河南副猪嗜血杆菌病流行病学调查

于2011年3月至2012年2月调查了河南周口、安阳、新乡、濮阳、驻马店、南阳等6个市60个猪场副猪嗜血杆菌病流行情况.采集病猪组织样品共96份进行副猪嗜血杆菌分离;对疑似菌株进行形态学观察、生化特性鉴定;最终分离鉴定到30株副猪嗜血杆菌,分离率为31.25％;对分离菌株进行血清型鉴定、致病性和药敏试验,结果表明,30株分离株中血清4型有6株,5型5株,9、11、13、14、15型各1株,其余13株未能鉴定出血清型.血清型5、13、14菌株和5个未能定型的菌株能引起小鼠全部死亡,其他菌株对小鼠致病性不强.药敏试验结果表明,63％以上的菌株除对庆大霉素、头孢喹诺高度敏感外,对其他药物敏感性不高.本调查结果将对河南副猪嗜血杆菌病的防治提供指导.     

锦州地区副猪嗜血杆菌病流行情况调查与药敏试验

为了解辽宁省锦州地区副猪嗜血杆菌病流行情况,对37个疑似样本进行分离鉴定。采用多重PCR方法进行血清型鉴定,并对分离菌株进行药敏试验,结果分离到23株副猪嗜血杆菌,分别为血清4型11株,5型6株,1、2、14型各1株,不可分型菌株为3株,对头孢噻肟、氟苯尼考、头孢曲松、阿莫西林、阿米卡星高度敏感。     

锦州地区副猪嗜血杆菌血清型鉴定

本研究先后从锦州市太和区、凌海、北镇、黑山、义县等地采集以多发性浆膜炎为主要特征的病猪病料13份,在被检的13份病料中分离到6株疑似副猪嗜血杆菌菌株,同时对所分离的菌株进行了形态学检查、分离培养、生化试验、PCR鉴定及血清型鉴定,最终证实6株为副猪嗜血杆菌,血清型分别为:血清4型1株、血清5型2株、血清12型1株、血清13型2株.本研究初步确定了锦州地区副猪嗜血杆菌流行菌株的血清型,为猪场制定副猪嗜血杆菌病的防控措施提供理论依据.     

副猪嗜血杆菌病的诊断

从江西省抚州市某规模化养猪场病猪肺脏分离到1株革兰阴性长丝状菌,经细菌生化鉴定、PCR检测鉴定为副猪嗜血杆菌。使用KRG方法对其进行血清分型鉴定,该株副猪嗜血杆菌的血清型为13型。抗生素药物敏感试验结果表明,分离菌株对氨苄西林、头孢类、苯唑西林、氯霉素、阿莫西林表现较高的敏感性。对强力霉素、青霉素G中度敏感,而对磺胺类、链霉素、庆大霉素表现出耐药性;致病性试验结果表明,分离菌株对小白鼠有强致病性,命名为JXFZHP01株。本结果为江西副猪嗜血杆菌病的防治提供了理论依据。     

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。     

辽宁地区副猪嗜血杆菌血清型的鉴定

为探明辽宁地区副猪嗜血杆菌菌株的血清型,先后从辽宁省的锦州、营口、沈阳、丹东、铁岭、阜新、抚顺、朝阳、葫芦岛等市县采集以多发浆膜炎、关节炎、脑膜炎以及急性死亡为主要特征病猪的病料样本236份,从被检的236份病料中分离到86株副猪嗜血杆菌菌株,最终有51株鉴定出副猪嗜血杆菌血清型,分别为:血清1型2株;血清4型5株;血清5型21株;血清12型3株;血清13型20株。通过本研究初步确定了辽宁省副猪嗜血杆菌主要优势血清型,为辽宁省规模化猪场制定副猪嗜血杆菌病的防控措施提供依据。     

血清6型副猪嗜血杆菌的分离鉴定和生物学特性研究

为了明确河南地区血清6型副猪嗜血杆菌流行菌株的生物学特性,试验从发病猪的肺脏、心血、关节液等病料中分离、纯化副猪嗜血杆菌,并对分离纯化的副猪嗜血杆菌进行PCR鉴定,同时进行血清型分型、致病性试验、毒力基因检测和药敏试验。结果表明：从肺脏中分离得到一株血清6型副猪嗜血杆菌,该菌株携带有vta1、vta2、vta3、wza、ompP2、nanH、cdtA、cdtC、espP2 9种主要毒力基因;对保育仔猪表现出轻微的致病性;对头孢噻呋、头孢他啶、阿莫西林等16种抗生素敏感,对氨苄西林、青霉素G、卡那霉素等8种抗生素耐药。说明分离得到的血清6型副猪嗜血杆菌毒力较弱,对大多数药物敏感。     

猪场副猪嗜血杆菌的分离鉴定及药敏试验

为查明贵阳市花溪区麦坪镇某猪场仔猪发生呼吸道疾病的病因,对送检的2头病猪采集病料进行细菌分离培养、染色镜检、生化试验、PCR扩增及测序、药敏试验。结果:从病料样本中分离得到1株细菌,根据形态学和生化试验初步鉴定为副猪嗜血杆菌;应用细菌16S rRNA序列分析技术从分子水平对分离细菌进行分型鉴定,运用DNAStar软件与不同血清型副猪嗜血杆菌基因序列进行比对,发现分离菌与不同血清型副猪嗜血杆菌菌株16S rRNA序列同源且相似性为97.4%～100%,其中与血清5型相似性最高;系统进化分析显示,分离菌株与血清5型副猪嗜血杆菌进化关系最近;分离菌株对利福平、头孢氨苄、阿米卡星、环丙沙星、万古霉素敏感。结论:综合分离细菌传统鉴定方法和分子生物学鉴定方法的实验结果,确定分离菌株属于血清5型副猪嗜血杆菌。     

副猪嗜血杆菌的分离鉴定及三价灭活疫苗免疫效力分析

副猪嗜血杆菌是猪上呼吸道早期定植菌也是格拉泽病的病原体,给猪场带来较严重的经济损失。为了调查副猪嗜血杆菌血清型的流行情况,采用细菌分离培养、玻板凝集法及PCR方法,从广东、广西、山东、江苏、江西45个规模化猪场送检的疑似样品中共分离鉴定了34株副猪嗜血杆菌,其中6株(17. 65%)血清4型、9株(26. 47%)血清5型、8株(23. 53%)血清12型、4株(11. 76%)血清13型以及7株(20. 59%)未定型菌株。为了有效地防控副猪嗜血杆菌病的流行,以4、5、12血清型优势流行血清型制备3价灭活苗,用仔猪免疫/攻毒试验评价3价灭活疫苗免疫保护力。动物实验表明,该疫苗对副猪嗜血杆菌4型、5型和12型具有较好的免疫保护能力。     

河南省平顶山地区副猪嗜血杆菌分离鉴定和外膜蛋白P5基因序列分析

为了解河南省平顶山地区副猪嗜血杆菌流行的血清型、菌株耐药性及其外膜蛋白ompP5基因的变异情况,采集疑似副猪嗜血杆菌病猪病料进行细菌分离鉴定、血清型鉴定、药敏试验和动物试验;测定5株不同血清型菌株的ompP5基因序列,分析其同源性。结果:分离鉴定了42株副猪嗜血杆菌,主要血清型为1(38.1%)、4(19.0%)、5(26.2%)、12(11.9%)和未定型(4.8%)。药敏试验显示菌株对磺胺甲氧嘧啶、磺胺-6-甲氧嘧啶和头孢喹肟耐药性较强,耐药率分别为85.7%、83.3%和64.3%,对氟苯尼考和青霉素高度敏感。基因序列分析显示菌株ompP5基因序列与其血清型、致病性无明显相关性。该研究为平顶山地区副猪嗜血杆菌病的防控提供了一定的理论依据。     

副猪嗜血杆菌流行病学调查及两株分离株耐药性和致病性分析

为了解山东地区副猪嗜血杆菌病的流行情况和流行菌株的生物学特性及致病性,将2016-2018年山东省12个地区送检的103个发病猪的病料进行细菌分离,并对疑似菌株进行形态学观察、PCR鉴定及血清型鉴定,对两株流行菌株进行了培养特性观察、生化特性鉴定、药敏试验及致病性研究。最终分离获得29株副猪嗜血杆菌,分离率为28.16%,其中血清型4型和5型最为流行,其次是1型和12型。该病多发于春秋两季,31~50日龄的仔猪感染率最高。两株流行菌株LZ株和LC株均对青霉素类、头孢类等药物高度敏感,LZ株对庆大霉素、卡那霉素等中度敏感,对林可霉素、链霉素不敏感,LC株对庆大霉素、林可霉素等中度敏感,对卡那霉素、链霉素不敏感;生化特性试验结果显示,LC株和LZ株的硝酸盐还原试验、接触酶试验、葡萄糖发酵试验以及果糖发酵试验的结果均为阳性,吲哚试验、氧化酶试验、甘露醇发酵试验的结果均为阴性;动物致病性试验表明LZ株和LC株均具有较强的毒力,最小发病剂量分别为4.5×10^9 CFU和6.0×10^9 CFU。该研究为副猪嗜血杆菌病的防治提供了重要的参考依据。     

Relationship between serotype, virulence and SDS-PAGE protein patterns of Haemophilus parasuis

141 Haemophilus (H.) parasuis and 8 H. parasuis-like strains from different farms were serotyped according to Morozumi and Nicolet (1986 b) as well as to Bakos et al. (1952). It was possible to classify 72.8% of the investigated strains. 7 out of 12 serotypes have been described for the first time. The high specificity in the agar gel precipitation test was not reproducible in the more sensitive dot-blot procedure. The dot-blot results point to a participation of non-immunogenic polysaccharides in the detection reaction. The serotypes SV 1, SV 5, SV Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were found to be nonvirulent. Unencapsulated strains and isolates of serotype SV 5 prevailed in animals with Glasser's disease. 23 H. parasuis and 3 H. parasuis-like strains were examined in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of protein profiles of whole-cell lysates, 23 of them could be assigned to 5 groups. Apart from the highly virulent strains of serovar 1, which belonged to PAGE type III, all other highly virulent strains of the serovars SV 5, SV Jena 6 and SV Jena 10 were grouped into PAGE type I. No correlation could be found between PAGE type on the one hand and virulence or origin of isolates on the other hand.     

副猪嗜血杆菌的分离鉴定及16S rRNA序列分析

从云南某规模化养猪场病猪肺脏分离到1株革兰氏阴性小杆菌,经细菌生化鉴定、PCR鉴定和16S rRNA序列比对鉴定为副猪嗜血杆菌。抗生素药物敏感试验结果表明,分离菌株对四环素、红霉素、氯霉素、头孢噻吩高敏;对庆大霉素、氧氟沙星、诺氟沙星中敏;对磺胺甲唑耐药。16S rRNA分析结果表明,该分离株与GenBank中的Hps参考株AB078973(基因登录号)同源性为100%,将分离菌株鉴定为副猪嗜血杆菌。16S rRNA遗传进化关系表明,分离株与副猪嗜血杆菌3株血清5型参考株AB078972、AB078973、AB078974的16S rRNA序列位于一个分支上,遗传进化关系最近,它们之间的核苷酸同源性在99.0%~99.4%之间,初步鉴定为血清5型副猪嗜血杆菌,致病性试验结果表明,分离菌株对小白鼠有强致病性,命名为YN-1株。     

Typing of heat-stable soluble Haemophilus parasuis antigen by means of agargel precipitation and the dot-blot procedure.

According to Morozumi's and Nicolet's (1986) investigations, a serological classification procedure for H. parasuis and to a certain extent, for H. parasuis-like strains was proposed on the basis of heat-stable cell antigens in the immunodiffusion test. It was possible to classify 72.8% of the investigated strains serologically using this procedure. 7 of 12 serotypes were described for the first time. 60.1% of the classified strains belonged to the already known serotypes SV 1 to SV 5, whereas the new serotypes SV Jena 6 to SV Jena 12 amounted to only 12.7% of the field isolates. The serotypes SV Jena 7 to 9 are represented by H. parasuis-like strains. Unencapsulated strains and isolates of serotype SV 5 dominate in animals with Glasser's disease. The serotypes SV 1, SV 5, Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were non-virulent. The high specificity in AGPT was not reproducible in the more sensitive dot-blot procedure. This must be taken into account, if the dot-blot is to be used for the classification of serotypes of H. parasuis. The results point to a participation of nonimmunogenic polysaccharides in the detection reaction.     

Development of an improved species specific PCR test for detection of Haemophilus parasuis

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.     

间接血凝方法检测副猪嗜血杆菌抗体

将副猪嗜血杆菌(Hps)4、5、13型分离菌株超声产物致敏经戊二醛和鞣酸处理的绵羊红细胞,建立了检测Hps抗体的间接血凝试验方法。对15种血清型Hps阳性血清进行检测,结果均为阳性,抗体效价达1∶2⁵~1∶2¹⁰,敏感性明显高于琼脂扩散试验。对其它16种病毒和细菌的阳性血清进行检测,结果除胸膜肺炎放线杆菌有凝集外,其余均为阴性。对620份临床血清进行检测,阳性率为67.58%。结果表明,该方法敏感性较高、特异性强,可用于Hps抗体水平的检测和流行病学调查。     

华南地区两株副猪嗜血杆菌的生物学特性与致病性研究

本试验旨在对来源于广东省的两株副猪嗜血杆菌的生化特性、血清型、耐药性、生长特性以及毒力和免疫原性进行研究。试验结果表明,两株菌分别为血清4型和5型;两株菌均具有较好的生长能力,培养10 h活菌含量都能达到10⁹以上;其LD₅₀分别是1.52×10⁹ CFU和9.3×10⁸ CFU;分离菌株对喹诺酮类和磺胺类抗生素有较强的抵抗力;免疫试验结果表明,两株菌具有较好的免疫原性。以上试验结果表明,这两株菌可以作为疫苗的基础候选菌株。     

First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae of swine from European wild boars

Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the etiological agent of Gl?sser's disease, which is characterized by a fibrinous polyserositis, meningitis and arthritis. Gl?sser's disease has never been reported in wild boar (Sus scrofa), although antibodies against H. parasuis have been detected. The goal of this study was to confirm the presence of this bacterium in wild boar by bacterial isolation and to compare the strains to H. parasuis from domesticated pigs. Therefore, nasal swabs from 42 hunted wild boars were processed for bacterial isolation and subsequent H. parasuis identification by specific PCR, biochemical tests and 16S rRNA gene sequencing. Two different strains of H. parasuis from two wild boars were isolated. These strains belonged to serotype 2 and were included by 16S rRNA gene sequencing and MLST analysis in a cluster with other H. parasuis strains of nasal origin from domestic pigs. During this study, Actinobacillus minor and Actinobacillus indolicus, which are NAD-dependent Pasteurellaceae closely related to H. parasuis, were also isolated. Our results indicate similarities in the respiratory microbiota of wild boars and domestic pigs, and although H. parasuis was isolated from wild boars, more studies are needed to determine if this could be a source of H. parasuis infection for domestic pigs.     

肉鹅大肠杆菌病的病原分离与鉴定

本试验旨在研究肉鹅大肠杆菌的生化特性、药敏情况、血清型和致病性。2010年12月江苏省某肉鹅场鹅群出现呼吸困难,死亡增多现象,对病死鹅剖检发现以气囊炎和肺部坏死为主,无菌采集病料分离到一株细菌,经分离培养、染色镜检、生化特性鉴定,确定为大肠杆菌,微量凝集试验表明其血清型为O₂。经过肺结节压片镜检,没有发现霉菌的菌丝;检测健康鹅和患病鹅各10只的鹅新城疫抗体,发现其抗体滴度比较高且整齐度较好,两者之间的抗体无明显差异。对分离菌株进行药敏试验,结果表明对氟苯尼考等7种药物敏感,对先锋Ⅴ等16种药物已产生了耐药性。将分离到的菌株接种21日龄雏鹅进行动物回归试验,引起80%死亡率,说明该分离菌株对雏鹅具有较强的致病性。