鸡卡氏杆菌GZ2017株的分离与16 S rRNA序列分析

对贵州省某白羽蛋鸡养殖场患病鸡进行临床和实验室诊断,以期明确病因,有效控制感染。通过病理剖检观察、分离培养、染色镜检、生化试验和16SrRNA序列分析等方法对分离菌株进行鉴定。结果显示,该分离菌的培养特性、菌体形态特征、生理生化特征均与文献报道的卡氏杆菌相同。药敏试验结果表明,该分离菌株对米诺环素和万古霉素极度敏感,对多西环素和头孢哌酮高度敏感。16SrRNA序列分析结果显示,该分离菌与鸡卡氏杆菌(Gallibacterium anatis)同源性最高,在98.6%~99.2%之间,遗传进化树结果表明分离菌与Gallibacterium属的细菌同属于一个分支,与日本分离菌株在同一小分支上,属于鸡卡氏杆菌。本试验成功分离到1株鸡卡氏杆菌,并将其命名为GZ2017,试验结果为鸡卡氏杆菌病的治疗与防控提供理论依据。     

鸡卡氏杆菌的分离与16S rRNA基因的分析鉴定

四川雅安某种鸡场种鸡出现消瘦,腹部膨大,肝脏肿大破裂出血,腹膜和输卵管炎为主要特征的疾病。为诊断和预防此病的发生,通过对细菌分离纯化、形态学观察、生化试验、药物敏感性试验鉴定出1株致病菌,应用通用引物对细菌的16S rRNA基因进行克隆和测序,测序结果于NCBI上进行BLAST比对,选择同源性较高的巴氏杆菌科15个属的29株细菌16S rRNA序列进行比对分析,构建遗传进化树。结果表明,分离纯化的细菌能在血平板上生长,革兰氏染色阴性短杆状,能发酵大多数糖和醇类,对头孢类抗生素敏感,16S rRNA序列与卡氏杆菌属细菌的同源性最高,在92.2%～99.5%之间,特别是与鸭卡氏杆菌同源在97.5%～99.5%之间。遗传进化树分析结果表明,该菌株与卡氏杆菌属细菌属于同一大分类群,与鸭卡氏杆菌位于同一个小分支,最终诊断该鸡场为卡氏杆菌感染。     

鸡卡氏杆菌的分离与16S rRNA基因的分析鉴定

四川雅安某种鸡场种鸡出现消瘦,腹部膨大,肝脏肿大破裂出血,腹膜和输卵管炎为主要特征的疾病.为诊断和预防此病的发生,通过对细菌分离纯化、形态学观察、生化试验、药物敏感性试验鉴定出1株致病菌,应用通用引物对细菌的16S rRNA基因进行克隆和测序,测序结果于NCBI上进行BLAST比对,选择同源性较高的巴氏杆菌科15个属的29株细菌16S rRNA序列进行比对分析,构建遗传进化树.结果表明,分离纯化的细菌能在血平板上生长,革兰氏染色阴性短杆状,能发酵大多数糖和醇类,对头孢类抗生素敏感,16S rRNA序列与卡氏杆菌属细菌的同源性最高,在92.2%～99.5%之间,特别是与鸭卡氏杆菌同源在97.5%～99.5%之间.遗传进化树分析结果表明,该菌株与卡氏杆菌属细菌属于同一大分类群,与鸭卡氏杆菌位于同一个小分支,最终诊断该鸡场为卡氏杆菌感染.     

禽卡氏杆菌分离鉴定及药敏试验

为了能更好地防治禽卡氏杆菌病,试验采用GAM培养基对病鸡输卵管进行了菌株分离,以革兰氏染色试验、生化试验、16S r DNA序列分析分子生物方法对该菌株进行鉴定,并对分离株进行了药敏试验。结果表明:该菌株为革兰氏阴性杆菌,生化试验结果与卡氏杆菌的生化特性一致。16S r DNA序列分析表明,分离株与Gen Bank上的卡氏杆菌16S r DNA序列同源性达到99%以上,最终确定分离株为卡氏杆菌,命名为Gallibacterium anatis C2-1-1。卡氏杆菌对青霉素、阿莫西林、米诺环素极度敏感,对氨苄西林、头孢氨苄、头孢曲松、氟苯尼考高度敏感,对头孢唑林、阿米卡星、头孢呋辛、四环素、环丙沙星中度敏感,对链霉素、林可霉素、红霉素等不敏感,该分离株具有一定耐药性。     

鸭疫里默氏杆菌的分离鉴定及生物学特性研究

从通辽地区养鸭场病死鸭的脑、肝、脾脏组织和心脏血中分离到4株细菌,经对分离菌进行染色、形态观察及生化鉴定,其中3株鉴定为Ⅰ型鸭疫里默氏杆菌,1株为Ⅱ型鸭疫里默氏杆菌。经动物回归试验,表明该菌对雏鸭有较强的致病力。对4株鸭疫里默氏杆菌进行了12种常用抗菌药物药敏试验,结果4株鸭疫里默氏杆菌分离株对头孢曲松、头孢唑啉、复方新诺明高度敏感,对强力霉素、新霉素、红霉素中度敏感,对庆大霉素、卡那霉素、四环素、环丙沙星、氨苄青霉素、丁胺卡那等均不同程度地产生耐药性。     

病死雏鸡的病原分离鉴定

从送检的３１只病死雏鸡中分离出３０株致病菌，其中沙门氏杆菌２７株，分离率９０％，大肠杆菌６株，分离率２０％。对其中５株沙门氏杆菌和４株大肠杆菌进行了系统生化鉴定及血清型鉴定，证实５株沙门氏菌中鸡白痢沙门氏杆菌占６０％（３／５），鸡伤寒沙门氏杆菌占４０％（２／５），４株大肠杆菌中Ｏ２２株，Ｏ６１株，Ｏ１１５１株。对分离菌进行了雏鸡致病力及抗菌药物敏感性试验，提出了相应的预防对策     

蛋鸡输卵管炎的防治

近期在哈市郊区售后服务时,了解到多数养殖户误认为产蛋鸡产软皮蛋、薄壳蛋、砂皮蛋、蛋褪色等,是由鸡输卵管炎引起的。本文就蛋鸡输卵管炎加以阐述,供养殖户参考。1病因输卵管炎多发生于初产、高产鸡,鸡舍不清洁,使沙门氏杆菌和大肠杆菌存在于鸡舍,并由泄殖腔侵入输卵管可引发     

绵羊肺脏中溶血性曼氏杆菌的分离鉴定及其药物敏感性分析

为了对引起绵羊肺炎的病原进行分离、鉴定和耐药性分析,本研究通过无菌采集绵羊肺脏并对细菌进行分离纯化、生化试验和PCR鉴定,然后对所得到的溶血性曼氏杆菌分离株进行药物敏感性研究。结果显示,分离纯化得到的细菌为革兰氏阴性短杆菌,具有弱溶血性,经生化试验和PCR鉴定为溶血性曼氏杆菌;耐药性分析显示该菌株对恩诺沙星、庆大霉素、四环素等大部分药物敏感。本研究为绵羊溶血性曼氏杆菌感染的有效防制提供有用的信息和数据。     

鸡波氏杆菌病的诊断

石家庄地区某肉鸡场暴发疫病。经流行病学调查、临床检查、病原分离培养与鉴定、病理学检查等方法诊断为鸡波氏杆菌病。人工复制试验结果表明，细菌分离物对鸡和小白鼠均具有致病性。药敏试验结果显示，磷霉素钠、氟苯尼考等抗生素对分离物抑菌效果最佳。     

两株羊溶血性曼氏杆菌的分离及其生物学特性研究

为了分离新型曼氏杆菌并研究其生物学特性,试验对从两例山羊病例中分离出的相似细菌进行纯化培养,通过革兰氏染色镜检、生化试验、16S rDNA基因序列测定、多位点序列分型法(MLST)鉴定、血清型鉴定、药敏试验等方法研究其生物学特性。结果表明:分离得到2株革兰氏阴性菌,2株细菌的生化试验和16S rDNA基因序列分析鉴定符合溶血性曼氏杆菌的特点,将其分别命名为201705YF和201805YS;对2株细菌进行血清型1型、2型和6型的多重PCR鉴定,201705YF为血清型2型,而201805YS的1型、2型和6型血清型检测为阴性;对2株细菌的7个管家基因adk、aroE、deoD、gapDH、gnd、mdh和zwf进行扩增并测序,菌株201705YF对应的7个管家基因的序列号为50,20,15,19,15,17,19,菌株201805YS对应的7个管家基因的序列号为1,2,20,2,1,1,1,这2株细菌的基因型为新基因型,即ST50和ST51;2株细菌对左氧氟沙星、卡那霉素、多黏菌素B、氟苯尼考、诺氟沙星、多西环素、美洛西林、环丙沙星、头孢哌酮等抗生素敏感,但201705YF对丁胺卡那耐药,201805YS对庆大霉素、新霉素、妥布霉素耐药。说明从送检病死山羊中分离出的2株细菌为不同血清型和新ST型的溶血性曼氏杆菌,为溶血性曼氏杆菌进化和流行病学关系研究提供了新的相关研究数据。     

豫陕两省14株鸡杆菌的gyrB、16S rRNA和rpoB基因序列比较分析

分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。     

鸡杆菌中国分离株RAPD分型方法的建立与应用

对9株鸡杆菌进行了血清分型,并应用8条随机引物对该9株鸡杆菌和3个参考菌株(1株巴氏杆菌、1株大肠杆菌和1株链球菌)共12株细菌做随机扩增多态性DNA(RAPD)分型研究。结果显示,9株鸡杆菌分别属于血清Ⅰ型(1/9)、血清Ⅱ型(3/9)和血清Ⅳ型(5/9)。所用8条随机引物中有3条呈现良好的多态性和稳定性,可产生差异显著的指纹图谱。采用SPSS13.0软件分析了不同菌株间的遗传距离,并依此绘制出菌株间的亲缘关系树状图。12株细菌共分为4个聚类群,其中9株鸡杆菌位于同一聚类群中,且又可分为4个聚类亚群。3个参考菌株各自形成一个独立的聚类群。不同血清型的鸡杆菌菌株具有相似的指纹图,同一血清型的菌株指纹图存在差异。结果表明,RAPD基因分型是目前鸡杆菌分子流行病学调查及病原分离鉴定比较理想的方法之一。     

Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes

Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods.     

The rarely reported tet(31) tetracycline resistance determinant is common in Gallibacterium anatis

The present investigation was undertaken to identify and characterize the tetracycline resistance determinant in 22 Gallibacterium anatis strains for which no determinant was identified using primers specific for tet(A, B, C, D, E, G, H, K, L, M, O). A recent study found tet(B) to be the most prevalent tetracycline resistance determinant in a larger collection of G. anatis field strains from Mexico and Denmark. However, in 41% of the tetracycline resistant strains no determinant could be assigned. Here we demonstrate that tet(31) is a common determinant in G. anatis originating from chickens from very different production systems and localities. In addition, tet(31) was identified in strains isolated over a 30-year period. This is the first report on tet(31) since its original identification in Aeromonas salmonicida.     

不同培养条件对鸭源鸡杆菌生物被膜形成的影响

采用体外定量法检测20株鸭源鸡杆菌的被膜形成能力,并对被膜形成能力较强的1株鸭源鸡杆菌在不同环境（培养时间、培养基质、营养条件）下产生生物被膜（BF）的差异进行了研究。结果显示,大部分鸭源鸡杆菌均具有不同程度形成被膜的能力,其中3株被膜形成能力中等,2株形成能力较强;鸭源鸡杆菌在银染法、试管法和微量板定量检测法中形成BF的最佳培养时间分别是24、60、24h。其生长周期为8h开始起始黏附、20h大量黏附阶段、24h时形成了完整的生物被膜、32h生物被膜老化散播,之后起始新一轮的被膜周期。葡萄糖、蔗糖及NaCl的加入均在一定程度上抑制了被膜的形成,且随着加入量增加NaCl对被膜形成抑制更显著,而低质量浓度的MgSO4在一定程度上促进BF的形成。扫描电镜观察发现,生物被膜内鸭源鸡杆菌聚集成团状、相互黏连形成致密的三维立体结构。     

Histopathologic findings in chickens experimentally infected with Gallibacterium anatis by nasal instillation

Histologic findings in chickens experimentally infected by nasal instillation with reference strains of Gallibacterium anatis are described. No clinical signs were observed in experimentally infected birds; however, sequential histologic examinations of trachea, lung, air sacs, and liver revealed lesions in all infected chickens. Our observations suggest that the reference strains of G. anatis used in this experiment are capable of causing primary infection in chickens. Despite that the experimental birds were inoculated by intranasal route, lesions were detected in the liver, suggesting a probable bacteremia. Because several degrees of severity were established in histopathologic lesions, probable variations in virulence, among the experimental strains, also are discussed.     

Development and preliminary application of a quantitative PCR assay for detecting gtxA-containing Gallibacterium species in chickens

A quantitative PCR (qPCR) assay using SYBR Green I was developed based on the published sequence of the gtxA gene from Gallibacterium anatis. This method produced reliable specificity, sensitivity, and repeatability. The detection rate of Gallibacterium in 181 clinical samples was 36.5% (66/181) by qPCR, which was superior to the detection rate of Gallibacterium-specific PCR (0/181) and an isolation and identification assay (18.2% or 33/181). No association was found between the prevalence of Gallibacterium and the age of the chickens. Gallibacterium infection was detected in one 4-day-old chicken, showing that infection can occur much earlier than the previously stated fourth week of life. Tissue sample analysis showed that Gallibacterium is mainly located in the trachea and ovaries, based on results from three groups of chicken with different health statuses. Furthermore, a titer analysis suggested that Gallibacterium loads in different organs may correlate with different clinical manifestations of disease. Thus, the qPCR assay developed in the present study is useful for identification and quantitative analysis of gtxA-containing Gallibacterium in various tissue samples from birds and for the assessment of the pathogenic mechanisms of Gallibacterium.     

鸭源鸡杆菌对SPF雏鸡的感染特性研究

为研究鸭源鸡杆菌的流行病学特性,本研究采用从自然病例分离得到的鸭源鸡杆菌人工感染4日龄SPF雏鸡,通过形态学、PCR、荧光定量PCR和组织学等方法分别对感染后SPF雏鸡的组织脏器进行了细菌分离、检测和鉴定,用ELISA对其血清抗体水平进行检测.结果表明,人工感染鸡无临床症状,组织学检查感染鸡的肝脏、气管和肺脏组织均有损伤.人工感染后第3d和同居感染第2d鸡体内可分离出鸭源鸡杆菌,人工感染后第96d仍能够分离到鸡杆菌.ELISA方法证实人工感染后47 d和同居后32 d出现抗体高峰,持续2～3周,人工感染组和同居组抗体水平均高于对照组(p＜0.05);荧光定量PCR检测病料结果显示人工感染组和同居组的气管组织中细菌含量最高.本研究表明雏鸡在4日龄即可感染鸭源鸡杆菌,并能通过同居传播,长期带菌、排菌;感染后抗体产生缓慢、持续期短.本研究为鸭源鸡杆菌的流行病学、致病机理研究及防治措施的制定等提供了参考依据.