection, and were therefore indicative of the infection status. Anti-D. nodosus antibodies were also transferred to kids via colostrum, but these antibodies did not persist and therefore were unlikely to interfere with the diagnostic ELISA after 3 months of age. Though these ELISA tests were highly specific, their sensitivity was rather low. Therefore, they are only suitable for a herd diagnosis of footrot in goats and are dependent on the development of advanced under-running infections in a proportion of affected goats.     

The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales

Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.     

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.