Evaluation of canine Bordetella bronchiseptica isolates using randomly amplified polymorphic DNA fingerprinting and ribotyping

Bordetella bronchiseptica is a respiratory tract pathogen in a variety of species. Previous studies suggest little genetic variation among canine B. bronchiseptica isolates. The degree of genetic diversity in 26 canine B. bronchiseptica strains was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprinting and ribotyping. Strains evaluated include historic reference strains (N=3). vaccine strains (N=5) and clinical isolates (N=18). RAPD fingerprinting with the 10-nucleotide primer OPA-4 resulted in four distinct fingerprint patterns. RAPD fingerprinting consistently separated four previously characterized electromorphotype (EMT) 6 strains into two fingerprint types. Ribotyping, using the restriction endonuclease PvuI, resulted in six distinct ribotypes. With the exception of vaccine strains, considerable genetic diversity exists in the canine B. bronchiseptica isolates examined. These findings indicate the genetic variability within canine strains of B. bronchiseptica is greater than appreciated previously. Additionally, OPA-4 RAPD fingerprinting and PvuI ribotyping will be useful tools in epidemiologic studies of canine B. bronchiseptica isolates.     

Antimicrobial susceptibility of Bordetella bronchiseptica isolates from cats and a comparison of the agar dilution and E-test methods

One hundred and fifty-two predominantly feline isolates of Bordetella bronchiseptica were tested for their susceptibility to seven antimicrobial agents using an agar dilution method. The majority of isolates tested by the agar dilution method were resistant to trimethoprim (MIC₉₀ 500 μg/ml) and ampicillin (MIC₉₀ > 32 μg/ml) but sensitive to tetracycline, doxycycline and enrofloxacin (MIC₉₀ 2 μg/ml for all three agents). The isolates showed a spectrum of susceptibility to sulphadiazine and clavulanate potentiated amoxycillin. The MIC's of twenty-nine of the 152 isolates were then compared for five of the antimicrobial agents using the E-test (AB Biodisk, Sweden), a recently introduced method for measuring the MIC's of antimicrobial agents based on the diffusion of a pre-defined antibiotic gradient from a plastic strip. Comparisons with the E-test demonstrated an overall agreement (±1 log₂ dilution) with the agar dilution method of 79.4% and an agreement within ±2 log₂ dilutions of 96.2%.     

Diversity of swine Bordetella bronchiseptica isolates evaluated by RAPD analysis and PFGE

The degree of genetic diversity in 45 Bordetella (B.) bronchiseptica strains comprised of a vaccine strain (N = 1), reference strains (N = 3) and field isolates (N = 41) was evaluated using random amplified polymorphic DNA (RAPD) fingerprinting and pulsed-field gel electrophoresis (PFGE). Three candidate primers were selected for RAPD analysis after screening 20 random decamer oligonucleotides for their discriminatory abilities. The OPA-07, OPA-08 and OPA-18 primers yielded 10, 10, and 6 distinct fingerprint patterns, respectively. The most common identical RAPD pattern was produced by OPA-07 which was shared by 32 isolates (71.1%), the pattern produced by OPA-08 was shared by 26 isolates (57.8%), and the pattern produced by OPA-18 was shared by 40 isolates (88.9%). The RAPD patterns of the vaccine strain and the 3 reference strains did not match any of the patterns produced by the field isolates when primers OPA-07 and OPA-08 were used. PFGE using the restriction endonuclease XbaI produced a total of 15 patterns consisting of 4 PFGE types (A, B, B1 and C, differing by ≥ 4 bands) and 11 A subtypes (differing by ≤ 3 bands). Most of the field isolates exhibited identical type A and B patterns, suggesting that they were related. The vaccine strain and the three reference strains showed different PFGE patterns as compared to the identical type A strains.     

Polymorphism of pertactin gene repeat regions in Bordetella bronchiseptica isolates from pigs

Bordetella bronchiseptica pertactin (prn) is an outer membrane protein which has been implicated as both an adhesin and a protective antigen that induces immunity against atrophic rhinitis in pigs. Previous studies demonstrated extensive heterogeneity of the prn sequence within two distinct regions of amino acid repeats for B. bronchiseptica isolated from the United States and Europe. By deducing the amino acid sequences of the repeat regions of the prn gene from recent isolates from Korea, two region 1 variants and five region 2 variants were identified. Five pertactin types were distinguished based on combinations of variants of both regions. Interestingly, none of the field isolates have the same pertactin type as the B. bronchiseptica P4 strain widely used to vaccinate pigs.     

Studies on the antigenic relationships of six adherent isolates of Bordetella bronchiseptica

Six isolates of Bordetella bronchiseptica recovered from swine with atrophic rhinitis were studied. All hemagglutinated swine red blood cells, autoagglutinated in saline and showed fimbriae by electron microscopy. Hyperimmune sera against each were produced in rabbits and the antigenic relationships between the isolates were studied by cross-absorption and by the determination of the cross-reactivity indices of pairs of sera. Three isolates seemed to be identical by both methods, while 2 others showed close antigenic relationships. Hemagglutination titers with heterologous antigens and cross-reactivity indices greater than 0 suggest some degree of cross-immunity among the isolates studied, even when antigenic heterogeneity was demonstrated.     

Detection of type III secretion system genes in animal isolates of Bordetella bronchiseptica

A cosmid clone bank of Bordetella bronchiseptica genomic DNA was screened for the presence of type III secretion (TTS) genes using a probe derived from the TTS system genes of Ralstonia solanacearum. A 3.35kb PstI fragment, sub-cloned from a hybridising cosmid clone, was sequenced and found to contain a 97bp overlap with the previously reported B. bronchiseptica bscIJKLNO TTS gene cluster. DNA and predicted protein homology analysis suggests that a bscPQRST cluster lies immediately downstream of bscIJKLNO. A PCR amplification assay indicated that the bscT locus was present in 27 B. bronchiseptica animal isolates tested (100%). Dot-blot DNA hybridisation using probes for bscT and bscP confirmed the presence of these loci in six canine isolates associated with a variety of clinical signs. Although TTS has been implicated in the pathogenicity of B. bronchiseptica, it is likely that different clinical manifestations may be due to variations in gene expression or host factors, rather than the absence or presence of TTS genes.     

Seroepidemiological survey of Bordetella bronchiseptica and canine parainfluenza-2 virus in dogs in Sweden

The prevalence of antibodies against Bordetella bronchiseptica and canine parainfluenza-2 virus (CPiV-2) was investigated in a population of 302 pet dogs in Sweden. Sera were analysed for B bronchiseptica-specific immunoglobulin G by means of an ELISA, and for CPiV-2 specific neutralising antibody by means of a haemagglutination inhibition test. B bronchiseptica had a seroprevalence of 22 per cent and CPiV-2 had a seroprevalence of 28 per cent. The two pathogens did not appear to circulate together. The crowding of dogs together was significantly associated with the seroprevalence of CPiV-2, but not with the seroprevalence of B bronchiseptica. The dogs' ages, gender or their Fédération Cynologique Internationale breed group affiliation was not correlated with the seroprevalence of either pathogen.     

猪萎缩性鼻炎支气管败血波氏杆菌PCR检测方法的建立

对表现猪萎缩性鼻炎临床症状的猪群中分离得到的34株支气管败血波氏杆菌(Bordetella bronchiseptica,Bb),采用针对Bb flagellum gene的一对引物进行PCR扩增,结果所有分离物均能扩增出237bp特异性DNA条带,与传统生化鉴定结果相一致,且其最小检出量为0.64pg;而猪鼻腔和肺组织中常见的多杀性巴氏杆菌、金黄色葡萄球菌、枯草芽胞杆菌、铜绿假单胞菌、变形杆菌及大肠埃希氏菌均未能出现任何DNA条带。这表明,本试验建立的PCR方法具有特异性强、灵敏度高、可靠性好等特点,可用于猪萎缩性鼻炎的临床诊断。     

Bordetella bronchiseptica infection in the cat

Feline Bordetella bronchiseptica infection had received little consideration until recent years when it has been increasingly documented in association with respiratory disease. This article reviews current knowledge on the organism; its epidemiology, pathogenesis, and clinical, diagnostic and therapeutic features.     

猪波氏杆菌病的诊治

1998年 3— 7月份我县某集约化种猪繁殖场 60～ 90日龄生猪发生了一种慢性支气管肺炎为主要特征的传染病。临床上以咳嗽、喘气为主 ,尤其咳嗽较为明显 ,像咽喉或气管内有异物咳出来的连声长咳 ;对抗生素耐药性强 ,一旦发病病情顽固 ,很难彻底治愈 ;经流行病学、临床症状、剖检变化、病原分离、鉴别诊断等 ,确诊为猪败血波氏杆菌病。该病在我国生猪发病病例少见报道 ,介绍如下。1　流行情况　 1 998年 3月某集约化种猪繁殖场引进 40头长白母猪 ,推广经济杂交组合 ,改良生猪品种。不久猪场部分断奶仔猪和小架子猪出现咳嗽、喘气症状 ,经抗生素…     

兔波氏杆菌病的诊断与综合防治

兔波氏杆菌病是由支气管败血波氏杆菌(B.bronchiseptica)引起的兔的呼吸道传染病。该病以发生慢性鼻炎和支气管肺炎为主要特征,近几年来,在我省的多个兔场中常有发生。本文对某兔场波氏杆菌病的诊断及综合防治措施简报如下。1 发病情况及临床症状 广州某兔场饲养近1 000只种兔和肉兔,2001年1月中旬,部分兔只出现打喷嚏、咳嗽、流鼻涕     

猫源支气管败血波氏菌的分离与鉴定

支气管败血波氏菌是引起猫呼吸道疾病的主要病原之一,而对猫源支气管败血波氏菌的分离鉴定目前国内鲜有报道。本研究对上海市1例有咳嗽症状的德文猫鼻拭子样品进行呼吸系统相关病原PCR检测,并对鼻拭子样品进行细菌分离培养,对分离菌株采用K-B纸片法进行药敏试验,同时检测分离菌株对小鼠的致病性。结果显示：猫杯状病毒、猫疱疹病毒、猫衣原体、猫支原体均为阴性,支气管败血波氏菌为阳性,并分离到1株支气管败血波氏菌;分离菌株对头孢拉定、氨苄西林、强力霉素等9种抗生素耐药,对阿莫西林、卡那霉素、氟哌酸、复方新诺明、美罗培南等15种抗生素敏感;用分离菌株接种ICR小鼠,发现小鼠出现精神沉郁症状,但未出现死亡。结果表明：上海市猫群中存在支气管败血波氏菌感染,需关注免疫力低下人群被感染的风险;分离株对小鼠致病力不强,但对首选治疗支气管败血波氏菌病的强力霉素耐药,建议选用敏感药物进行防治。本研究为猫源支气管败血波氏菌病的诊断与防治提供了参考。     

Detection of urease-negative Bordetella bronchiseptica from the field

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs? assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.     

猪源支气管败血波氏杆菌河南株的分离鉴定

从河南省多个养猪场采集126份有呼吸道症状猪的肺脏组织样品或鼻拭子,进行支气管败血波氏杆菌的分离鉴定。根据细菌培养特性、革兰氏染色镜检、生化试验和PCR鉴定,确认分离出11株支气管败血波氏杆菌(Bordetella bronchisepti-ca,Bb)。对Bb最重要的3种毒力因子fha、prn、dnt基因进行PCR检测,除2株为prn基因阴性外,其它PCR结果均为阳性。红细胞凝集试验显示各菌株均能不同程度的凝集猪和羊的红细胞。乳鼠皮肤坏死试验表明,各菌株均能不同程度的造成乳鼠皮肤坏死。通过小鼠毒力试验,筛选到4株强毒菌株(HN0710、HN0806、HN0922、HN0827)。