胸膜肺炎放线杆菌地方分离株和参考菌株的生物被膜形成

通过玻璃试管和聚苯乙烯微孔板结晶紫染色法分别对胸膜肺炎放线杆菌(APP)的15个血清型国际参考菌株和22个中国地方分离菌株在体外形成生物被膜的能力进行了定性和定量分析.在15个血清型(无14型)的参考菌株中,只有5b、6和11型能在聚苯乙烯微孔板上形成生物被膜;而59%的地方分离株(涵盖13种血清型,无14和15型)在聚苯乙烯微孔板上表现出产生生物被膜的能力.结果表明,大多数血清型的APP能在体外形成生物被膜,尤其在地方分离株中更普遍;与低毒力血清型的菌株相比,高毒力和中等毒力血清型的菌株更倾向于形成生物被膜.     

副猪嗜血杆菌ERIC-PCR指纹图谱多样性研究

建立了副猪嗜血杆菌ERIC-PCR分型技术,并应用于临床分离菌株的流行病学调查。试验结果表明,47株副猪嗜血杆菌产生28种不同的指纹图谱,分离自同一猪场和相同血清型的菌株表现出不同的指纹图谱,不能进行血清分型的副猪嗜血杆菌应用该方法可得到充分区分,证实副猪嗜血杆菌ERIC指纹图谱存在丰富的多样性,ERIC-PCR方法是副猪嗜血杆菌流行病学规律分析的有效方法。     

副猪嗜血杆菌野外分离株的ERIC-PCR 及其外膜蛋白图谱分析

为了解副猪嗜血杆菌(HPS)现地分离株的流行情况及其进化来源,本研究采用肠杆菌科基因间重复一致序列PCR(ERIC-PCR)分型和外膜蛋白(OMP)分型技术对采集自3个猪场的24株HPS分离株进行分型.结果表明,以ERIC-PCR法分析24个分离株共产生10种DNA指纹图谱,依次分别包含5株、3株、1株、7株、1株、2株、1株、1株、2株和1株分离株.其中一个猪场的分离株仅为图谱Ⅰ和Ⅱ,另外两个猪场分别为图谱Ⅲ、Ⅳ、V和Ⅵ、Ⅶ、Ⅷ、Ⅸ、X.试检测结果表明同一猪场存在的菌株有两种或两种以上基因型,并且不同猪场流行的基因型不同.采用OMP分型也表现出与ERIC-PCR分型一致的结果.因此,ERIC-PCR和OMP分型均可以用于HPS的流行病学调查.     

Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping

OBJECTIVE: To characterize the genetic diversity of Haemophilus parasuis field isolates with regard to serovar, herd of origin, and site of isolation. SAMPLE POPULATION: Isolates of H parasuis obtained from pigs in 15 North American herds and multi-farm systems. PROCEDURE: 98 H parasuis isolates were genotyped with the enterobacterial repetitive intergeneic consensus based-polymerase chain reaction (ERIC-PCR) technique and serotyped via agar gel precipitation test. Genomic fingerprints were analyzed and dendrograms were constructed to identify strains from the same serovar group, herd of origin, or isolation site and to evaluate the genetic variability within these categories. RESULTS: Serovar 4 (39%) and nontypeable (NT) isolates (27%) were most prevalent. Thirty-four distinct strains were identified among the 98 isolates, using a 90% similarity cutoff. Strains from serovar 4 and NT isolates had high genetic diversity (12 and 18 strains, respectively). One to 3 major clusters of prevalent strains could be identified in most of the evaluated herds. Haemophilus parasuis strains isolated from the upper respiratory tract were either serovar 3 or NT isolates. Potentially virulent strains (isolated from systemic sites) were either serovars 1, 2, 4, 5, 12, 13, or 14, or NT isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Although H parasuis had high genetic diversity overall, only a few strains caused disease in these herds. The ERIC-PCR technique was more discriminative than serotyping, and a broad genetic variety was observed within particular serovar groups.     

Serological characterization of Danish Haemophilus parasuis isolates

A total of 103 Danish Haemophilus parasuis field isolates was collected from diseased pigs in connection with routine diagnostics. The isolates were serotyped using indirect haemagglutination (IHA) and for 57 of the isolates the serotyping was also performed by immunodiffusion. Serovar 5 was the most prevalent (36%), followed by serovar 4 (13%) and serovar 13 (22%), whereas 15% of the strains were nontypeable by IHA. Serovars 1, 2, 6, 7, 9, 12, 14, and 15 were only represented by a small number of isolates. Most of the Danish isolates belong to serovars, which earlier have been shown to be virulent. The strains could be divided into two groups depending on whether they were isolated from cases with systemic disease (polyserositis, arthritis or meningitis) or if they only were found in the lower respiratory tract. The most marked differences were observed for serovar 4, which had a higher prevalence in respiratory disease compared to systemic infection, and for the nontypeable isolates, which were mainly found in cases of systemic infection.     

中国东南部地区副猪嗜血杆菌分离株ERIC-PCR指纹图谱分析

采用肠杆菌基因间重复一致序列PCR方法,在对15种副猪嗜血杆菌血清型参考株鉴定获得15种不同ERIC-PCR指纹的基础上,对分离自中国东南部发生Glasser's病的不同猪场的111株副猪嗜血杆茵进行了指纹鉴定.结果显示:111株分离株显示出23种指纹图谱,前3种最流行的指纹图谱为ERIC-PCR X X(20/111),X X ⅢⅠ(9/111)和Ⅳ(8/111).且在111株分离株中,来自不同地区的分离株分别表现出不同种类的指纹图谱.该试验表明,ERIC-PCR方法可适用于对某一地区的副猪嗜血杆菌进行分子流行病学的研究和基因型的鉴定;试验结果还揭示了副猪嗜血杆茵在中国东南部地区已广泛存在并具有多样的基因型.     

Serological characterization of Haemophilus parasuis isolates from China

From September 2002 to December 2004, a total of 281 strains of Haemophilus parasuis were isolated from 17 provinces of China. All these isolates were serotyped by both the gel diffusion (GD) and the indirect haemagglutination (IHA) tests. By combining the GD and IHA results, serovar 4 (24.2%) and serovar 5 (19.2%) were the most prevalent serovars, followed by serovars 13 (12.5%), 14 (7.1%) and 12 (6.8%), while 12.1% of the isolates could not be assigned to a serovar (nontypable). A comparison of the number of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovar 4 and a significantly decreased frequency of serovar 13 among isolates from the respiratory tract of swine without polyserositis, whereas the frequency of isolation of serovars 5, 12, 14 and nontypable from swine with or without polyserositis were similar. Co-infection of H. parasuis and other bacterial agents was studied in 183 cases examined from June 2003 to December 2004. Streptococcus suis (30.6%; 56), Escherichia coli (21.9%; 40), Bordetella bronchiseptica (21.3%; 39) and Pasteurella multocida (14.2%; 26) were the bacterial agents frequently co-isolated with H. parasuis in China.     

Biofilm formation is prevalent among field isolates of Actinobacillus pleuropneumoniae

A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.     

运用免疫蛋白质组学方法筛选副猪嗜血杆菌血清4型菌株的免疫反应性蛋白

选取2株副猪嗜血杆菌血清4型菌株(A、B菌株),采用蛋白提纯和双向电泳技术获得高分辨率的蛋白胶。通过比对蛋白胶和免疫印迹膜上的蛋白反应点,并切取相应蛋白点进行质谱鉴定。根据质谱鉴定结果,菌株A和B有13个相同的蛋白点,其中8个蛋白点是新发现的具有免疫原性的蛋白点,它们分别是二氢硫辛酰胺脱氢酶(DLDH)、dppA、过氧化氢酶(CAT)、蛋白酶(P)、转酮醇酶(TK)、延长因子(EF-Ts)、周质丝氨酸蛋白水解酶(SP)、2,3-二磷酸甘油酸变位酶(BPGM)。在2株副猪嗜血杆菌血清4型菌株中发现有13个相同的免疫反应性蛋白,DLDH、dppA、CAT、P、TK、EF-Ts、SP和BPGM是新发现的免疫原性蛋白,它们作为疫苗候选抗原有待进一步研究。     

Serological characterisation of Haemophilus parasuis isolates from Australian pigs

A total of 31 isolates of Haemophilus parasuis obtained from Australian pigs were serotyped by the Kielstein-Rapp-Gabrielson scheme. The isolates were assigned to serovar 1 (1 isolate), serovar 2 (1 isolate), serovar 4 (4 isolates), serovar 5 (7 isolates), serovar 9 (2 isolates), serovar 10/7 (4 isolates), serovar 12 (1 isolate) and serovar 13 (6 isolates). The remaining 5 isolates could not be assigned to a serovar. Two different serovars (5 and 13) were detected in one herd. The only 2 isolates obtained from clinically normal pigs (from the same herd) were serovar 9. The common serovars were isolated from pigs with pneumonia as well as from pigs with conditions of the Glässer's disease type. The serological heterogeneity amongst Australian isolates of H parasuis has important implications for the use of vaccines to control Glässer's disease.     

Differences in phagocytosis susceptibility in Haemophilus parasuis strains

Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains.     

Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene

On the basis of a species-specific PCR assay, a RFLP analysis for typing of Haemophilus parasuis strains was developed and evaluated. Amplification was based on the gene tbpA, encoding a transferrin-binding protein. RFLP analysis of the 1.9-kb tbpA-amplicon using TaqI, AvaI and RsaI endonucleases produced 12 different patterns for the reference strains of the 15 known H. parasuis serovars, and showed a high heterogeneity (33 RFLP groups) for 101 H. parasuis clinical isolates tested. The sensitivity, typeability (100% versus 65% for immunodiffusion), high degree of discrimination (0.93 versus 0.84 for immunodiffusion), simplicity and low cost per test make this PCR-RFLP assay a useful method for typing H. parasuis and, therefore, for studying the epidemiology of outbreaks of Gl?sser's disease.     

副猪嗜血杆菌分离株耐药性、血清型及RAPD基因型分析

为弄清某规模化养猪场感染和流行的副猪嗜血杆菌(HPS)菌株的耐药性、血清型及基因型,本试验自该猪场发生浆膜炎和关节炎病猪的不同组织样品中分离到6株HPS菌株,通过细菌培养特性、生化试验和PCR扩增细菌16S rRNA基因片段并测序对分离株进行鉴定;进而鉴定分离株对不同类别抗生素的耐药性;最后对分离株进行血清分型和RAPD基因分型并分析两者的相关性。结果显示,自病猪不同组织脏器中分离鉴定了6株HPS菌株,体外培养具有典型卫星生长现象;生化试验结果符合HPS反应特性;PCR均可扩增出821 bp的HPS 16S rRNA基因片段,且序列与HPS一致。药敏试验结果显示分离株对头孢菌素类、青霉素类、氨基糖苷类、大环内酯类和四环素类抗生素敏感,而对喹诺酮类、磺胺类、林可霉素类和氯霉素类抗生素产生耐药性。琼脂扩散试验结果显示HPS分离株均为血清5型;RAPD分析显示HPS分离株与15个血清型参考菌株可分为4个基因型。本试验结果为HPS的流行病学调查及防控具有一定的借鉴价值。     

Prevalence of Haemophilus parasuis serovars among isolates from swine.

Two hundred sixty Haemophilus spp isolates that had been obtained from the respiratory tract and other sites of swine were acquired from diagnostic laboratories, primarily in the United States and Canada. The majority of isolates (243/260) were biochemically characterized as H parasuis; however, a few isolates of taxa distinct from H parasuis (taxa "minor group," D, E, and F) were identified. Fourteen H parasuis serovars were identified, and of those previously described, the most prevalent were 5 (24.3% of isolates), 4 (16.1%), 2 (8.2%), and 7 (3.7%). Three new serovars that were also prevalent included ND4 (11.1%), ND3 (8.6%), and ND5 (6.6%). Serovars 1, 3, 6, C, D, and new serovars ND1 and ND2 were infrequently identified, and 15.2% of isolates were nontypeable. It was not uncommon to isolate multiple serovars from swine of the same herd or related herds. Distribution of serovars among isolates from the United States and Canada was generally similar; however, a higher prevalence of serovar 5 and a lower prevalence of serovars 2, ND3, and ND5 were evident in isolates from Canada. Comparison of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovars 4 and 5, and a decreased frequency of serovar 2, among isolates from swine with polyserositis. However, all prevalent serovars were isolated from swine with polyserositis, and data were not indicative of an association between serovar, site of isolation, or pathogenic potential.     

副猪嗜血杆菌的分离鉴定及药敏试验

从病猪中分离到4株细菌,经染色镜检、生化试验以及PCR等方法鉴定为副猪嗜血杆菌,。通过药敏试验确定其对药物的敏感性,以期对临床用药有一定的指导作用。     

副猪嗜血杆菌流行优势菌株调查和耐药性分析

为调查研究河南规模化猪场副猪嗜血杆菌(HPS)流行优势菌株及其耐药性情况,2017-2018年从河南规模化猪场分离到30株HPS,根据菌株分离部位统计,肺脏、气管是HPS分离的首要组织部位,肺脏分离14株,占46.67%;气管分离11株,占36.67%。通过PCR分子血清型鉴定,河南流行的优势菌株为血清5,7,4型,其中血清型5型有9株,占30%;血清型7型有5株,占16.67%;血清型4型有4株,占13.33%。通过K-B纸片琼脂法药敏试验,30株分离菌株除对头孢噻呋100%敏感外,对其他17种常用抗生素都有不同程度的耐药现象,多重耐药现象突出,预防控制HPS病选药、用药方面更需慎重和规范。     

6株副猪嗜血杆菌基因组DNA的PCR指纹图谱研究

根据肠道菌基因闻重复一致序列，设计了一对特异性引物，采用ERIC-PCR和RAPD技术，研究了副猪嗜血杆菌6个分离菌株的指纹图谱和DNA多态性。结果表明，6个分离株的PCR指纹图谱与15个标准血清型指纹图谱相比较可分辨出4种血清型；6个分离株的RAPD研究结果均表现出多态性。有意义的是，6个菌株的多态性DNA片段也能明显将其分为4种类型的副猪嗜血杆菌，与特异性引物PCR结果相一致。该研究可作为流行病学调查和该菌的分子分型快速诊断方法的基础。     

Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis

Objective To validate a polymerase chain reaction (PCR) based method, Enterobacterial Repetitive Intergenic Consensus‐PCR (ERIC‐PCR), for the fingerprinting of Haemophilus parasuis strains and to use that method to differentiate isolates from apparently related outbreaks of Glässers disease on three pig farms. Design ERIC‐PCR was evaluated by comparing 15 different strains that represented all 15 recognised serovars in the Kielstein‐Rapp‐Gabrielson (KRG) scheme for serotyping H parasuis. Next, ERIC‐PCR was used to examine 14 Australian field isolates of H parasuis; 12 collected from three farms suffering apparently related outbreaks of Glässers disease and two from two other farms with no known connection. Results The 15 serovar reference strains all gave unique, reproducible ERIC‐PCR fingerprints. The 12 isolates from the three apparently related outbreaks all gave a single fingerprint, which was distinct from any seen in the 15 serovar reference strains and the two other Australian field isolates in the studied farms. The confirmation that all 12 isolates were the same strain allowed the development of a prevention and control program that has prevented the emergence of any further outbreaks of Glässer disease on the three farms. Conclusion ERIC‐PCR is a suitable technique for the differentiation of unrelated strains of H parasuis. The finding that the 12 field isolates of H parasuis all shared the same fingerprint is strong evidence that there was a common source of infection on all three farms. This study has shown, for the first time, that ERIC‐PCR is a suitable technique for the sub‐typing of H parasuis and useful for studying the epidemiology of outbreaks of Glässers disease.     

新疆北部部分地区副猪嗜血杆菌的ERIC-PCR指纹聚类分析

为了解新疆北部部分规模化养猪场副猪嗜血杆菌(H.parasuis)分离株的基因型,本实验采用ERIC-PCR方法结合统计学分析软件,对来源不同的12株H.parasuis进行分子指纹图谱分析.结果表明12株分离株分别位于4个聚类中,各个菌株之间的遗传距离较近,并且含有长度为1 000 bp的相同条带,相同血清型的分离株在其分子指纹聚类上均位于同一个分支中.试验结果表明基于ERIC-PCR的分子指纹聚类分析结合传统的琼脂免疫扩散法可以准确地对不同分离株H.parasuis进行分型.试验结果显示H.parasuis在该地区广泛存在并具有多种不同的基因型,同时为该地区H.parasuis的免疫防治提供了重要的实验依据.