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.     

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.     

Serological classification of Australian isolates of Haemophilus paragallinarum

Thirty-nine Australian isolates of Haemophilus paragallinarum were compared serologically with 3 reference serotype strains of H. paragallinarum using a plate agglutination test. Twenty-eight of the isolates were serotype C, 5 were serotype A, while the remaining 6 isolates could not be assigned to a serotype.     

Serological characterisation of Australian isolates of Actinobacillus pleuropneumoniae

SUMMARY: Using hyperimmune rabbit antiserums to 8 reference serovar strains, a total of 51 Australian isolates of Aclinobacillus pleuropneumoniae were serotyped by either a rapid slide agglutination test or a gel diffusion test. The results were: serovar 1 — 24 isolates; serovar 2 — 6 isolates; serovar 3 — 5 isolates; serovar 7 — 15 isolates; nontypable — 1 isolate. The rapid slide agglutination test was suitable for screening field isolates, as 73% of those tested reacted with only 1 of the 8 antiserums and were assigned to a particular serovar of the basis of this test alone.     

Serovar profiling of Haemophilus parasuis on Australian farms by sampling live pigs

Objective Investigate the diversity of serovars of Haemophilus parasuis (Hps) present in Australian pig herds. Design Nasal swabs were used to obtain multiple isolates of Hps, which were grouped first by genotyping and then by serotyping representative isolates. Procedure Swabs were taken from the nasal cavity of just-weaned healthy pigs from multiparous sows on 12 farms and from post-weaned pigs of multiparous sows on 1 farm. On 5 of the 13 farms, nasal swabs were also obtained from pigs showing clinical signs suggestive of Glässer's disease. On a further 7 farms, nasal swabs were obtained only from pigs with clinical signs suggestive of Glässer's disease. Results A total of 556 Hps isolates were genotyped, and 150 isolates were serotyped. Hps was detected on 19 of the 20 farms, including 2 farms with a long history of freedom from Glässer's disease. Isolates of Hps belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glässer's disease outbreaks. Sampling 213 sick pigs yielded 115 isolates of Hps, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these 213 sick pigs were of a serovar known to be non-pathogenic. Conclusion Healthy pigs contain a range of Hps serovars, even on farms free of Glässer's disease. Nasal swabbing of both healthy and sick pigs seems a useful method of serovar profiling of farms.     

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.     

Antimicrobial susceptibility and PFGE genotyping of Haemophilus parasuis isolates from pigs in South China (2008-2010)

H. parasuis isolates (n=112) from pigs were tested for antimicrobial susceptibility against 23 antimicrobial agents by the disk diffusion method. All isolates were sensitive to Florfenico and most strains were sensitive to Cefotaxime (103/112; 92%), Ceftazidime (99/112; 88.4%), Chloramphenicol (90/112; 80.4%) and Gentamicin (85/112; 75.9%). High resistance levels to Nalidixic acid (84.8%), TMP (67.9%) and Trimethoprim+Sulfamethoxazole (58%) were observed. Genomic DNA extracted from 52 isolates resistant to at least seven antimicrobial agents was analyzed by PFGE and 46 distinct PFGE patterns identified. Diverse variation was observed between the drug-resistant H. parasuis isolates examined, suggesting that resistance traits were acquired independently by the respective isolates.     

豚鼠副猪嗜血杆菌感染动物模型的建立

为建立副猪嗜血杆菌(Hps)的感染动物模型,本试验用Hps血清5型标准菌株(Nagasaki),以2.0×10~9CFU剂量腹腔感染豚鼠,观察豚鼠发病及死亡情况.取死亡豚鼠的主要器官组织,观察其病理和组织病理变化,与猪Classer's病痛变进行比较.并同时对死亡豚鼠进行细茵分离,分离菌经PCR鉴定.实验结果显示:在接种14 h后试验组豚鼠(5/8)出现死亡,死亡豚鼠剖检时出现了与猪Classer's病相似的病变;主要组织器官组织学变化以炎性细胞浸润、纤维蛋白和红细胞渗出等变化;并通过细茵分离培养,在豚鼠大脑、心血、肺、肝、脾和肾主要器官中分离到Hps血清5型茵.实验结果表明豚鼠可以作为Hps的感染动物模型.这一结果为研究其致病机制、诊断和免疫研究奠定基础.     

Differences in susceptibility to Haemophilus parasuis infection in pigs

In animal breeding programs, deoxyribonucleic acid (DNA) markers can be used to identify sires that are less susceptible to disease. These DNA markers are typically discovered in populations that display differences in susceptibility. To find those differences, it was hypothesized that sires influence their offspring responses to infection with H. parasuis. To identify differences in susceptibility, colostrum-deprived pigs derived from 6 sires were inoculated with a virulent strain of H. parasuis serovar 5. Pigs were infected at 21-d of age and euthanized 1, 2, or 3 days post-infection. Rectal temperatures, bacterial detection, clinical signs, and lesions were measured by comparing disease susceptibility in the offspring from each sire. The effect of the sire on the severity of disease in the offspring was statistically analyzed using to a 2-way ANOVA with sire and test day as fixed effects. Significant differences among sires were found for lesions, rectal temperatures from days 0-1 and 0-2 (P < 0.05) and marginal effects for clinical signs (P = 0.08). On average, the offspring of sire H94 was the most susceptible to challenge. Responses to infection were categorized to determine the clinical responses and analyzed by Chi square. Overall, 10% of all pigs infected were fully resistant to H. parasuis infection. Boar H94 didn't produce any fully resistant offspring. Differences in susceptibility to H. parasuis were observed, and the results support the hypothesis that sires influence their offspring's response to infection. Tissues from this population could be used to identify DNA markers for genetic selection of sires that produce offspring more resistant to H. parasuis 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.     

Prevalence and characterization of genotypic diversity of Haemophilus parasuis isolates from southern China

From September 2008 to December 2010, 112 Haemophilus parasuis strains were isolated from 536 pigs with clinical signs of Glässer’s disease in South China, for a frequency of 21%. The 112 strains were subjected to serovar analysis by gel diffusion (GD) and indirect hemagglutination (IHA) tests and to genotype analysis by means of pulsed-field gel electrophoresis (PFGE). With a combination of the GD and IHA results, serovars 5 and 4 were found to be the most prevalent, at 23% and 17%, respectively, followed by serovars 2 (8%), 15 (7%), 13 (6%), and 12 (5%); 20% of the strains were nontypeable. The 112 strains were genetically diverse, with 85 genotypes identified (discriminatory index 0.992). The 89 typeable isolates belonged to 15 H. parasuis serovars displaying 63 different PFGE profiles. The 23 nontypeable strains displayed 22 different PFGE profiles. These findings confirmed that 15 serovars and diverse genotypes of H. parasuis were widely distributed in southern China.     

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.     

副猪嗜血杆菌对豚鼠和小鼠的致病力研究

为了验证副猪嗜血杆菌分离株FS0307株、XX0306株和标准株HS1079株的致病力,本文以豚鼠和Balb/c小鼠作为模型动物,分别腹腔接种副猪嗜血杆菌FS0307株、XX0306株和HS1079株,观察临床症状和死亡情况,并进行Hps的再分离和血清抗体检测。结果显示,2株副猪嗜血杆菌分离株均能引起豚鼠和Balb/c小鼠100%发病,以及对豚鼠和小鼠的致死率均分别达到100%和20%;标准株HS1079株能引起豚鼠和Balb/c小鼠100%发病,并对豚鼠有80%的致死率,而不致死小鼠。可见,副猪嗜血杆菌FS0307株、XX0306株和HS1079株的致病力均较强,这为副猪嗜血杆菌病疫苗的研究提供了可靠的技术参数。     

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.     

Analysis of Haemophilus parasuis isolates from southern Ontario swine by restriction endonuclease fingerprinting.

To study the occurrence and distribution of various strains of Haemophilus parasuis in southern Ontario swine, organisms isolated from healthy swine, from specific pathogen-free and conventional herds, and from disease cases were examined using restriction endonuclease fingerprinting analysis. In most herds, several strains of H. parasuis could be detected although one or two strains usually predominated. Individual animals were colonized by a single or limited number of strains. In several cases, the same strains were isolated from more than one specific pathogen-free herd. Conventional herds carried a more heterogeneous population of H. parasuis. Only one strain was isolated which was common to more than one conventional herd. No strains were isolated which were common to both specific pathogen-free and conventional herds. None of the strains isolated from disease cases were found in healthy conventional or specific pathogen-free swine examined in this study.     

Biofilm formation by field isolates and reference strains of Haemophilus parasuis

The ability to form biofilms for a total of 80 field isolates and 15 reference strains of Haemophilus parasuis, the etiological agent of Glasser's disease, was tested by glass tube and polystyrene microtiter plate assays. A total 43% of field isolates, including strains representing 13 serovars (except serovars 3 and 8) and non-typable strains, exhibited the ability to form biofilms at different levels via polystyrene microtiter plate assays. Among the reference strains representing 15 serovars, only serovars 2, 9, 12, 13 and 15 could not form biofilms on the polystyrene surface. A total of 85% of the strains forming biofilms at air-liquid interfaces in glass tubes also formed biofilms on polystyrene surfaces. Generally, non-virulent serovars showed a higher degree of biofilm formation than virulent serovars. The biofilm formation phenotype of most strains was maintained when cultures were passaged on agar and in broth. H. parasuis from the nasal cavities of pigs experimentally infected with biofilm-positive bacteria maintained the biofilm formation phenotype, whereas bacteria recovered from the lung and brain lost the ability to form biofilms. The biofilm-negative strains did not recover the ability to form biofilms via experimental infection. Our data indicate that most serovars of H. parasuis could form biofilms in vitro, and the biofilm formation phenotype is associated with the recovery site of the strains and is maintained when bacteria are passaged in vitro and in the upper respiratory tract.