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.     

Serum resistance and virulence of Escherichia coli isolated from turkeys

Twenty-five strains of Escherichia coli isolated from turkeys were characterized for their serum resistance and virulence. An in vitro bactericidal assay was used to determine the serum resistance of E coli. Virulence was determined by survival time after IV inoculation of each strain into 3-week-old turkeys. Serum-resistant E coli strains were generally found to be virulent for turkeys, whereas serum-sensitive E coli strains were avirulent. Of the 25 strains, 18 strains were placed in the 2 categories of serum-resistant/virulent and serum-sensitive/avirulent. Five strains were serum-resistant and avirulent, and 2 strains were serum-sensitive and virulent. Serum resistance appears to be an important determinant of virulence for E coli in turkeys; however, the requirement for other virulence factors, in addition to serum resistance, was suggested by the finding that 5 serum-resistant strains were avirulent in turkeys.     

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.     

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.     

Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells

Cytolethal distending toxin (CDT) is proposed to be an important virulence determinant of many pathogens. Although two cdt gene cluster loci have been identified in Haemophilus parasuis strain SH0165, the characteristics of CDTs associated with pathogenesis remain unclear. In this study, three CDT-deficient mutants, cdt-1, cdt-2 and the double-knockout cdt-1cdt-2 (Δcdt-1, Δcdt-2 and Δcdt-1Δcdt-2, respectively), were obtained in the H. parasuis serovar 4 clinical strain SC096 using a natural transformation method. Compared to the wild-type SC096 strain, the Δcdt-1, Δcdt-2 and Δcdt-1Δcdt-2 mutants showed subtle growth defects and clearly exhibited an increased sensitivity to the bactericidal action of porcine and rabbit sera. Additionally, these mutants had a significantly reduced ability to adhere to and invade porcine umbilicus vein endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15). These findings suggest that both CDTs in the H. parasuis SC096 strain are involved in serum resistance and adherence and invasion of host cells.     

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.     

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.     

Survival of avian strains of Pasteurella multocida in chicken serum

The ability of bacteria to survive in serum is considered a likely virulence determinant in diseases where the infective bacteria become septicaemic. Optimal conditions were established to test the survival of Pasteurella multocida in chicken serum. Serum was used at 90%, the inoculum was 10(3)-10(4)cfu in phosphate buffered saline pH 7.4. Survival was measured after incubation for 2-4 h; if survival was <50% the strain was considered serum susceptible. Susceptible strains were either killed or their growth was inhibited. Some resistant strains not only survived but grew rapidly in unheated serum. Thirty-five strains, all originally isolated from clinical fowl cholera, were tested; eight were susceptible, of which three were killed and five inhibited, and the remainder (27) were resistant. Ten serum-resistant P. multocida serogroup A strains were grown in hyaluronidase to remove the capsule and survival in chicken serum was re-tested. Three strains became susceptible, while seven strains remained resistant. Three serum susceptible strains were then tested in the presence of cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). This substance is present in the human serum, and is known to mask the effect of complement on Neisseria gonorrhoeae rendering susceptible strains resistant. Two of the three serum susceptible strains became resistant in the presence of CMP-NANA. Serum susceptibility/resistance was more complex than that of Escherichia coli, and the role of resistance to avian complement in the pathogenesis of fowl cholera remains to be determined.     

Anti-capsular antibodies activate killing of Escherichia coli O8:K87 by the alternate complement pathway in porcine serum

Enterotoxigenic Escherichia coli (ETEC) strains that produce K88 (F4)+ fimbria are important causes of diarrhea and post-diarrheal septicemia in swine. ETEC O8:K87, a serotype represented by a number of these strains, is typically serum resistant. Strain-specific antibodies are known to activate alternative C pathway-mediated killing of other serum-resistant E. coli [Hill, A.W., Shears, A.L., Hibbitt, K.G., 1978. The requirement of specific antibody for the killing of E. coli by the alternate complement pathway in bovine serum. Immunology 34, 131-136], but their antigenic targets have not been determined. We tested the hypothesis that anti-K87 antibodies activate alternative pathway-mediated killing of ETEC O8:K87. Pigs were immunized with ETEC O8:K87 strain 2534-86 cells or purified K87 polysaccharide. Post-, but not pre-immunization sera killed 2534-86 cells, and absorption with 2534-86 cells or by K87 affinity chromatography eliminated bactericidal activity. Complementation of absorbed serum with anti-K87 antibodies restored bactericidal activity, confirming the ability of these antibodies to activate C-mediated serum killing. Serum from age-matched, non-vaccinated control pigs also killed 2534-86. This activity was eliminated by absorption with 2534-86 cells, but not K87 affinity chromatography, indicating that specific non-capsular antibodies are also able to activate C-mediated killing. In all cases, Mg-EGTA-treated serum was as effective as non-treated serum in killing, suggesting that bactericidal activity was mediated predominantly if not exclusively via the alternative C pathway.     

Development of an improved species specific PCR test for detection of Haemophilus parasuis

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.     

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.     

The virulence characteristics of strains of Escherichia coli isolated from cases of bovine mastitis in England and Wales

Of 470 Gram-negative facultative anaerobes isolated from cases of bovine mastitis in England and Wales, 422 were identified as Escherichia coli. The characteristics of 237 of these were investigated. Guinea-pig red cell haemagglutinins were possessed by 86% of strains and 12% were resistant to D-mannose. None of the strains tested invaded Vero cells. Haemolysin, Vero toxin and enterotoxin were produced by 5, 0.5 and 1% of strains, respectively. Twenty-two percent were resistant to one or more antibiotics and 4% to sodium arsenate. Transfer ability was possessed by 41% and lysogenic phage by 27% of strains; 62% possessed either one or the other and 12% possessed both. Colicin production was detected in 18% of strains; 5% produced Colicin V. Ninety-nine percent of strains were serum-resistant, while only 6% were able to grow well in bovine serum. A microscopically visible capsule was seen in 75% of strains. All strains possessed at least one of the potential virulence factors or markers studied. Several strains which possessed one characteristic only (mannose-sensitive haemagglutination or serum resistance), possessed one or more large molecular weight plasmids. None of the strains was particularly virulent for chickens following intramuscular inoculation. Of the strains which possessed one virulence characteristic, only those which were serum-resistant were re-isolated from expressed milk following intramammary inoculation of lactating cows.     

Interaction of turkey complement with Escherichia coli isolated from turkeys

The role of turkey complement in a serum bactericidal reaction was determined using serum-sensitive and serum-resistant Escherichia coli isolated from turkeys. Inactivation of complement by heating serum (56 C for 40 minutes) or by treating serum with 10 mM EDTA eliminated bactericidal activity. Serum-sensitive E coli organisms were killed by turkey serum treated with 10 mM ethylene glycol-bis-beta-(aminoethyl ether)-N,N,N',N'-tetraacetic acid and 5 mM MgCl2. Exposure of normal turkey serum to serum-sensitive or serum-resistant E coli resulted in equivalent reductions in hemolytic activity of serum. Treatment of serum-resistant E coli with antibody rendered the bacteria sensitive to bactericidal effects of normal turkey serum. Serum-sensitive E coli organisms were readily killed by an alternative complement pathway, serum-sensitive and serum-resistant E coli activated the complement system equally well, and antibody was required for complement-mediated killing of certain serum-resistant E coli organisms from turkeys.     

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.     

Serum resistance is correlated with encapsulation of avian strains of Pasteurella multocida

Encapsulated avian strains of Pasteurella multocida possessing an A-type capsule were shown to be resistant to the bactericidal action of turkey serum, whereas unencapsulated variants as well as other unencapsulated strains were not. Removal of the capsule from serum-resistant strain P1059-1 resulted in this strain becoming susceptible to the bactericidal effects of turkey serum. Since complement was consumed when encapsulated or unencapsulated strain P1059-1 was incubated in turkey serum, we conclude that the capsule acts to shield the outer membrane rather than prohibiting the generation of an effective membrane attack complex.     

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.     

Complement resistance in Actinobacillus (Haemophilus) pleuropneumoniae infection of swine

The possible role of the complement-mediated bactericidal system in protection of swine against contagious pleuropneumonia was investigated. Strains of Actinobacillus (Haemophilus) pleuropneumoniae representing serotypes 2, 3 and 5 were found to be fully resistant to the bactericidal action of porcine serum from precolostral, clinically normal adult, and chronically infected pigs. All strains were also resistant to hyperimmune rabbit serum, but 3 of 4 strains were sensitive to normal human serum. This bactericidal effect was lost when human serum was previously absorbed with the homologous bacteria, indicating that antibody was necessary for killing. Addition of human serum to porcine serum or to absorbed human serum did not restore the bactericidal system. Pretreatment of the bacteria with undiluted heat-treated human serum also failed to sensitize the bacteria to the absorbed serum, indicating that a heat-labile, absorbable factor may have been required for killing of A pleuropneumoniae. None of the strains was sensitized to porcine serum by sublethal treatment with polymyxin B, a treatment that is known to disrupt the integrity of the outer membrane and induce serum sensitivity in gram-negative bacteria. The ability of A pleuropneumoniae to resist complement killing in vitro may reflect a virulence mechanism in vivo that assists bacteria in avoiding the pulmonary defenses of swine and promotes bacterial invasion of the lungs.     

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.     

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.