Update on the diagnosis of Haemophilus parasuis infection in pigs and novel genotyping methods

Haemophilus parasuis causes Gl?sser's disease as well as a number of other diseases in pigs. The diagnosis of H. parasuis-associated disease is usually established by clinical signs, pathological findings and bacterial isolation but diagnosis is complicated by the existence of non-virulent strains and the early colonisation of the upper respiratory tract of healthy piglets. Moreover, several strains can be found on a farm and even within a single animal so it is important to determine the specific strain that is causing the clinical outbreak. Recently, genotyping methods have been developed with the goal of correlating genotype with the degree of virulence of H. parasuis strains. The association between genotype and virulence in H. parasuis is challenging due to the lack of knowledge of the complete genomic sequence and virulence factors of this bacterium.     

我国部分地区副猪嗜血杆菌病的流行病学调查

采用细菌分离鉴定、结合PCR方法,对江苏、上海、安徽、广西、浙江、江西、山东、河南、湖北等省市2003年11月～2005年4月送检的159个患病猪场仔猪肺脏进行了副猪嗜血杆菌检测,结果阳性猪场为76个,阳性率48%,临床症状主要为呼吸困难和体温升高;病理变化主要有肺炎病变和淋巴结肿大或出血。通过数据统计分析,发现该病已在我国不同地区广泛流行,且多发于秋、冬季节。该研究为副猪嗜血杆菌的流行病学研究奠定了基础,也为该病的临床预防提供了理论依据。     

Vaccination of gnotobiotic primary specific pathogen-free pigs against Haemophilus parasuis.

Three trials were conducted to establish if young primary specific pathogen free (SPF) pigs could be protected from Glasser's disease by vaccination. Three age groups of cesarean-derived isolator-reared gnotobiotic pigs were vaccinated twice at 4 and 6, 3 and 5, and 2 and 4 wk of age respectively with a formalin killed aluminum hydroxide adsorbed bacterin prepared from three strains of Haemophilus parasuis isolated from Ontario pigs affected with Glasser's disease. When challenged two weeks later with the homologous strains of virulent bacteria, all the vaccinated pigs remained healthy, while 17/18 nonvaccinated pigs became severely sick or died between three and seven days postchallenge. The one surviving nonimmunized pig was retarded in growth. All of the nonimmunized pigs had visible lesions of polyserositis, the most common being polyarthritis (14/18). Other lesions were fibrinous meningitis, pericarditis, pleurisy and/or peritonitis. Two of the pigs died with a septicemia. Haemophilus parasuis was isolated from 15/18 nonimmunized pigs, usually from several of the affected sites. The organisms were not isolated from the immunized pigs, nor from the surviving nonimmunized pig. Attempts to detect the presence of specific antibodies against the H. parasuis strains in the sera of the immunized or exposed pigs by the passive hemagglutination test or by enzyme linked immunoassay were unsuccessful. The results of this work indicate that primary SPF pigs can be protected from Glasser's disease by vaccination as early as 2 and 4 wk of age. The nature of this protective mechanism was not established in this study.     

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.     

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.     

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).     

Preliminary assessment of a Haemophilus parasuis bacterin for use in specific pathogen free swine.

A whole cell formalin killed trivalent Haemophilus parasuis bacterin was tested for efficacy in four week old, weaned specific pathogen free pigs challenged under laboratory conditions. The vaccine contained three field strains of H. parasuis selected from confirmed cases of Glasser's disease. Two different formulations were evaluated in separate trials. In trial 1, ten pigs received 5 mL of bacterin subcutaneously in the neck, followed by a second 5 mL dose two weeks later. Another ten pigs served as nonvaccinated controls. One week after the second dose, all pigs were subjected to an aerosol challenge containing the strains of H. parasuis present in the vaccine. In trial 2, a broth rather than a saline based vaccine was prepared, and tested as in trial 1. In both trials, the vaccinated pigs remained healthy postchallenge, while eight of nine (Trial 1) and eight of ten (Trial 2) nonvaccinated pigs succumbed to Glasser's disease.     

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.     

某规模化猪场猪伪狂犬病的诊断

试验采用流行病学调查、临床观察和实验室检查对华北地区某疑似伪狂犬病发病猪场进行了诊断。猪场发病情况主要为母猪流产,新生仔猪死亡率较高,肥猪死亡率低。发病或死亡仔猪扁桃体充血出血、溃疡,肺脏出血、水肿,心肌色淡、心包积液,脑膜充血;表现为坏死性扁桃体炎、出血性淋巴结炎、出血性间质性肺炎、间质性心肌炎和非化脓性脑炎。发病猪扁桃体、肺脏、脑组织匀浆上清液接种家兔后出现奇痒等典型症状并很快死亡。发病猪脑组织可检出猪伪狂犬病毒gD基因特异性目的片段。猪场伪狂犬病野毒感染抗体阳性率为63%（63/100）。本试验结果表明,确诊该猪场发生了伪狂犬病。     

Glasser's disease and prevalence of subclinical infection with Haemophilus parasuis in swine in southern Ontario

Glasser's disease accounted for less than 1% of total swine mortalities in an 11 year retrospective postmortem survey of swine submissions at three provincial government diagnostic laboratories in southern Ontario. However, Glasser's disease was suspected in 17 of 83 boar mortalities at the Record of Production Boar Test Station between 1983 and 1985 and was much more common in specific-pathogen-free (SPF) boars than in conventional boars. The prevalence of the causative organism, Haemophilus parasuis, was determined for 19 SPF herds in Ontario classified as "Excellent" under the Ontario Swine Herd Health Policy. Nasal swabs from two-month-old pigs were cultured on chocolate agar containing 1.5 mug/mL lincomycin, 5 mug/mL bacitracin, and 0.1 mug/mL crystal violet. Three herds were negative for H. parasuis infection; 16 herds contained clinically healthy carrier pigs.     

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.     

Isolation and Identification of Haemophilus parasuis SD02 strain

A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province,and identified through morphological observation,culture traits,biochemical characteristics and PCR amplification.Additionally,primers were de-signed according to the 16S rRNA sequence of Haemophilus parasuis,and the bacterial strain was amplified by PCR.The amplified fragments of approximately 1 400 bp was sequenced,and aligned with the sequence in Gen Bank.The results showed that it shared the homology of 97%-99%with the 16S rRNA sequence of foreign H.parasuis,and confirmed as H.parasuis(HPS).The strain was determined as serotype 4 through serotype identification.The strain was named SD02.     

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.     

An investigation of enzootic Glasser's disease in a specific-pathogen-free grower-finisher facility using restriction endonuclease analysis

Enzootic Glassers's disease was investigated to study the epidemiology of the disease strains on a farm where it presented a problem. Restriction endonuclease fingerprinting (REF) analysis technique was used, as all strains of Haemophilus parasuis are biochemically similar and many strains are biochemically untypable. After young weaned pigs were moved from farm A to farm B, Glasser's disease routinely occurred despite the use of antibiotics and a commercial bacterin. Isolates were taken from the nasal passages and from carcasses of clinically affected cases and subjected to REF analysis. Haemophilus parasuis was not isolated from any of the pigs on farm A, but it was isolated from 7/10 and 5/10 nasal swabs taken from farm B. Two H. parasuis strains isolated from clinical cases of Glasser's disease from farm B had an identical REF pattern, but were different from the nasal swabs and the H. parasuis strain contained in the bacterin. The subsequent use of a custom autogenous bacterin made from a clinical isolate of H. parasuis reduced the mortality rate on farm B. This investigation indicates that nasal isolates of H. parasuis are different than those causing clinical disease, and not all bacterin strains are cross protective for other strains.     

Naturally-farrowed, artificially-reared pigs as an alternative model for experimental infection by Haemophilus parasuis

The use of naturally-farrowed, artificially-reared piglets as an alternative model to study Haemophilus parasuis infections was evaluated. Two trials were performed in order to evaluate the proposed model. In trial 1, animals were vaccinated and challenged with H. parasuis. Results showed that the proposed model was effectively used to evaluate protective immunity against this organism. In trial 2, animals were challenged with different doses of H. parasuis. Results showed that the severity of clinical signs and lesions tended to increase with higher doses. The reproduction of clinical signs and lesions characteristic of H. parasuis systemic infection was successful in both trials, proving that this model is a viable alternative to specific-pathogen free and cesarean-derived, colostrum-deprived pigs.     

Development of a PCR test to diagnose Haemophilus parasuis infections.

A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.     

Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS)

The lymphoid, renal, pulmonary, and hepatic lesions of naturally occurring postweaning multisystemic wasting syndrome (PMWS) affected pigs have been studied by means of immunohistology. Ten conventionally reared pigs showing acute clinical signs of PMWS were selected from a farm on which animal were seronegative to porcine reproductive and respiratory virus and to Aujeszky's disease virus. All pigs were positive in tests for porcine circovirus type 2 by ISH and IHC. Monoclonal and polyclonal antibodies to CD3, CD79alpha, CD45RA (3C3/9), lysozyme, SLA-II-DQ (BL2H5), and MAC387 were used to characterise cells in PMWS lesions. The most relevant changes were reduction or loss of B and T lymphocytes, increased numbers of macrophages, and partial loss and redistribution of antigen presenting cells throughout lymphoid tissues compared to uninfected controls. The characteristics of lymphoid lesions in the present study strongly suggest an immunosuppressive effect of PMWS in affected pigs.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.     

副猪嗜血杆菌分离鉴定及药敏试验

副猪嗜血杆菌能够引起猪的多发性浆膜炎、关节炎和脑膜炎等,是影响猪的最重要细菌之一,目前在所有的主要养猪国家均有存在。为了弄清河南省副猪嗜血杆菌病流行情况,2012年-2015年,从河南不同地区猪场送检的疑似病料,进行副猪嗜血杆菌分离和鉴定,共分离到5株细菌,通过细菌形态观察、培养特征鉴定、生化试验、PCR检测,鉴定为副猪嗜血杆菌,分别命名为A6-fei、C3-xin、C12-xin、D2-fei和E1-fei。采用纸片扩散法,对分离5株副猪嗜血杆菌进行药敏试验,其结果表明所分离的5株副猪嗜血杆菌的药物敏感性不尽相同,各分离菌株对头孢噻肟、氟苯尼考星最敏感,对复方新诺明敏感性最差,其中菌株C3-xin对复方新诺明、庆大霉素、卡那霉素、青霉素完全耐药。表明副猪嗜血杆菌病在河南省依然存在,并且不同地区菌株对常用药物的敏感性各不相同,应当引起养猪场重视。     

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.