Development of a new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America.
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Haemophilus parasuis causes polyserositis in swine. Fifteen serovars have been characterized by immunodiffusion test, but many field strains are not typeable. Isolates (n = 300) of H. parasuis from animals in North America were serotyped by a new indirect hemagglutination test. The test was rapid and effective for serotyping of H. parasuis, and serovars 4, 5, 13, and 7 were the most prevalent serotypes. (+info)
Computer-based analysis of Haemophilus parasuis protein fingerprints.
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The present study aimed to compare the whole-cell protein profiles of Haemophilus parasuis field isolates by using a computer-based analysis, and evaluate the relationship between polyacrylamide gel electrophoresis (PAGE) type and virulence potential based on isolation site. A dendrogram clustering isolates with similar protein profiles was generated. Haemophilus parasuis isolates were grouped into 2 major PAGE type groups. The PAGE type II isolates were characterized by the presence of major proteins with molecular weights varying from between 36 and 38 kDa and included 90.7% of the isolates recovered from systemic sites, such as pleura, pericardium, peritoneum, lymph nodes, joints, and brain. Isolates classified as PAGE type I were characterized by the absence of this group of proteins and included 83.4% of the isolates recovered from the upper respiratory tract of healthy animals. The present study further corroborates the existence of a unique group of major proteins in potentially virulent H. parasuis isolates. (+info)
Development of polymerase chain reaction and comparison with in situ hybridization for the detection of Haemophilus parasuis in formalin-fixed, paraffin-embedded tissues.
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DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation. (+info)
Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protective role in mice.
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Monoclonal antibodies (MAbs) against Haemophilus parasuis were obtained by the fusion of SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with a whole-bacterial-cell suspension (WC) of H. parasuis strain SW124 (serotype 4). Two MAbs showing strong reactivity in ELISA were further characterized using SDS-PAGE and Western-blot assays. Different treatments of the WC indicated that MAbs 4D5 and 4G9 identified epitopes of proteinic and polysaccharidic nature, respectively. Electron microscopic examination revealed that, unlike the proteinic epitopes, the lipopolysaccharidic epitopes were exposed on the surface of the cell. Using coagglutination, Western-blot and dot-blot assays it was found that both MAbs recognized common epitopes of all the reference strains and field isolates of H. parasuis. None of the other bacteria tested reacted with the MAbs. These results indicated that both the proteinic and polysaccharidic antigens carried species-specific epitopes. It is suggested that these MAbs may potentially be useful for identification of H. parasuis isolates as well as for developing serological diagnostic tools. MAbs 4D5 and 4G9 were unable to kill H. parasuis in vitro in the presence of complement. However, an enhanced bacterial clearance from blood was observed in mice inoculated with either of the MAbs. Highly significant protection was observed in mice using MAb 4G9. This is believed to be the first report of MAbs capable of identifying common species-specific antigens of H. parasuis and of their implication in protection against challenge infection in mice. (+info)
Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor.
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Bacteria have evolved a set of highly specialized proteins to capture iron in iron-depleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Glasser disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under iron-restricted conditions, however, it is one of the targets of the humoral immune response. (+info)
Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glasser's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis. (+info)
Genotypic diversity of Haemophilus parasuis field strains.
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Haemophilus parasuis is the cause of Glasser's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains. (+info)
Study of the population structure of Haemophilus parasuis by multilocus sequence typing.
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Haemophilus parasuis is the aetiological agent of Glasser's disease in swine. In addition, this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they had resolution or implementation problems. To overcome these limitations, a multilocus sequence typing (MLST) system, using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB, was developed. Eleven reference strains and 120 field strains were included in this study. The number of alleles per locus ranged from 14 to 41, 6pgd being the locus with the highest diversity. The high genetic heterogeneity of this bacterium was confirmed with MLST, since the strains were divided into 109 sequence types, and only 13 small clonal complexes were detected by the Burst algorithm. Further analysis by unweighted-pair group method with arithmetic mean (UPGMA) identified six clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation (putative non-virulent), while another cluster showed a significant association with isolation from clinical lesions (putative virulent). The remaining clusters did not show a statistical association with the clinical background of the isolates. Finally, although recombination among H. parasuis strains was detected, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. Interestingly, one branch included almost all isolates of the putative virulent UPGMA cluster. (+info)