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.     

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.     

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.     

Comparison between Haemophilus parasuis infection in colostrums-deprived and sow-reared piglets

The aim of this study was to compare the development of Glasser's disease in sow-reared and colostrum-deprived piglets. Ninety piglets from a commercial pig farm in Spain were used. The farm was positive for Haemophilus parasuis. Fifty-two pigs were sow-reared (SR) and 38 were colostrum-deprived (CD) piglets. The animals were intratracheally inoculated with H. parasuis serovar 5 and sacrificed at 1, 2 and 3 days post-infection. To assess the development of disease, antibody titers, clinical signs, pathological lesions, microbiological isolation and PCR amplification were compared between the groups. Inoculation of SR pigs did not cause clinical signs or lesions of Glasser's disease. In SR pigs, H. parasuis isolation and specific PCR amplification from tissues showed a very low number of positive samples. In contrast, in CD pigs, inoculation resulted in the typical signs and lesions of Glasser's disease. Positive microbiological isolation and specific PCR products were obtained from the majority of the tissues tested, and no antibodies against H. parasuis were detected. The experimental infection using CD pigs describes a successful method to study this microorganism and confirms the important role that maternal antibodies play in protection against clinical signs and disease.     

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.     

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.     

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.     

Protection of vaccinated pigs against experimental infections with homologous and heterologous Haemophilus parasuis

The efficacy of a new Haemophilus parasuis vaccine for pigs was investigated. The vaccine contains H parasuis serotype 5 cells and is adjuvanted with Diluvac Forte (Intervet). Groups of pigs were vaccinated at five and seven weeks with 2 ml and their littermates served as unvaccinated controls. The vaccinated pigs were protected against a challenge with another strain of Hparasuis serotype 5 at two, eight and 17 weeks after the second vaccination, whereas the controls became very ill. The susceptibility of the pigs to the infection decreased with increasing age. After a heterologous challenge with H parasuis serotypes 1, 12, 13 and 14, two weeks after the second vaccination, the vaccine also gave clear protection. The severity of the illness among the control pigs differed with the different serotypes.     

Prior infection with Bordetella bronchiseptica increases nasal colonization by Haemophilus parasuis in swine

The objective of this study was to determine whether Bordetella bronchiseptica would predispose to colonization or disease with Haemophilus parasuis. Three experiments were completed. In the first experiment, three groups of pigs (10 pigs/group) were inoculated intranasally with either B. bronchiseptica, H. parasuis, or with B. bronchiseptica followed by H. parasuis 1 week later. A fourth group of 10 pigs served as a non-infected control group. The second experiment was like the first, except that there were only five pigs per experimental group. The third experiment consisted of only two groups (10 pigs/group), one of which was inoculated intranasally with H. parasuis, whereas the other was inoculated with B. bronchiseptica followed by H. parasuis 1 week later. Pigs were necropsied 1-2 weeks after inoculation with H. parasuis. Mean nasal colonization by H. parasuis was significantly higher in the coinfected groups compared to the groups infected with H. parasuis alone. Pneumonia was present in 9/25 pigs coinfected with B. bronchiseptica and H. parasuis, 5/25 pigs infected with H. parasuis alone, 1/15 pigs infected with B. bronchiseptica alone, and in none of the pigs in the non-inoculated groups. Thus, B. bronchiseptica increased colonization of the upper respiratory tract with H. parasuis.     

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.     

Immunological Identification and Characterization of Extracellular Serine Protease-Like Protein Encoded in a Putative espP2 Gene of Haemophilus parasuis

Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESP-like protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 10(9) bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection.     

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.     

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.     

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.     

Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP+EF+) was compared with the efficacy of a vaccine containing formalin-killed bacterin of S. suis serotype 2 (MRP+EF+). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [WO] and an aluminium hydroxide-based adjuvant [AH]) and their side effects were also studied. The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and six weeks of age and challenged intravenously with virulent S. suis serotype 2 strains (MRP+EF+) at eight weeks of age. At challenge, the pigs vaccinated with MRP+EF/WO had high anti-MRP and anti-EF titres and were protected as effectively as pigs vaccinated with WO-formulated vaccines with bacterin. Eight of the nine pigs survived the challenge and almost no clinical signs of disease were observed. The titres obtained with the MRP+EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP or EF were less well protected; three of the four pigs died after challenge but the clinical signs of disease were significantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of the bacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis and arthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRP and EF, formulated with the WO adjuvant, protected pigs against challenge with virulent S. suis type 2 strains.     

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.     

Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype

The efficacy of a commercial swine influenza vaccine based on A/New Jersey/8/76 (H1N1) and A/Port Chalmers/1/73 (H3N2) strains was tested against challenge with an H1N2 swine influenza virus. Influenza virus-seronegative pigs were vaccinated twice with the vaccine when they were four and eight weeks old, or with the same vaccine supplemented with an H1N2 component. Control pigs were left unvaccinated. Three weeks after the second vaccination, all the pigs were challenged intratracheally with the swine influenza strain Sw/Gent/7625/99 (H1N2). The commercial vaccine induced cross-reactive antibodies to H1N2, as detected by the virus neutralisation (VN) assay, but VN antibody titres were 18 times lower than in the pigs vaccinated with the H1N2-supplemented vaccine. The challenge produced severe respiratory signs in nine of 10 unvaccinated control pigs, which developed high H1N2 virus titres in the lungs 24 and 72 hours after the challenge. Vaccination with the commercial vaccine resulted in milder respiratory signs, but H1N2 virus replication was not prevented. Mean virus titres in the pigs vaccinated with the commercial vaccine were 1-5 log10 lower than in the controls at 24 hours but no different at 72 hours. In contrast, the H1N2-supplemented vaccine prevented respiratory disease in most pigs. There was a 4-5 log10 reduction in the mean virus titre at 24 hours in the pigs vaccinated with this vaccine, and no detectable virus replication at 72 hours. These data indicate that the commercial swine influenza vaccine did not confer adequate protection against the H1N2 subtype.     

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.     

Evaluation of a Killed Vaccine Against Porcine Pleuropneumonia Due to Haemophilus pleuropneumoniae

A bacterin containing serotypes 1 and 5 of Haemophilus pleuropneumoniae was developed for the prevention and the control of porcine pleuropneumonia. It was injected intramuscularly into three groups of ten piglets, the first group with one dose, the second one with two doses and the third one with three doses at two-week intervals. Another group of ten piglets did not receive the vaccine. All the piglets were then challenged by an aerosol of mixed suspensions of H. pleuropneumoniae serotypes 1 and 5. Two and three injections of vaccine completely prevented mortality, whereas half of the control piglets and of those receiving only one dose of vaccine died. All surviving piglets, both control and vaccinated, had severe signs of respiratory disease for at least 36 hours after exposure to challenge. Moreover, vaccination did not induce the production of antibodies at high titers. Local reactions were not noted after vaccination and at postmortem; ten weeks after the challenge, there were no signs of abscess formation or induration.     

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.