水貂绿脓杆菌分离株的生物学特性和血清型分析

为了解国内水貂出血性肺炎病原绿脓杆菌的生物学特性和血清型分布,2002—2010年间从国内5个省水貂养殖场暴发出血性肺炎的水貂肺中分离获得45株绿脓杆菌,测定了分离株的生化特性、血清型、抗生素敏感性和对小鼠和水貂的半数致死量(LD50)。结果表明:分离菌株的主要生化指标除甘露醇和ONPG与人医临床分离株的不同外,其余均与绿脓杆菌的生理生化特性相同。45株菌共分为3种血清型:G型(42/45),B型(2/45)和I型(1/45),此结果与国外近几十年流行的水貂出血性肺炎和人医临床分离的绿脓杆菌主要流行血清型相同。45株菌对5种抗生素均较敏感,敏感性与分离的年份和菌株的血清型无关系。经动物试验证明,所选的4株菌毒力均不同,2株B型菌株毒力均较G型弱,4菌株对水貂的毒力均强于对小鼠的毒力,说明菌株对水貂较易感。     

貂源肺炎克雷伯氏菌的分离与鉴定

2013~2016年山东省、河北省、黑龙江省、吉林省的部分水貂养殖场发生了严重的出血性肺炎,导致水貂大量死亡,为了客观评估水貂出血性肺炎的危害及为综合防控提供参考依据,试验对423个水貂病例样本进行分离鉴定并且对所分离的细菌进行了形态特征观察、16SrRNA基因测序、血清型鉴定、小鼠毒力试验测定、生化试验鉴定及药敏试验测定。结果表明:423个样本中分离到68株肺炎克雷伯氏菌,其中最为流行的血清型为K2型(35株);并筛选出4株菌株对小鼠有很强毒力性;生化试验结果表明同一血清型的不同菌株之间也存在明显差异;药敏试验结果表明,4株K2型肺炎克雷伯氏菌强毒菌株对氧氟沙星、阿米卡星2种抗生素均为敏感或介于中间;对强力霉素、环丙沙星等8种抗生素均耐药;对诺氟沙星、庆大霉素等4种抗生素均为耐药或介于中间。     

貂源绿脓杆菌的分离鉴定及其生物学特性研究

2013～2016年山东省、河北省、黑龙江省、吉林省的部分水貂养殖场发生了严重的水貂出血性肺炎,导致水貂大量死亡,为了客观评估水貂出血性肺炎的危害及为综合防控提供参考依据,试验对423个水貂病例样本进行分离鉴定并且对所分离的细菌进行了形态特征观察[1]、16SrRNA基因测序、血清型鉴定、小鼠毒力试验测定、生化试验及药敏试验测定.结果表明:423个样本中分离到90株绿脓杆菌,其中最为流行的血清型为G型(25株)、B型(21株)、I型(17株)、C、E型及未定型(27株);并筛选出12株菌株对小鼠有很强的毒力性.生化试验结果表明:同一血清型的不同菌株之间也存在明显差异.药敏试验结果表明:12株菌株对强力霉素、林可霉素均耐药;对阿米卡星为敏感;对庆大霉素等4种抗生素均为耐药或介于中间;对环丙沙星等6种抗生素的敏感性不同;对氧氟沙星为敏感或介于中间.     

水貂绿脓杆菌(山东株)血清分型和ERIC-PCR分型的研究

为了探明水貂出血性肺炎病原和流行特点,2011-2012年对山东省15个水貂养殖场进行水貂出血性肺炎流行病学调查。从病貂中分离出19株绿脓杆菌,分别测定了分离株的血清型,同时应用肠杆菌科基因间重复序列聚合酶链反应(ERIC-PCR)对菌株进行DNA分型。结果 19株分离株中血清型G型15株,血清型B型2株,血清型E型和I型各1株;ERIC-PCR谱型表现为7种基因型,其中15株G型株存在于谱型Ⅰ~Ⅶ中(Ⅵ除外),B型株属于谱型Ⅱ,I型株属于谱型Ⅳ,E型株属于谱型Ⅵ。表明山东地区水貂出血性肺炎由绿脓杆菌引起,其分离株血清型以G型为主,基因型呈现多样性。     

山东省貂源铜绿假单胞菌分离株的血清型和致病性

为了解山东省貂源铜绿假单胞菌(PA)的血清型分布,本研究从部分市县采集的山东77份水貂肺脏病料样品中分离PA,并进行生化鉴定、分子生物学鉴定、血清学鉴定、生长曲线测定和半数致死量(LD_(50))测定。结果显示,共分离获得53株PA,分别属于11个血清型,其中G型27株、B型5株、D型4株。其他血清型分离株较少。其中G、B和D型分离株SD004、SD050、SD052生长性能良好,对小鼠的LD50分别为1.99×10~(10) cfu、2.51×10~(10)cfu、5.62×10~(10)cfu。该研究为水貂出血性肺炎灭活疫苗菌株的选择提供实验依据。     

水貂绿脓杆菌的分离与鉴定

2011~2013年间,从山东省、河北省和江苏省等地的多个貂场疑似水貂出血性肺炎病貂的肺脏、肝脏和脾脏中分离到425株疑似绿脓杆菌。用绿脓杆菌的OprF和PEA基因的PCR引物对分离菌株的目的基因扩增,经核苷酸测序确定其均为绿脓杆菌序列。通过日本生研株式会社血清学分型系统进行分型：G型376株,占88.5%;B型34株,占8%;C型12株,占2.8%;E型3株,占0.7%。取其中6株细菌（G血清型3株,其他血清型各1株）进行详细生化试验,结果显示该6株菌均符合绿脓杆菌的生化特性,并且从这6株中选4株细菌（每血清型各1株）腹腔接种小白鼠和气管内接种水貂,结果表明4株菌对其均有致病性。     

辽宁地区貂源铜绿假单胞菌分离鉴定与药敏试验

为了解辽宁地区水貂铜绿假单胞菌的流行状况及耐药情况,对从辽宁地区暴发出血性肺炎养貂场分离的16株分离菌进行了分离纯化、生化鉴定、基因鉴定、血清学分型和药敏试验。结果显示,分离株包括G型和B型两种血清型,其中G型是主要流行血清型,占分离株的75%,其余是B型。分离株对阿米卡星、头孢呋肟、阿莫西林和亚胺培南的敏感率较高,对氨苄青霉素的敏感率中等,对氯霉素、氨曲南和左氟沙星具有一定的耐药性。     

牦牛源鲍氏志贺菌的鉴定及对BALB/c小鼠的致病性

本试验旨在对从待屠宰健康牦牛中分离的志贺菌进行鉴定及确定分离株的致病性.通过形态染色、培养特性、生化特性、血清型鉴定、16S rRNA基因序列分析以及BALB/c小鼠的LD50测定等方面对分离株进行系统鉴定,并对分离株进行电镜观察和药敏试验.结果显示,分离株形态染色、培养和生化特性符合志贺菌的特点,血清型鉴定为鲍氏6型志贺菌,16S rRNA基因序列系统演化分析表明,分离菌株与人源鲍氏志贺菌和宋内志贺菌亲缘关系近,而与其他动物志贺菌亲缘关系较远,分离株对BALB/c小鼠具有致病性,其LD50值为2.5×107.5 CFU,药敏试验结果显示分离菌株对常用抗生素无耐药性.本次试验首次从健康牦牛中分离的鲍氏6型志贺菌具有较强的致病性,与人源菌株亲缘关系较近,提示牦牛中分离的鲍氏志贺菌具有公共卫生学意义.     

貂源绿脓杆菌的分离鉴定及生物学特性

为了解国内貂出血性肺炎病原绿脓杆菌的生物学特性,2012年从山东省发病的水貂肺内分离得到了22株绿脓杆菌,测定了分离株的生化特性、血清型、基因分型以及抗生素敏感性。结果显示,分离株对阿米卡星、环丙沙星、庆大霉素、头孢他啶敏感率分别为97%、87%、78%、81%,对头孢哌酮中敏率为87%,对多黏菌素B耐药率为68%。22株分离株可以分为G、B2种血清型,比例分别为82%、18%。     

大肠杆菌和绿脓杆菌混合感染引起的水貂出血性肺炎的鉴定

为了鉴定引起水貂发生出血性肺炎的病原体,试验采用病原分离鉴定、分离株16S rRNA扩增与序列分析、分离株对小鼠致病力试验、分离株血清分型鉴定、大肠杆菌H基因型鉴定、分离株药敏试验和分离株动物感染试验对来自山东省、辽宁省、河北省和黑龙江省的28份具有典型出血性肺炎症状死亡水貂的病料进行鉴定。结果表明:混合感染12例,单一病原菌感染16例,镜检可见短小的阴性杆菌和两端钝圆的阴性长杆菌; 16S rRNA扩增与序列分析共分离出22株绿脓杆菌和18株大肠杆菌,其中有4株对小鼠致死率达到50%以上,9株对小鼠致死率在10%～40%;从22株绿脓杆菌中共分离出8株G型、7株E型和7株D型,未检测出大肠杆菌血清型,大肠杆菌H基因型为H11;绿脓杆菌分离株对环丙沙星、妥布霉素、阿米卡星等6种药物敏感,大肠杆菌分离株对环丙沙星、妥布霉素、阿米卡星等10种药物均耐药;大肠杆菌和绿脓杆菌分离株均可导致水貂死亡并造成肺泡壁毛细血管瘀血、脾脏淋巴细胞数量减少、肺脏上皮细胞坏死、红髓内巨噬细胞数量增多等明显病理变化。说明目前引起水貂出血性肺炎的病原体已由传统的绿脓杆菌单一感染逐渐转变为大肠杆菌和绿脓杆菌的混合感染。     

Isolation,Identification and Drug Sensitivity Analysis of Pseudomonas aeruginosa from Minks

In order to provide therapeutical guidance for drug admistration,the bacteria from five dead minks suffering with typical symptoms provided by mink farms in Shandong province were isolated and identified,and the drug sensitivity was tested.The bacteria were isolated with TSA plates,and identified using biochemical methods and PCR assay.The drug sensitivity of the isolates to antimicrobial agents was investigated using K-B method.PCR was used to detect the resistance genes.A total of 5 Pseudomonas aeruginosa isolates were obtained from sick minks.The drug sensitivity results showed that 5 strains were sensitive to fluoroquinolone,the third and fourth generations cephalosporin drugs,but were resistant to aminoglycoside,tetracycline,chloramphenicol,penicillin,the first and second generations cephalosporin drugs.There were six resistance genes were detected,aadA1,aac(3')-Ⅱc,aac(6')-Ⅰb,tetA,tetK and cat2.All of the isolates were detected more than three kinds of resistance genes.The result showed that 5 strains were all Pseudomonas aeruginosa,and Pseudomonas aeruginosa was the main cause of mink hemorrhagic pneumonia.The resistance of 5 strains were very serious and mainly for multiple drug resistance phenomenon.The resistance genes detected in the mink were various,and could cause the resistance to the drugs.     

Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis

Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P. aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. P. aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other. However, PFGE types of strains causing clusters of farm outbreaks changed from year to year. These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P. aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment.     

水貂绿脓杆菌分离鉴定及药物敏感性分析

本试验旨在通过对山东某水貂养殖场送检的5只具有典型出血性肺炎症状的病死水貂进行细菌分离鉴定及耐药情况分析,为临床用药和治疗提供依据和参考。通过细菌分离纯化、生化鉴定和PCR方法对分离菌株进行鉴定,采用K-B药敏法检测分离菌株对常用药物的敏感性,并通过PCR方法检测分离菌株耐药基因的携带情况。经鉴定共分离得到5株绿脓杆菌,药物敏感性试验结果显示,5株绿脓杆菌对氟喹诺酮类药物、第3代和第4代头孢类药物较敏感,对氨基糖苷类、四环素、氯霉素、青霉素类、第1代和第2代头孢类药物耐药。耐药基因检测结果显示,5株绿脓杆菌共检测出6种耐药基因aadA1、aac(3')-Ⅱc、aac(6')-Ⅰb、tetA、tetK、cat2。且每株分离菌均检测出3种以上耐药基因。结果表明,分离的5株菌株均为绿脓杆菌,主要引起水貂出血性肺炎。分离菌株耐药性较严重,主要表现为多重耐药现象。菌株携带的耐药基因呈多样性,可引起菌株对相应药物产生耐药现象。     

北美水貂源沙门氏菌的分离鉴定及耐药性检测

为调查秦皇岛地区北美水貂沙门氏菌的检出率及耐药情况,对65份水貂样品中的细菌进行分离纯化,采用细菌的鉴定培养、革兰氏染色、生化鉴定和16S rDNA鉴定分离菌,采用小鼠致病性试验研究分离菌株的致病性,采用KB纸片法检测分离菌的耐药性。结果显示,经鉴定共计分离出9株沙门氏菌,分离率13.85%(9/65);9株沙门氏菌均可致小鼠发病死亡,死亡率20%—100%;9株沙门氏菌分别对5—11种抗生素药物耐药,氟苯尼考、替米考星、磺胺间甲氧嘧啶、磺胺二甲氧嘧啶和复方新诺明的耐药率高达100%,土霉素敏感率最高,为88.89%(8/9)。上述研究结果表明,秦皇岛地区从水貂分离到的沙门氏菌均具有一定的致病力和多重耐药性,为该地区水貂沙门氏菌病的防治提供参考。     

昆明地区奶牛临床型乳房炎病原菌分离鉴定及药敏试验

对昆明地区多个奶牛场的奶牛临床型乳房炎病原菌进行分离、鉴定,从114份乳样中共分离出细菌13种305株,经培养特性、染色镜检、生化鉴定等,鉴定出金黄色葡萄球菌63株,占20.66%;表皮葡萄球菌26株,占8.52%;无乳链球菌53株,占17.38%;停乳链球菌23株,占7.54%;乳房链球菌19株,占6.23%;乳链球菌14株,占4.59%;粪链球菌15株,占4.92%;化脓链球菌14株,占4.59%;大肠杆菌48株,占15.74%;真菌12株,占3.93%。分别对分离菌株进行17种药物的敏感性试验,除真菌外,所有菌株对庆大霉素、新霉素、环丙沙星、诺氟沙星、林可霉素、乳肿康均高度敏感,对临床常用的青霉素、链霉素、鱼腥草等具有较强的耐药性。     

Isolation,Identification and Drug Sensitivity Analysis of Shigella from Minks

In order to provide therapeutical guidance for drug admistration, the bacteria of three sick minks suffering from typical diarrhea symptoms provided by mink farms in Jilin province were isolated and identified, and the drug sensitivity was tested. The bacteria were isolated with TSA plates, and identified using biochemical methods and PCR assay. The virulence of the isolates was determined by infecting BALB/c mice. The antimicrobial susceptibility of the isolates to antimicrobial agents was investigated using the K-B method. PCR was used to detect the resistance genes and Ⅰ integrons. A total of 3 Shigella isolates were obtained from sick minks. The virulent determination showed that all isolates could cause mice diarrhea. The drug sensitivity results showed that 3 strains were sensitive to fluoroquinolone, cephalosporin, florfenicol and polymyxin, but they were resistant to aminoglycoside, tetracycline, chloramphenicol, penicillin and ampicillin. There were seven resistance genes were detected,bla_TEM-1,aadA1, aac(3')-Ⅱc, aac(6')-Ⅰb, aph(3')-Ⅶ, tet(M), cat2 and three class Ⅰ integrons carrying aadA 1 gene cassette. All of the isolates were virulent and caused the mice diarrhea. The resistance of the 3 strains were very serious and mainly for multiple drug resistance phenomenon. The resistance genes detected in the mink were various, and could bring enormous implications for clinical treatment.     

水貂志贺氏菌分离鉴定及药物敏感性分析

试验旨在对吉林省某水貂养殖场送检的3只具有典型腹泻症状的病死水貂的小肠和肠内容物样品进行细菌分离鉴定及耐药情况分析,为临床治疗提供参考。通过细菌分离纯化和PCR方法对分离菌株进行鉴定,对BALB/c小鼠进行菌液注射来检测菌株的致病性。采用K-B药敏法检测菌株对常用药物的敏感性,并通过PCR方法检测其耐药基因和Ⅰ类整合子的携带情况。结果显示,分离得到3株志贺氏菌,致病性检测试验显示分离菌可引起小鼠腹泻。药物敏感性试验结果显示,3株志贺氏菌对氟喹诺酮类药物、头孢类药物、氟苯尼考和多黏菌素较敏感,对氨基糖苷类、四环素、氯霉素、青霉素、氨苄西林耐药。耐药基因检测结果显示,3株志贺氏菌共检测出7种耐药基因bla_TEM-1、aadA1、aac（3'）-Ⅱc、aac（6'）-Ⅰb、aph（3'）-Ⅶ、tet（M）、cat2及携带aadA1基因盒的Ⅰ类整合子。结果表明,分离的3株志贺氏菌均具有致病性,可引起小鼠腹泻,主要表现为多重耐药现象,携带的耐药基因呈多样性,为临床治疗带来巨大影响。     

Isolation,Identification and Genetic Evolution Analysis of Mink Serratia marcescens

In order to determine drug resistance, pathogenicity and genetic of the pathogenic bacteria in mink of Heilongjiang province, three strains were isolated from dead minks and identified. Based on colony morphology, microscopic characteristics, biochemical test and 16S rRNA sequencing. The antibiotic susceptibility to 24 antibiotics were detected by using standard K-B paper method, and virulence assay, phylogenetic analysis were carried out at the same time. The result showed that the isolates were highly tolerance to antibacterials, could cause 100% death of mice in experimental group and were closely related to strains from activated sludge and volcanic deposits. It showed that Serratia marcescens infection of mink existed in Heilongjiang province. Strains resistance phenomenon was serious and had a strong pathogenicity, and might be originated from the environment. It was the first time that the pathogenic Serratia marcescens isolated from the mink was reported. The results provided information for the detection, identification, diagnosis, treatment and prevention of related diseases.