Serum-resistance in Haemophilus parasuis is associated with systemic disease in swine

Haemophilus parasuis can cause pneumonia and systemic disease in swine but it is also a coloniser of the upper respiratory tract of healthy pigs. These differences in pathogenicity are probably the result of diverse mechanisms of virulence in different strains. Since serum-resistance is a feature frequently found in systemic pathogens, 31 H. parasuis strains of different clinical origin were tested and a variety of serum susceptibility levels detected. Nasal strains from healthy piglets were sensitive to the bactericidal effect of the serum, while systemic strains were mainly resistant. The pulmonary strains included both serum-sensitive and serum-resistant strains. Interestingly, the serum-resistant pulmonary strains were isolated from animals with systemic lesions. Heat-treatment of the sera abolished the bactericidal activity, indicating that complement is a key factor in this effect. Equivalent susceptibility was observed with rabbit and porcine sera, and the presence of H. parasuis specific antibodies did not increase the killing of the strains by serum. In an attempt to associate serum-resistance to a surface determinant of the bacteria, agglutination in acriflavine was tested but no direct link with serum susceptibility was found. The results indicate that serum-resistance is a virulence mechanism in H. parasuis.     

Comparative virulence of Haemophilus parasuis serovars 1 to 7 in guinea pigs.

Reference strains for Haemophilus parasuis serovars 1 to 7 were examined for virulence by inoculation of guinea pigs. Guinea pig response to intraperitoneal inoculation was similar for the 7 reference strains. However, apparent differences in virulence were detected after intratracheal inoculation. Cells of the references strains for serovars 1 and 5 were most invasive, causing moribundity or death at higher doses and a persistent septicemia at lower doses. Haemophilus parasuis could be isolated from respiratory and systemic sites; purulent bronchopneumonia, pericarditis, and pleuritis were apparent in infected guinea pigs. Inoculation of cells of the reference strains for serovars 2 and 6 also resulted in bronchopneumonia and moribundity or death in some guinea pigs; however, reisolation of H parasuis and microscopic lesions at necropsy were less pronounced than those observed with serovars 1 and 5. Inoculation of cells of serovars 3, 4 and 7 induced only transient clinical signs and minimal evidence of H parasuis infection at necropsy. The data from intratracheal inoculation of guinea pigs are similar to data from other investigations in swine, indicating differences in the pathogenic potential of H parasuis strains. Thus, guinea pigs may be useful as a laboratory animal model for examining cellular factors associated with virulence and immunogenicity of H parasuis.     

Haemophilus parasuis exhibits IgA protease activity but lacks homologs of the IgA protease genes of Haemophilus influenzae

Haemophilus parasuis, the bacterium responsible for Gl?sser's disease, is a pathogen of significant concern in modern high-health swine production systems but there is little information regarding the identity or function of its virulence factors. Several important human mucosal pathogens, including the closely related bacterium Haemophilus influenzae, utilize IgA proteases to aid in defeating the host immune response and facilitate disease but it is unknown whether H. parasuis synthesizes any product with IgA protease activity. To investigate potential virulence mechanisms of H. parasuis, we evaluated five strains for their ability to digest purified IgA. Western blotting demonstrated cleavage of swine IgA, but not human IgA1, following incubation with culture supernatants from three strains, two of which are known to cause invasive disease. No genes with homology to the H. influenzae IgA protease genes iga and igaB could be identified in any H. parasuis strain using either PCR or Southern blotting. These results demonstrate that a novel IgA protease produced by some strains of H. parasuis cleaves the swine IgA heavy chain at a site not found in human IgA1.     

Effect of marbofloxacin on Haemophilus parasuis nasal carriage

Haemophilus parasuis is a colonizer of the upper respiratory tract and the causative agent of Gl?sser's disease in swine. This study focused on the nasal carriage of H. parasuis after treatment with marbofloxacin. Three marbofloxacin treatments (three doses of 2mg/kg body weight [bw] every 24h, two doses of 4 mg/kg bw every 48 h and 8 mg/kg bw in one single shot) were used and all of them reduce significantly (p<0.05) the nasal carriage of H. parasuis as compared to control animals. Moreover, H. parasuis was not detected in the nasal cavities of piglets after administering the highest dose. The effect of a dose of 8 mg marbofloxacin/kg bw in one shot was further studied in a farm with clinical cases of Gl?sser's disease using a longitudinal study. Statistically significant reduction of nasal carriage of H. parasuis was detected during the first week after treatment in comparison with the control group. However, a clear relationship between the minimum inhibitory concentration (MIC) of the different strains, their putative virulence or the treatment group (antibiotic or control) from which they were isolated was not detected. Finally, the effect induced by the antibiotic treatment on the bacterial strains seemed to be transitory, since diverse H. parasuis strains (with high and low marbofloxacin MICs) were observed 7 days after finishing the treatment.     

Haemophilus parasuis: new trends on diagnosis, epidemiology and control

Haemophilus parasuis is a commensal organism of the upper respiratory tract of conventional pigs, but under appropriate conditions can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. Factors involved in systemic invasion by H. parasuis remain largely unknown. However, major advances in our knowledge of H. parasuis include (1) development of a species-specific PCR test to detect H. parasuis in clinical samples, (2) study of molecular epidemiology within and between herds, by use of a repetitive element-based PCR, (3) the proposal of an alternative serotyping technique, (4) development and testing of a new in vivo model for pathogenesis and virulence studies, and (5) use of controlled exposure of young pigs to low doses of live, virulent H. parasuis strains to reduce nursery mortality in affected swine herds.     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

Dynamics of Haemophilus parasuis genotypes in a farm recovered from an outbreak of Glässer's disease

Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs, although it is better known as the etiological agent of Gl?sser's disease. Interestingly, several strains can be isolated from a single farm, as determined by both genotyping and serotyping. However, it is not known how an outbreak and the subsequent treatment affect the population of H. parasuis strains. In this study, a farm was studied during an outbreak of Gl?sser's disease and 1 year after antimicrobial treatment and elimination of clinical signs. Bacterial isolation was attempted from nasal swabs and lesions. After isolation, antimicrobial susceptibility, serotype and genotype were determined. Two different genotyping techniques, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST) were used. The H. parasuis strain that was isolated from lesions during the disease outbreak clustered with other virulent strains by both MLST and serotyping analysis. Nasal isolates were included in the corresponding nasal cluster by MLST, but they presented high variability by serotyping. These nasal isolates included serotypes previously classified as virulent and non-virulent. Finally, we found that during the antimicrobial treatment the diversity of strains isolated in the farm was affected and just one strain, which was resistant to the treatment, was detected. One year after the treatment strain diversity was back to normal (three strains).     

副猪嗜血杆菌潜在毒力因子的综述

副猪嗜血杆菌是引起猪格氏病的病原,多继发感染引起全身性疾病,其高发病率和死亡率给养殖业造成严重损失。作者主要对其潜在毒力因子,包括外膜蛋白ompP5和ompP2、自转运蛋白vtaA、转铁蛋白、6-磷酸葡萄糖酸脱氢酶和毒素等的研究作一综述,并对应用于副猪嗜血杆菌的最新研究方法进行概述,从而形成对其毒力相关研究较全面的认识。     

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.     

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.     

Purification and renaturation of membrane neuraminidase from Haemophilus parasuis

Haemophilus parasuis, which causes polyserositis, polysynovitis, meningitis, septicemia, and pneumonia in pigs, has emerged as an increasing problem in modern swine production systems. Co-factors for and the pathogenesis of H. parasuis disease are not defined. One of the potential virulence factors of H. parasuis is its neuraminidase (sialidase). While purifying the H. parasuis neuraminidase from the membrane fraction, we developed a protocol to renature enzymatic activity after enzyme preparations were resolved electrophorectically in denaturing polyacrylamide gels. The H. parasuis neuraminidase co-resolved with recombinant neuraminidase of Vibrio cholera; thus its apparent molecular mass is 82 kilodalton (kDa). The H. parasuis neuraminidase was associated with the membrane fraction and the purification protocol removed over 99% of the H. parasuis cell protein while retaining over 90% of the neuraminidase activity. Purified protein will provide another avenue to clone the neuraminidase gene that has been refractory to cloning and the protocol will be a means to purify recombinant protein.     

Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE

Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5 (17.4%), 14 (8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil.     

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.     

Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs.

In an attempt to establish if cross protection can be induced by different strains of Haemophilus parasuis, three groups of 12 gnotobiotic pigs were immunized each with an aluminum hydroxide adsorbed whole cell bacterin of one of three H. parasuis strains. Two weeks later, four pigs within each vaccinated group were challenged with aerosols of live cultures of each of the three test strains and observed for response. Two virulent strains V1 and V2 protected all the vaccinated pigs, while all nonvaccinated controls succumbed to Glasser's disease when challenged with these strains. Vaccination with strain LV (of low virulence) protected the pigs against challenge with strain V2, but not against strain V1. Strain LV did not cause disease in the immunized animals and only in one of ten nonimmunized pigs upon second challenge. The results suggest that strains may differ in antigenicity and that virulence and immunoprotection are positively related. Strains to be used in commercial vaccines should therefore be selected carefully. Antibodies detected in the sera of vaccinated pigs were to outer membrane proteins of the bacteria, but not to lipopolysaccharides or capsular polysaccharides. This would suggest that for gnotobiotic pigs outer membrane proteins are more immunogenic than lipopolysaccharide or capsular antigens. Further work is needed to determine if outer membrane proteins also contribute protective immunogens.     

Genotyping of Haemophilus parasuis isolated from northwest China using PCR-RFLP based on the ompA gene

Outer membrane proteins (OMPs) are the major virulent factors of Haemophilus parasuis. PCR-RFLP targeting the ompA gene was conducted to investigate the possibility of genotyping H. parasuis in this study. Fifteen reference strains and 49 isolates from pig farms in northwest China were genotyped by PCR-RFLP with a pair of specific primers. The results indicated that both the 15 reference strains and 49 isolates could be classified into 8 different genotypes by PCR-RFLP, respectively. Seven genotypes including AA, BB, BA, CA, BC, BD and CD existed simultaneously in the reference strains and isolates, but genotype CB only existed in the isolated strains. Interestingly, genotypes BA, CD and CA were only found in diseased pigs and accounted for 38.8%, 22.4% and 18.4% of the isolates, respectively. On the other hand, strains isolated from apparently healthy pigs were classified into genotypes AA, BB, BC and CB. However, the virulent reference serovar 1 strain has an AA genotype, and the fact that nearly all strains from the healthy pigs belonged to serovars classed as virulent suggests that these genotypes might also include virulent strains; therefore, further validation with more field strains is needed. The capability of the RFLP-PCR method based on the ompA gene for genotyping H. parasuis isolates indicates that this method may be a useful tool for epidemiological study.     

副猪嗜血杆菌rfaD基因的克隆及原核表达

rfaD基因编码ADP-L-甘油-D-甘露庚糖-6-异构体酶,缺失该基因会导致LOS糖链缩短和疏水性增强,从而影响细菌的致病性。为进一步探索副猪嗜血杆菌(Haemophilus parasuis,Hps)ADP-L-甘油-D-甘露庚糖-6-异构体酶的功能,本研究对Hps SC096 株rfaD基因进行克隆及原核表达。根据GenBank上登录的NC_011852序列,设计引物扩增rfaD基因,获得927 bp目的片段,将其克隆至pMD19-T载体。经送样测序鉴定正确后,连接到pET-32a(+)上进行原核表达,并用IPTG诱导,将诱导产物进行SDS-PAGE和Western blotting分析。SDS-PAGE结果显示,H.parasuis rfaD基因能在E.coli BL21(DE3)中表达,重组蛋白分子质量约为50 ku,与预期分子质量大小一致。Western blotting分析结果表明,该蛋白质能与H.parasuis血清4型阳性高免血清产生特异性结合反应,具较好的反应原性。     

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.     

广西副猪嗜血杆菌病流行病学调查

本研究于2009年5月至2010年11月调查了广西南宁、桂林、玉林、钦州4个市60个猪场发生副猪嗜血杆菌病的情况。采集病猪组织样品共86份进行副猪嗜血杆菌分离;对疑似菌株进行形态学观察、培养特性、生化特性和PCR鉴定;最终分离鉴定到26株副猪嗜血杆菌,分离率为30.2%;对分离菌株进行血清型鉴定、致病性和药敏试验。结果表明26株分离株中血清4型有5株,5型3株,9、11、13、14、15型各1株,有1株与2、9、10、11型血清均有凝集,其余12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白鼠全部死亡,其他菌株对小白鼠致病性不强。药敏试验结果表明70%以上的菌株除对恩诺沙星和氟苯尼考高度敏感外,对其他药物敏感性不高。本调查结果将对广西副猪嗜血杆菌病的防治提供指导。     

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.