Isolation And Identification And Establishment Of Serological Typing Methods Of Haemophilus Parasuis

Haemophilus parasuis (H.parasuis/HPS) is a non-motile, pleomorphicrod-shaped， nicotinamide adenine dinucleotide (NAD)-dependent, gram-negativebacterium of Haemophilus genus of the family Pasteurellaceae. This bacterium is thepathogens of Gl sser’s disease in swine, the main features of which is polyserositis,arthritis, and meningitis. Haemophilus infection is causing a significant increase inmortality and morbidity in piglet. With the development of swine industry andimprovement of intensive, Haemophilus parasuis infection shows an increasing trend,and the disease is prevalent in the world. H.parasuis infection causes a high morbidityand mortality, which will bring a huge finance depression to farming businesses and beharm to the development of swine industry. It has become a huge worldwideproblem.There is no standard specific antiserum of H.parasuis in China to typing HPSisolates for the time being. Therefore, it is critical important to carry out typingantiserum preparation and establish typing method for rapid serotyping of isolates andtreatment.Firstly, Various disease materials were collected from suspected of H.parasuisinfection animals of north China. Nineteen strains were lsolated successfully by usingTSA medium. Bacterial morphological observation, culture and biochemicalcharacteristics and species specific PCR were conducted after purification, and expectedfragment of1090bp was amplified. The results showed that the19isolates wereHaempholius parasuis.The further studied outcomes of drug sensitivity, pathogenicity and virulence geneindicated that all isolates except GS-3have strong pathogenicity and multiple drugresistance, but they are sensitive to macrolide antibiotics which provides guidelines forprevention and control of this diease of north China area pig farms. The PCR results ofpotential virulence genes were consistent with mice infection results, which proved thatnhaC、hhdA and hhdB maybe related with virulence of H.parasuis, while fhuA gene isnot virulence gene.Secondly, in this experiment15reference strains of H.parasuis were grown onTSA solid medium and washed down with PBS after expanding culture. The inactived bacteria were used to immune rabbit. When the antibody is generated,15serotypesantiserum were prepared after blood collection.Cross-reaction and titer of antiserumwere tested by gel diffusion (GD) test and slide agglutination respectively.According tothe result of cross reaction, uni-factor serum was prepared by absorbing.19isolated strains were typed by making use of uni-factor serum, the resultsdemonstrated that19isolates could be typed accurately. Four strains of H.parasuis thatisolated early were typed with uni-factor serum GD and indirect haemagglutination(IHA). The results showed that the isolates were non-typable by IHA because ofcross-reaction, while all strans can typed by uni-factor serum. It is demonstrated that thetyping method with uni-factor serum GD has good specificity and has a higher typingrate than IHA.