猪源链球菌的分离鉴定及生物学特性研究

通过生化试验、药敏试验、PCR分型、毒力基因的PCR检测及动物试验对分离的猪源链球菌进行药物敏感性、血清型和分子流行病学初步研究。从不同省份猪链球菌病疑似病例猪的心血、肝、淋巴结、脑和关节液组织分离出97株链球菌,药敏试验结果表明各菌株对13种抗菌素的耐药谱不同,但对先锋霉素V和环丙沙星的耐药率均低于5%。通过对分离菌株进行PCR鉴定和分型,确认26株为猪链球菌,其中1株为1型,16株为2型,4株为7型,没有9型,另5株为其它型。进一步对1型、2型和7型猪链球菌mrp、epf和sly3种毒力基因的分布情况进行了PCR检测。动物试验表明能100%致死小白鼠的猪链球菌基因型均为epf^＋mrp^＋sly^＋2型猪链球菌,1株2型和1株7型猪链球菌均能复制出典型的猪链球菌病例。     

屠宰场猪链球菌分离菌株分型及其致病性和耐药性分析

为了解吉林省猪链球菌的流行情况,从屠宰场采集的猪咽拭子和鼻拭子样品中分离鉴定猪链球菌,并进行菌株血清型、基因型和毒力表型的鉴定,以及致病性和耐药性的分析。结果表明,从100份样品中共分离鉴定猪链球菌104株,其中29株鉴定为血清2型、9型和1型等几种常见的致病性血清型,其他75株不属于常见的致病性血清型。血清2型的菌株中,2株经鉴定为ST1基因型和mrp+epf+sly+毒力型,并经动物试验鉴定为强毒菌株;其他菌株均为ST28基因型和mrp+epf-sly-毒力型,具有中等毒力。血清9型的菌株,均为mrp-epf-sly-毒力型,动物试验鉴定均为强毒菌株。根据Kirby-Bauer纸片扩散法的药敏试验结果,98%的猪链球菌分离株对四环素耐药;对大环内酯类、克林霉素和链霉素的耐药率都在50%以上;对β-内酰胺类、氯霉素和喹诺酮类抗生素的耐药率小于20%。总体分析,从屠宰场分离的猪链球菌强毒菌株所占的比例并不高,但是菌株多重耐药的情况非常严重。     

9型猪链球菌广东株的分离鉴定和基因序列分析

为了解9型猪链球菌广东分离株毒力因子基因型、基因变异和进化情况,本试验采集发病猪脑脊液、关节液、脾脏、肝脏、淋巴结进行细菌分离培养和生化鉴定,采用PCR方法鉴定其血清型及部分毒力基因,对cps9D、gdh、gapdh与orf2基因进行序列测定和分析,并构建系统发育树。结果显示,分离菌镜检为革兰氏阳性链状球菌,在鲜血琼脂平板中呈β溶血,可发酵大多数糖类,能成功扩增cps9D基因,含重要的保守基因gdh及毒力基因gapdh和orf2,表明分离菌株为9型猪链球菌。分离菌株cps9D、gdh、gapdh、orf2基因核苷酸序列与GenBank上国内外参考菌株的同源性分别为95.9%~100.0%、98.6%~99.8%、98.7%~99.6%和95.5%~99.9%,说明分离菌株与国内外其他9型猪链球菌毒株核苷酸同源性都较高,且与国内外的流行毒株基本一致,未发生太大的变异。系统发育树表明,分离株与国内外来源不同的猪链球菌分离株同源性高,亲缘关系密切。     

9型猪链球菌广东株的分离鉴定和基因序列分析

为了解9型猪链球菌广东分离株毒力因子基因型、基因变异和进化情况，本试验采集发病猪脑脊液、关节液、脾脏、肝脏、淋巴结进行细菌分离培养和生化鉴定，采用PCR方法鉴定其血清型及部分毒力基因，对cps9D、gdh、gapdh与orf2基因进行序列测定和分析，并构建系统发育树。结果显示，分离菌镜检为革兰氏阳性链状球菌，在鲜血琼脂平板中呈β溶血，可发酵大多数糖类，能成功扩增cps9D基因，含重要的保守基因gdh及毒力基因gapdh和orf2，表明分离菌株为9型猪链球菌。分离菌株cps9D、gdh、gapdh、orf2基因核苷酸序列与GenBank上国内外参考菌株的同源性分别为95.9%~100.0%、98.6%~99.8%、98.7%~99.6%和95.5%~99.9%，说明分离菌株与国内外其他9型猪链球菌毒株核苷酸同源性都较高，且与国内外的流行毒株基本一致，未发生太大的变异。系统发育树表明，分离株与国内外来源不同的猪链球菌分离株同源性高，亲缘关系密切。     

新疆石河子部分猪场链球菌2型的分离鉴定与毒力因子检测

为了解石河子垦区具有呼吸道病症状的病死猪感染猪链球菌2型的情况，对采集的53份肺脏组织进行了猪链球菌的分离培养、生化鉴定、PCR鉴定以及cps2J、ef、mrp毒力因子的检测。分离出猪链球菌2型21株，其中8株毒力因-T-为cps2J．10株毒力因子为ef，3毒力因子为cps2J／ef；小鼠试验表明cps2J毒力因子的菌株不具有致病性，毒力因子为ef、cps2J／ef的菌株具有致病性。得出结论，石河子部分规模猪场存在链球菌2型感染情况，且部分感染菌株具有致病性，应引起有关生猪养殖场的高度重视。     

猪链球菌14型的分离鉴定及生物学特性研究

为了检测确定2019年5月河南某规模化猪场一栋保育仔猪发病猪群的病原,本研究从送检的发病猪关节液中分离获得1株细菌。通过细菌纯化培养、革兰氏染色、形态学观察及猪链球菌gdh基因PCR扩增,确定该分离菌株为猪链球菌。用猪链球菌分型引物对该菌株进行PCR扩增分型鉴定及软件比对分析,结果表明该分离菌株为猪链球菌14型,与猪链球菌JS14株(GenBank登录号:CP002465.1)同源性为100%。毒力基因检测结果表明,该菌株的同时携带有epf、mrp、sly、fbps、orf2毒力基因,属于高致病性菌株。小鼠致病性试验结果也证明该菌株是一株高致病性猪链球菌。药物敏感性试验结果显示,该菌株对β内酰胺类和喹诺酮类药物敏感,对氨基糖苷类、四环素类、大环内酯类和磺胺类高度耐药,表现出多重耐药现象。对该菌株进行5大类24种耐药基因检测,该菌株同时携带有bla_(TEM)、aadA1、strA、strB、aacC2、aphA1、tet(B)、gyrA、parC、sul2耐药基因。该研究为后续进一步开展猪链球菌14型流行特点和致病机制研究奠定了基础,为猪链球菌14型临床防控提供了理论依据,同时具有重要的公共卫生意义。     

一例猪链球菌病的诊断及其病原分离鉴定

对某猪场送检的一份出现神经症状的保育仔猪病料进行了细菌分离,并对该分离菌株进行了染色镜检、生化鉴定、PCR检测和药敏试验。革兰氏染色结果表明从病料中分离到1株革兰氏阳性链状球菌,其生化鉴定结果与猪链球菌生化特性一致。分离菌株16srRNA基因测序结果表明其与2型猪链球菌亲缘关系最近,同源性达100%,确定该分离菌株为2型猪链球菌。药敏试验结果表明该分离菌株对青霉素、恩诺沙星、氨苄西林、氟苯尼考、头孢噻肟、阿莫西林、氧氟沙星、诺氟沙星等高度敏感;对复方新诺明、多西环素、土霉素、头孢曲松、丁安卡那等中度敏感,对磺胺异恶唑耐药。     

河南省规模化猪场猪链球菌2型的分离鉴定和药敏试验

从2006-2009年河南省发生猪高热综合征的113家规模化猪场291份病料中分离革兰氏阳性球菌,通过培养特性试验、生化试验、猪链球菌和猪链球菌2型的PCR试验,结果鉴定125株为链球菌,其中35株为猪链球菌2型。对35株猪链球菌2型进行小鼠毒力试验及药敏试验,结果显示,35株猪链球菌2型对小鼠均有毒力,均对头孢噻肟、头孢唑啉、头孢他啶、阿莫西林、氯霉素、环丙沙星、氧氟沙星敏感。     

猪链球菌14型的分离鉴定及生物学特性研究

为了检测确定2019年5月河南某规模化猪场一栋保育仔猪发病猪群的病原,本研究从送检的发病猪关节液中分离获得1株细菌。通过细菌纯化培养、革兰氏染色、形态学观察及猪链球菌gdh基因PCR扩增,确定该分离菌株为猪链球菌。用猪链球菌分型引物对该菌株进行PCR扩增分型鉴定及软件比对分析,结果表明该分离菌株为猪链球菌14型,与猪链球菌JS14株（GenBank登录号：CP002465.1）同源性为100%。毒力基因检测结果表明,该菌株的同时携带有epf、mrp、sly、fbps、orf2毒力基因,属于高致病性菌株。小鼠致病性试验结果也证明该菌株是一株高致病性猪链球菌。药物敏感性试验结果显示,该菌株对β内酰胺类和喹诺酮类药物敏感,对氨基糖苷类、四环素类、大环内酯类和磺胺类高度耐药,表现出多重耐药现象。对该菌株进行5大类24种耐药基因检测,该菌株同时携带有bla_TEM、aadA1、strA、strB、aacC2、aphA1、tet（B）、gyrA、parC、sul2耐药基因。该研究为后续进一步开展猪链球菌14型流行特点和致病机制研究奠定了基础,为猪链球菌14型临床防控提供了理论依据,同时具有重要的公共卫生意义。     

2型猪链球菌河南株的生物学特性研究

从河南省豫东某规模化猪场保育猪群发生疫情的病死猪脑组织分离得到一株细菌,通过对该株细菌纯化鉴定、血清型分型鉴定、致病性试验及毒力基因检测、药敏试验和耐药基因检测等一系列研究,结果表明,该株细菌鉴定为2型猪链球菌,毒力较强,为携带胞外因子epf基因、溶血素sly基因、溶菌酶释放蛋白mrp基因的3个毒力基因的强毒力菌株;该菌株对头孢他啶、阿莫西林、氨苄西林、青霉素、复方新诺明、左氟沙星、氧氟沙星等β-内酰胺类、磺胺类和喹诺酮类药物敏感;对氨基糖苷类、四环素类和大环内酯类药物高度耐药,携带有β-内酰胺类耐药基因blaTEM和氨基糖苷类耐药基因strB。该研究对临床防控猪链球菌病具有重要意义。     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.     

副猪嗜血杆菌的分离鉴定及相关特性研究

本试验从2013年北京地区疑似副猪嗜血杆菌病病料中分离到19株革兰氏阴性短小杆菌,对分离株进行培养特性、荚膜染色、生化特性、血清型分型及PCR鉴定,结果显示分离的19株细菌均为副猪嗜血杆菌（Haemophilus parasuis,Hps）,分别属于血清4、5、7、12及13型。对分离株进行药敏试验及致病性试验,结果表明各分离株均有多重耐药性,且各分离株除GS-3外均有较强毒力。     

Evaluation of Pasteurella multocida isolated from rabbits by capsular typing, somatic serotyping, and restriction endonuclease analysis.

The goal of this study was to characterize Pasteurella multocida isolated from rabbits. Five hundred and fifty-three apparently healthy rabbits were sampled for this study. Nasal swabs were collected from each rabbit for P. multocida isolation and identification. Isolates were further characterized by capsular and somatic antigens and genomic DNA fingerprinting. Thirty-nine P. multocida isolates were recovered from 553 rabbits (7%). Capsular typing was done by depolymerization of P. multocida capsule by Staphylococcus aureus hyaluronidase and by disc diffusion with mucopolysaccharidase enzymes (heparinase III, chondroitinase AC, and hyaluronidase). Thirty-one (79%) of the isolates were capsular type A, and 8 isolates (21%) had untypable (UT) capsules. The gel-diffusion precipitin test was used to determine the somatic type of P. multocida isolates. Nineteen isolates were somatic serotype 3 (49%), 12 were serotype 1 (31%), 1 was serotype 2, 2 were serotype 5, 2 were serotype 12 with a weak reaction to antiserum raised against serotype 7 (5%), and 1 was serotype 4. Two of the isolates (5%) were UT. Restriction endonuclease analysis of the DNA of the isolates revealed 7 distinct profiles by digestion with HindIII, and 12 profiles were obtained with HpaII, whereas digestion with EcoRI did not differentiate between any of the P. multocida DNA isolates studied. The DNA restriction endonuclease enzyme HpaII was found more useful for differentiating between DNA fingerprints of P. multocida rabbit isolates. However, no correlation between capsular type, somatic serotypes, and DNA fingerprints was seen in this study.     

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。     

Serotypes and mating types of clinical isolates from feline cryptococcosis in Japan

Most isolates of Cryptococcus neoformans (teleomorph: Filobasidiella neoformans) from human patients and from environmental materials in Japan have been identified as serotype A mating type a by the seroagglutination test and mating experiments. A PCR method using the mating type alpha allele-specific primer of the STE12 gene and the serotype- and mating type-specific primers of the STE20 gene for identification of C. neoformans has been developed. Using the PCR method, conserved strains and clinical isolates from feline cryptococcosis were examined for serotype and the mating type. The results showed that all clinical isolates examined were identified as serotype A, MATalpha, indicating that feline cryptococcsis cases in Japan are caused by C. neoformans serotype A, MATalpha, as is the case in humans.     

DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae

A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expceted hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.