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At least 19 recordsLinked to original sources
PubMed2004

Development of a genetic manipulation system for Haemophilus parasuis.

Haemophilus parasuis is a member of the family Pasteurellaceae and an important respiratory-tract pathogen of swine, which is the etiological agent of Glasser's disease. Because no genetic manipulation system is available for H. parasuis so far, in vivo studies about the role of its genes involved in virulence are unfeasible. Here we demonstrate that H. parasuis has a cyclic AMP (cAMP)-dependent natural transformation system that enables the uptake of DNA in which the ACCGAACTC sequence signal must be present. After improving DNA transformation parameters, such as cAMP and DNA concentration and exposition time of the exogenous DNA, a knockout mutant of H. parasuis defective in the thy gene, encoding the thymidylate synthase enzyme, has been constructed. Data presented in this work open the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.

Animals↗
PubMed2005

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis.

Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.

Bacterial Proteins↗
PubMed1992

Effect of oxygen supply during growth on the production of cytochromes, enzymes, and acid end products by Haemophilus parasuis.

Haemophilus parasuis, grown under conditions of high aeration, was found to lack a tricarboxylic acid cycle but to possess phosphoenolpyruvate carboxylase and a reductive pathway leading to the production of succinate. Such organisms contained approximately equal quantities of b-, c-, and d-type cytochromes and excreted acetate. When the oxygen supply for growth was either reduced or eliminated, the specific activities of phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and NADH: fumarate oxidoreductase were increased substantially, and the acid products were succinate, acetate, and formate. Organisms grown under the latter conditions also contained increased quantities of b- and c-type cytochromes, some of which were low-potential cytochromes. These low-potential cytochromes were reduced by NADH and oxidized by fumarate, and hence, appeared to be components of NADH: furmarate oxidoreductase. Our results indicate that in H. parasuis, growing aerobically in medium containing glucose, the sole function of the reductive pathway is to provide intermediates for biosynthetic processes, and oxygen is the preferred electron acceptor. As the supply of oxygen is reduced or eliminated, the reductive pathway becomes more involved in NAD+ recycling and fumarate becomes the acceptor. In effect, irrespective of the oxygen supply, the growth of H. parasuis is absolutely dependent upon the presence of an electron transport system.

Acetates↗
PubMed2003

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.

Animals↗
PubMed1981

Glässer's disease in piglets produced by intraperitoneal inoculation with Haemophilus parasuis.

Haemophilus parasuis was inoculated into the abdominal cavities of piglets. Its infective doses varied from 1.4 X 10(6) to 1.0 X 10(9) organisms. When inoculated with 1.0 X 10(9) or 1.6 X 10(8) organisms, piglets were affected with fibrinous polyserositis associated with arthritis and purulent meningitis. The three disorders were considered to be characteristic of Glässer's disease. Septicemia was also found in many of the infected piglets, some of which died within a few days after inoculation. No lesions were observed in piglets inoculated with 1.4 X 10(7) or 1.4 X 10(6) organisms. It was difficult to reisolate H.parasuis from lesions, especially those in the abdominal cavities of piglets killed on the 6th day after inoculation. Agglutinating and complement-fixing antibodies were present in the piglets with Glässer's syndrome examined on the 6th day after inoculation. Neither of them was detected in any piglet manifesting no clinical signs, except transient pyrexia.

Animals↗
PubMed1986

Morphological variations of Haemophilus parasuis strains.

Haemophilus parasuis strains isolated from the noses of apparently healthy animals and from animals with pathological conditions were examined for the presence of a capsule, for their ability to agglutinate in acriflavine or after boiling, and for their peptide profile after polyacrylamide gel electrophoresis (PAGE). The capsule was identified by precipitation against hexadecyl trimethylammonium bromide (Cetavlon), by demonstration of iridescence, and by means of a capsule-staining method. We found a group of capsulated strains showing a rather coccobacillary morphology compared with the morphology with polymorphism, varying from rod-like to filamentous, in strains without detectable capsules. The strains of the latter group were agglutinated by acriflavine or by boiling. Soluble antigens of capsulated strains reacting with Cetavlon were thermostable and resisted proteolytic enzymes, thus suggesting the presence of an acidic polysaccharide. A few of the capsulated strains did not precipitate with Cetavlon, which indicated that their chemical composition was different. Acriflavine-positive strains belonging to a definite PAGE pattern (type II) seemed to be associated with pathological conditions more frequently than were capsulated strains which were mostly isolated from nasal cavities of apparently healthy pigs. We put forward the hypothesis that the agglutinability in acriflavine, together with the PAGE profile type II, may be associated with particular structures responsible for virulence.

Acriflavine↗
PubMed1991

[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.

Animals↗
PubMed2006

Genotypic diversity of Haemophilus parasuis field strains.

Haemophilus parasuis is the cause of Glässer'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.

Animals↗
PubMed2006

Isolation and characterization of the P5 adhesin protein of Haemophilus parasuis serotype 5.

Haemophilus parasuis is a Gram-negative respiratory pathogen of young pigs that colonizes the upper respiratory tract and produces a number of symptoms collectiviely described as Glässer's disease. Recently, an H. parasuis P5-like outer membrane adhesin protein homologous to H. influenzae P5 was identified. The P5 adhesin was partially purified by anion exchange and size-exclusion chromatography. Final purification for functional studies was performed by elution of the protein from a polyacrylamide gel. Identification of the protein as a P5 adhesin homolog of H. influenzae was confirmed by N-terminal sequencing. The P5 protein had a molecular mass of 32,000 and a pI of 5.5. Unlike the H. influenzae P5 adhesin, the H. parasuis P5 protein did not bind carcinoembryonic antigen.

Adhesins, Bacterial↗
PubMed1997

Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase.

Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD+ oxidoreductase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific activity of 222 units/mg protein. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic parameters for all four substrates in the forward and reverse reactions indicated a sequential mechanism for this enzymic process. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH the second product released. Protection against thermodenaturation of the enzyme by NAD and not by malate was supportive of this mechanism. A pronounced product inhibition by NADH (K(i) = 9.0 microM) was observed. Although NADP did not serve as a coenzyme, a number of analogs of NAD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the purified malate dehydrogenase. Coenzyme-competitive inhibition of the malate dehydrogenase was observed with five adenosine derivatives and six structural analogs of NAD. Of the NAD analogs studied as inhibitors, 3-pyridylcarbinol adenine dinucleotide was the most effective (K(i) = 18 microM). Although inhibition of growth of H. parasuis by this analog was observed, it was less effective (K(i) = 136 microM) than the inhibition of the purified dehydrogenase.

Adenosine↗
PubMed1982

Experimental infections of mice and guinea pigs with Haemophilus parasuis.

The pathogenicity of Haemophilus parasuis for mice and guinea pigs was examined. Mice were inoculated intraperitoneally with 2 X 10(7) to 2 X 10(9) organisms suspended in saline or in broth containing fresh yeast extract. Most of them survived after inoculation. Death occurred only in mice inoculated with 2 X 10(9) organisms suspended in broth. The recovery rate of H. parasuis from all the dead mice varied from 28.9% of the brains to 71.4% of the lungs. There were no lesions observed in any mouse, except one. Guinea pigs were inoculated intraperitoneally with 1 X 10(8) to 1.6 X 10(10) organisms suspended in saline. Many of them died after showing septicemia and serofibrinous serositis, which were associated with purulent leptomeningitis or meningoencephalitis in some of them. H. parasuis was recovered abundantly from many organs, including the brain, in the guinea pigs. It was also recovered from guinea pigs inoculated with 1.8 X 10(9) organisms by various routes. Serositis was observed in guinea pigs inoculated intramuscularly or intrapulmonarily. These results suggested that guinea pigs might be available for investigation of the pathogenicity of H. parasuis.

Animals↗
PubMed1995

Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis.

Two strains of Haemophilus parasuis, namely, the type strain (ATCC 19417) and strain E751, were investigated with respect to iron acquisition. Both strains produced iron-repressible outer membrane proteins and could acquire iron from porcine transferrin but not from porcine lactoferrin. Neither strain used bovine transferrin, and human transferrin was used to only a very limited extent, if at all. In all cases, iron acquisition from transferrin required direct contact between the organisms and the protein. An affinity isolation technique based on biotinylated porcine transferrin plus streptavidin-agarose, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine transferrin binding polypeptides (94 and 60 kDa) from total membranes derived from the type strain grown under iron-restricted conditions but only one (96 kDa) from strain E751. Each of these polypeptides was iron repressible and was not isolated when biotinylated human or bovine transferrin was used instead of biotinylated porcine transferrin. It is concluded that both strains acquire transferrin-bound iron by means of siderophore-independent mechanisms and that the isolated polypeptides represent porcine transferrin receptor components.

Animals↗
PubMed1995

Experimental reproduction of Haemophilus parasuis infection in swine: clinical, bacteriological, and morphologic findings.

Haemophilus parasuis is a common cause of polyserositis and polyarthritis in swine. Little is known about the mucosal and systemic sites of replication and lesions which follow an aerosol exposure to H. parasuis. In this experiment 5-week-old cesarean-derived, colostrum-deprived (CDCD) pigs were inoculated intranasally with an inoculum containing 2 x 10(9) colony-forming units of H. parasuis. Two principals and one control pig were necropsied at 12, 36, 84, and 108 hours postinoculation (PI) and samples obtained for bacteriologic culture and microscopic examination. Inoculated pigs developed clinical signs of inappetence, reluctance to move, lameness, and a serous nasal discharge. Macroscopic findings included a fibrinous polyserositis and polyarthritis 36 hours PI which became progressively more severe at 84 and 108 hours PI. No lung lesions were grossly visible. Microscopic lesions included a mild purulent rhinitis at each post inoculation interval and fibrinous to fibrinopurulent synovitis and serositis at 36, 84, and 108 hours PI. A focal suppurative bronchopneumonia was observed in one pig examined at 36 hours PI. The nasal cavity and trachea were the only mucosal sites from which H. parasuis was reisolated. Haemophilus parasuis was isolated from the blood and systemic sites at 36, 84, and 108 hours PI. Findings presented indicated that intranasal inoculation of 5-week-old CDCD pigs with H. parasuis results in clinical signs and lesions of polyserositis and polyarthritis typical of field cases and is a useful model for the study of H. parasuis pathogenesis. The results also suggest that H. parasuis initially colonizes the nasal mucosa.

Animals↗
PubMed2006

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.

Animals↗
PubMed2005

aroA gene PCR-RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species.

The Haemophilus parasuis aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase and participates in the aromatic amino acids and the folic acid universal metabolic pathway of bacteria. The application of aroA-based PCR-RFLP methodology yields a significant degree of diversity in H. parasuis and Actinobacillus species. PCR amplification of the aroA gene rendered a 1,067-bp fragment in all 15 H. parasuis serovars, and also in Actinobacillus pleuropneumoniae serotypes 1-12, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus suis, Actinobacillus ureae, Actinobacillus minor and Actinobacillus indolicus. Sau3AI and RsaI digestions of the aroA PCR products rendered seven different restriction fragment length polymorphism (RFLP) patterns: group I (H. parasuis serovars 1, 2, 4-6, and 8-15, A. porcinus and A. ureae), group II (H. parasuis serovars 3 and 7, and A. pleuropneumoniae serotypes 1, 4, 5, 9, 11 and 12), group III (A. lignieresii), group IV (A. pleuropneumoniae serotype 7), group V (A. pleuropneumoniae serotypes 2, 3, 6 and 8, A. equuli, A. rossii, A. minor and A. indolicus), group VI (A. suis) and group VII (A. pleuropneumoniae serotype 10). This is the first report describing the presence of aroA gene in H. parasuis, A. lignieresii, A. porcinus, A. rossii, A. suis, A. ureae, A. minor and A. indolicus and the data presented here demonstrates a significant degree of aroA genetic diversity in H. parasuis and species of the genus Actinobacillus.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Study of the population structure of Haemophilus parasuis by multilocus sequence typing.

Haemophilus parasuis is the aetiological agent of Glässer'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.

Alleles↗
PubMed2001

Experimental colonization of piglets and gilts with systemic strains of Haemophilus parasuis and Streptococcus suis to prevent disease.

Haemophilus parasuis and Streptococcus suis are both major causes of losses during the nursery period, especially in herds using the segregated early weaning system. In this system, only a few piglets may be colonized with the herd's prevalent systemic strain, which results in infection of naive penmates late in the nursery. In view of these factors, the objectives of this study were: (1) to evaluate the early colonization of piglets with the farm's prevalent systemic strain of H. parasuis and S. suis as an alternative method for disease prevention; and (2) to evaluate 2 different protocols for experimental colonization: direct colonization of piglets and colonization of piglets through nose-to-nose contact with inoculated sows. Haemophilus parasuis and S. suis isolates recovered from diseased nursery pigs were characterized by the rep-PCR technique and the herd's prevalent strains were used for colonization. Piglets in the experimentally colonized groups were inoculated at 5 days of age by the oral route using a spray pump. Sows were colonized at 2 weeks prior to farrowing using a similar protocol. Although both colonization protocols were successful in getting the piglets colonized, direct inoculation of 5-day-old piglets with the herd's systemic strains of H. parasuis and S. suis tended to be more effective in reducing the morbidity and the mortality than the colonization of piglets by nose-to-nose contact with inoculated sows.

Animals↗
PubMed2006

[Detection of Haemophilus parasuis and Mycoplasma hyorhinis in swine and association of those pathogens with clinical and pathological-anatomic findings].

In routine diagnostic it is difficult to detect a Haemophilus parasuis infection causing a polyserositis. In the presented study, a detection method by means of PCR, from collective swabs of the serosal surfaces from animals, with and without fibrinous serositis, was investigated. A significant association was detected between post mortal findings of fibrinous serositis and molecular biological detection of Haemophilus parasuis and Mycoplasma hyorhinis. However, no significant association existed between cultural detection of Haemophilus parasuis in bronchoalveolar lavage fluid and pathological-anatomical ascertainable fibrinous serositis. A significant association between Haemophilus parasuis and Mycoplasma hyorhinis could be determined in the animals that were included in this study.

Animals↗