| Harmophilus parasuis is the etiological agent of Glasser's disease in pigs, which is characterized by serofibrinous to fibrinopurulent polyserositis, arthrites, and meningitis. 15 serovars of H.parasuis strains have been defined by traditional immunological meoths (based on heat-stable somatic antigen and using immunodiffusion). The corss-protection between different serotypes is variable or not prensent. However,15-20% of the stains remained non-typeable. To overcome those limitations, several genotyping methods based on DNA fingerpring (Restriction endonuclease pattern and ERIC-PCR) have been developed. However, all the typing techniques developed for H. parasuis are based on defferent band size patterns comparisons. They do not get the fact that the data generated is difficult to share and compare globally, and that they are poorly protable and provide little information on the relationship between clusters. Moreover, there are no still specified antiserums using for serovar typing and serology research at present in our country. Thus, it is probably the best choice that develops a multilocus sequence typing method based on DNA sequecing. It is significant to find the correlation between specific genomic clusters and population structure of H. parasuis, to understand the relationship between different stratins genotype with virulence potental and protective immunity, and to control glasser's disease.Seven selected genes (βchain of ATP synthase,atpD, translation initiation factor IF-2,infB, malate dehydrogenase gene,mdh, ribosomal proteinβsubunit, rpoB,6-phosphogluconate dehydrogenase,6pgd, glyceraldehyde-3-phosphate dehydrogenase, g3pd and fumarate reductase. B ,frdB) were amplified and sequenced from 86 H.parasuis isolaties. We analysed the sequence types and genetical relitionship between strains, and constructured NJ tree and unweighted-pair group method with arithmetic mean (UPGMA) tree. The results were following:1. Seven pairs of primers were used for PCR amplifications.450-600pb fragments were amplified from every housekeeping gene under the same PCR reaction conditions.2. Seven selected genes were sequenced from the 46 H. parasuis isolates. The Bioedit, Mega 4.0 and START II softwares were used to edit, assemble and align, and to define MLST schemes and allele squences profile for all squences including 42 Hp isolates from PUBMET net and 8 reference strains. We constuctrued H. parasuis PubMLST isolates database and H. parasuis PubMLST locus/sequence difinitions database, which was cooperated with Keith Jolley in department of zoology, university of Oxford. The allelic profiles, or sequence types (STs) of these isolates were stored on the Internet, forming a virtual isolate collection which could be continually expanded. Application of this approach to further isolate collections will enable an integrated global picture of H. parasuis epidemiology to be established and will permit more detailed studies on the population genetics of this organism.3. After START analysis, we confirmed the high heterogeneity of H parasuis, ie.86 isolates studied were assigned to 74 STs, with a high level of diversity. Allele average genetic distance was 0.674, IA=0.804. The numbers of alleles per locus are ranged from 64 to 76. The locus with the highest diversity were frdB (325) and 6pgd (219), and locus with the lowest polymorphy was infB (77).4. Five different clusters were defined by use of UPGMA.5. It was found that more than one strain can be isolated in a herd (up to 10 strains in a single farm) and even from a single animal, and that contained the virulent strains of H. parasuis in the upper respiratory tract of pathogenetic pigs or healthy pigs.
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