猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。     

2014年猪群常见细菌性疫病的分离鉴定与流行病学调查

2014年华中农业大学动物疫病诊断中心对湖北、湖南、河南、广东、浙江等21个省市送检的12 452份临床病料进行细菌分离、生化试验和PCR方法鉴定,共分离出3 926株致病菌,并对其中所分离到的225株副猪嗜血杆菌和231株链球菌采用玻片凝集方法进行血清型分型。结果表明链球菌、副猪嗜血杆菌、巴氏杆菌、波氏杆菌、致病性大肠杆菌和猪丹毒丝菌等9种病原菌已严重影响我国规模化养猪业的健康生产。     

鸭源多杀性巴氏杆菌的分离鉴定及耐药性分析

为鉴定临床疑似鸭多杀性巴氏杆菌感染肉鸭的病原菌，本试验通过细菌分离培养、菌体形态观察、细菌生化鉴定、16S rRNA基因测序分析、细菌种特异性鉴定、荚膜分型鉴定和动物回归试验进行鉴定，并通过药敏试验和耐药基因检测进行耐药性分析。结果显示，从患病鸭肝脏组织分离到的细菌在鲜血琼脂培养基中呈现表面光滑凸起、灰白色菌落，为革兰氏阴性短小杆菌，瑞氏染色呈两极浓染；生化鉴定结果显示，分离菌能发酵葡萄糖、蔗糖和甘露醇，硫化氢、氧化酶和吲哚等试验阳性；16S rRNA基因序列系统进化树分析显示，该分离菌与多杀性巴氏杆菌聚为一支，同源性 > 99%；细菌种特异性鉴定结果与多杀性巴氏杆菌相符；荚膜分型鉴定结果仅扩增到约为1 050 bp的目的基因片段，与荚膜血清A型相符；动物回归试验显示，该分离菌有较强的致病性；药敏试验结果显示，该分离菌对羧苄西林、氨苄西林、复方新诺明和四环素等12种药物耐药；经耐药基因PCR检测显示，该分离菌携带Sul1、Sul3、tet（X）和Intl1 4种耐药基因，与药敏表型相符。本试验成功分离到1株鸭源荚膜血清A型多杀性巴氏杆菌，为鸭多杀性巴氏杆菌病的防治提供参考依据。     

胸膜肺炎放线杆菌研究进展

胸膜肺炎放线杆菌 (Actinobacillus pleuropneumoniae,APP,也有简写为 Ap) ,原称胸膜肺炎嗜血杆菌(H aemophiluspleuropneumoniae,Hp) ,属于巴氏杆菌科嗜血杆菌属 ,后又根据其表型 (phenotype)和 DNA杂交水平均与放线杆菌属模式种密切相关 ,归属为巴氏杆菌科放线杆菌属 ,命名为猪胸膜肺炎放线杆菌 [1 ] 。由本菌引起的猪接触传染性胸膜肺炎是猪的呼吸道传染病 ,各种年龄的猪均易感染 ;常与巴氏杆菌等混合感染 [1 ]。病猪发热 (可达 4 2℃ ) ,呼吸困难 ,食欲不振 ;剖检可见纤维素性胸膜肺炎 ,多感染两侧 ,6 5 %的肺叶病变严重 ;发病率 8.…     

猪传染性胸膜肺炎的诊治

正该病是由胸膜肺炎放线杆菌引起的猪的一种呼吸道传染病,以急性出血性纤维素性胸膜肺炎或慢性纤维素性坏死性胸膜炎为特征。随着规模化集约化养猪业的发展,该病的发生率有逐年增高的趋势。1病原该病的病原为胸膜肺炎放线杆菌,属于巴氏杆菌科、放线杆菌属,是一种革兰氏阴性小球杆菌,具有典型的球杆菌形态。在血琼脂上的溶血能力是鉴别特性。在诊断该病菌时,需与猪的其他嗜血杆菌相区别,如副猪嗜血杆菌、"小群"嗜血杆菌、"分类C"嗜血杆     

副猪嗜血杆菌的分离与签定

副猪嗜血杆菌是猪革氏病的病原菌,属于巴氏杆菌科嗜血杆菌属。副猪嗜血杆菌主要感染小猪,特别是4～6周龄的断奶仔猪,临床多表现为急性败血症,或者转为慢性,表现为跛行、食欲减退等。为有效地开展本病的防治研究,进行了副猪嗜血杆菌的分离工作。笔者从浙江某养殖场的“猪高热综合征”样病例中分离到1株细菌,经PCR扩增和16SrRNA鉴定,确定为副猪嗜血杆菌。现将临床发病及分离鉴定情况报道如下。     

猫口腔炎的细菌分离鉴定

通过采样拭子采集患口腔炎的猫口腔黏膜、舌头、牙龈分泌物样本,分离到23株菌,经致病性试验证实有3种菌具有致病性。根据形态、生长特性和生化特性,11株被鉴定为葡萄球菌,9株被鉴定为链球菌,3株被鉴定为巴氏杆菌;从5只健康猫样本中分离到大肠杆菌5株。细菌分离结果表明,大肠杆菌为常在非致病菌,葡萄球菌、链球菌及巴氏杆菌为致病菌。药敏试验结果表明,分离的3种致病菌对头孢噻肟高度敏感。因此临床上治疗应首选头孢类抗生素。     

副猪嗜血杆菌病病原菌的分离鉴定

更有效地控制和防治副猪嗜血杆菌病,本试验从山西省晋中市疑似猪嗜血杆菌病感染的一个大型规模化猪场采集病料,选择改良巧克力琼脂培养基,从9头有关节炎、胸膜炎、腹膜炎等症状的疑似副猪嗜血杆菌病的临床病例中分离到5株疑似副猪嗜血杆菌菌株。生长特性观察发现分离株为多形性、革兰氏阴性小杆菌,生长缓慢(24～48h)。菌落呈光滑露珠样,不溶血。葡萄糖、树胶醛糖、乳糖、甘露醇、蜜三糖发酵和脲酶实验呈阴性,过氧化氢酶实验阳性,该结果符合副猪嗜血杆菌的生化特性,表明该分离菌株为副猪嗜血杆菌。     

努比亚山羊巴氏杆菌羊创伤球菌混合感染的诊治

<正>巴氏杆菌属为无芽孢,不运动,兼性厌氧,菌体两端常染色浓重的革兰阴性小杆菌。本属已确定的种有多杀巴氏杆菌、嗜肺巴氏杆菌、溶血巴氏杆菌、尿巴氏杆菌和鸭疫巴氏杆菌等。在猪、牛~([1])、羊~([2])等动物常有巴氏杆菌感染的报道。创伤球菌属是1993年Collins等提议设立的一个菌属,目前该属的主要成员有孔兹创伤球菌、羊创伤球菌、瑞典创伤球菌和化脓创伤球菌。只有羊创伤球菌分离自动物,其他均分离自人的临床样品。羊创伤球     

猪胸膜肺炎放线杆菌和副猪嗜血杆菌的鉴别诊断及血清型鉴定

利用PCR技术、琼脂扩散试验、糖发酵试验和间接血凝试验(IHA)对野外分离到的可疑猪胸膜肺炎放线杆菌和副猪嗜血杆菌进行了诊断和血清型鉴定.PCR鉴定分离物HS1580为副猪嗜血杆菌,分离物HS1582为猪胸膜肺炎放线杆菌;生化试验表明分离物HS1581和HS1582为猪胸膜肺炎放线杆菌;血清型鉴定分离物HS1580不属于被检的14个血清型之列,HS1581为App血清15型,HS1582为App血清7型.试验结果表明,综合使用PCR等技术可快速、准确地对这两种传染性细菌进行鉴别诊断和血清型鉴定.     

Lack of evidence for the occurrence of Pasteurella ureae in rodents

The taxonomy of five typical human isolates of Pasteurella ureae, one strain of Actinobacillus hominis, and three murine isolates which had been designated as Pasteurella ureae in published reports were re-examined. Their taxonomic relationships were investigated by both conventional phenotypic characterization and by DNA/DNA hybridization using the renaturation method. The human Pasteurella urea strains were highly homogeneous in their phenotypes and in their DNA reassociation. The strain of Actinobacillus hominis studied was genetically distinct from Pasteurella ureae, but was located, like Pasteurella ureae, in the Actinobacillus group. The remaining strains exhibited only low DNA relatedness with Pasteurella ureae and each other; this agreed with their phenotypic divergence. Two of the murine isolates were identified as indole-negative variant strains of Pasteurella pneumotropica sensu stricto (i.e., type Jawetz), or of the type Heyl of Pasteurella pneumotropica, respectively. The remaining murine isolate appears to represent a hitherto unrecognized species of Pasteurellaceae. So far, there is no evidence for the occurrence of Pasteurella ureae outside the human host.     

The genetic classification of the Pasteurella pneumotropica complex

Pasteurella pneumotropica with its biotypes Jawetz and Heyl are the most common bacterial pathogens associated with diseases in rodents. 23 P. pneumotropica biotype Jawetz, biotype Heyl and P. pneumotropica-like rodentia isolates have been investigated phenotypically by characterization of their micromorphology and biochemical fermentation reactions. The taxonomic position within the family Pasteurellaceae has been examined by DNA:DNA hybridisation (optical method). It could be shown that P. pneumotropica biotype Jawetz represents a genus-like cluster containing several species including the V-factor dependent Haemophilus Taxon B and the avian P. pneumotropica-like organism and therefore resembles a new species of the new genus. It is concluded that the biotype Heyl of P. pneumotropica taxonomically remains as a species within the family Pasteurellaceae, however without further relationship to other known genera or genus-like groups.     

Confirmation that PCR can be used to identify NAD-dependent and NAD-independent Haemophilus paragallinarum isolates.

Seventy five bacteria tentatively identified as Haemophilus paragallinarum (the causative agent of infectious coryza), eight identified as Ornithobacterium rhinotracheale and 13 identified as NAD-independent Pasteurella species were isolated from chickens with respiratory infection in various provinces in South Africa. The isolates were characterized by conventional biochemical and serological methods. A polymerase chain reaction (PCR) assay specific for H. paragallinarum was used to identify the cultures directly from colonies. The PCR assay gave positive results for all isolates that were identified by conventional methods as H. paragallinarum, irrespective of whether they were nicotinamide adenine dinucleotide (NAD)-dependent (43 isolates) or NAD-independent (32 isolates). The eight isolates that were identified by conventional methods as O. rhinotracheale and the 13 isolates identified as various Pasteurella species gave negative results in the PCR assay. This study has demonstrated that colony PCR is a rapid method for uniquely identifying both NAD-dependent and NAD-independent strains of H. paragallinarum and distinguishing them from other bacteria, such as O. rhinotracheale and Pasteurella species.     

Bacterial Identity and Characteristics in Healthy and Unhealthy Respiratory Tracts of Sheep and Calves

Barbour, E.K., Nabbut, N.H., Hamadeh, S.K. and Al-Nakhli, H.M., 1997. Bacterial identity and characteristics in healthy and unhealthy respiratory tracts of sheep and calves. Veterinary Research Communications, 21 (6), 421-430The aim of this study was to compare different bacteriological aspects of the respiratory systems of healthy (H) versus unhealthy (UH) animals with respiratory signs. The prevalence of different bacterial species was determined in the upper and lower respiratory tract of H and UH Najdi sheep, Somali sheep and Holstein calves. The characteristics of Pasteurella spp. isolates, and the biotype of Pasteurella haemolytica were identified in H and UH animals. Eighteen out of 28 (64.3%) of the identified bacterial species in the upper respiratory tract were more prevalent in the nasal cavities of UH Najdi and Somali sheep and Holstein calves with respiratory signs than in apparently healthy animals; four of the most prevalent bacteria in the upper respiratory system of UH sheep were Moraxella spp., Pseudomonas pseudomallei, Erysipelothrix spp., and Pasteurella multocida, while three of the most prevalent bacteria in UH calves were Pasturella haemolytica, Actinomyces spp., and Pseudomonas aeruginosa. The prevalence of six different bacterial species was greater in the lungs of UH animals, namely Actinomyces pyogenes, Erysipelothrix spp., P. haemolytica, Pasteurella ureae, Staphylococcus aureus, and Staphylococcus epidermidis, which could be risk factors in the complexity of the prevalent respiratory diseases of the animals surveyed.Of the biochemical, cytological and colonial characteristics studied in the identified P. haemolytica and P. multocida, two characters were significantly different (p < 0.05) in organisms isolated from UH as compared to those from H animals. These were the higher loss of haemolytic power by the strains of P. haemolytica and the decreased fermentation of trehalose by all the strains of P. multocida recovered from healthy animals.The only biotype of P. haemolytica isolated from H animals was biotype A, while both biotypes A (88.0% of the isolates) and T (12.0% of the isolates) were recovered from UH animals.     

Abortion of a sow caused by Pasteurella aerogenes.

Three strains of the Pasteurella aerogenes complex were isolated as sole pathogens from aborted fetuses of a sow aborted at the 12th week of gestation on a farm of 600 sows. Gross pathology showed no characteristic lesions. The isolates were biochemically identical and resembled P. pneumotropica on the basis of their strong indole and urease positivity but they produced gas, were ornithine decarboxylase negative and fermented mannitol but not trehalose. Only a few differences were apparent in biochemical characteristics between the isolated strains and P. aerogenes. They differed from the type strain of P. aerogenes in ornithine decarboxylase activity, indole production and lactose and mannitol fermentation; however, such strains do occur within this heterogeneous species. At the time of abortion the antibody titre of the aborted sow was 1 in 16 when examined with live bacterial suspension and 1 in 128 if boiled antigen was used. Similar strains could not be isolated from the vaginas of aborted sows or pregnant and newly farrowed sows in the same group. The bacteriological, serological and histological findings support the opinion of other workers on the occasional pathogenic nature of P. aerogenes.     

Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.     

Capsular serogroups of Pasteurella multocida isolated from animals in Zimbabwe

Pasteurella multocida is isolated from a variety of disease conditions from different animal species in our diagnostic laboratory. In order to determine serogroup distribution among the isolates, an indirect haemagglutination test using glutaraldehyde-fixed sheep red blood cells was employed. A serological examination of 79 isolates revealed that 47/79 were of capsular serogroup A, 11/79 capsular serogroup D, 4/79 capsular serogroup B and 17/79 were untypable strains. None of the isolates belonged to either serogroup E or F. All those from cases of classical pasteurellosis could be grouped, but a significantly high proportion of those which originated from companion animals were untypable. The significance of these results is discussed. This report appears to be the first detailed information on the prevalence of various serogroups of P. multocida in animals in southern Africa.     

Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis

DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.     

Domestic sheep (Ovis aries) Pasteurellaceae isolates from diagnostic submissions to the Caine Veterinary Teaching Center (1990-2004)

A retrospective study of Pasteurellaceae isolated from domestic sheep (Ovis aries) was conducted. The aim was to identify Pasteurellaceae present in animals that were clinically healthy and others with evidence of respiratory disease. The bacteria had been isolated from samples submitted to the University of Idaho Caine Veterinary Teaching Center as part of disease diagnostic testing. The 844 isolates identified mainly three species of Pasteurellaceae: Mannheimia haemolytica, Pasteurella multocida, and Pasteurella (Bibersteinia) trehalosi. A total of 114 biovariants were identified among these three species. Individual biovariants were identified 1-180 times. Two of those (M. haemolytica 1 and P. (B.) trehalosi 2) constituted 36% of the isolates, and were the only biovariants sufficiently numerous to account for >7% of the total isolates. Samples were primarily submitted from sheep with signs of respiratory disease. Eighty percent of biovariants were identified most often in animals with signs of respiratory disease, but 26% of biovariants were isolated from both sheep with respiratory disease and apparently healthy sheep. P. multocida constituted 4.7% of isolates, and were exclusively associated with animals with respiratory disease. The ability of isolates to produce beta-hemolysis on culture media was not associated with animals with respiratory disease (odds ratio 0.77, 95% CI 0.50-1.19). The inference of this study is limited due to the retrospective study design. However, it is the first study that provides an extensive baseline list of biovariants associated with respiratory disease in domestic sheep.     

禽巴氏杆菌Pm-HB株的分离与鉴定

湖北省某鸡场疑似巴氏杆菌(Pasteurella)感染,从送检病鸡的肺脏和肝脏中分离到1株细菌,通过菌落形态、培养特性、生化试验、16S rRNA序列比对鉴定为禽多杀性巴氏杆菌,并命名为Pm-HB株。动物回归试验表明,10 CFU细菌即可100%致死30日龄SPF鸡,可确诊为巴氏杆菌病。