Isolation And Identification Of Haemophilus Parasuis And Determination Of Their Biological Characterization

Haemophilus parasuis was widely prevalent on pig farms in China in recentyears.In our research, H. parasuis was isolated from57/137(41.6%) clinical cases fromHenan and several other provinces, the strains were identified by colony morphology,Gram-staining characteristics (Gram-negative bacilli), Nicotinamide adeninedinucleotide (NAD) dependence, biochemical tests and polymerase chain reaction(PCR). The isolation rate in lung (39.7%) was highest in different tissues, followed byheart-blood (27.9%), brain (17.4%), joint (14.3%), lymph nodes (8.8%) and spleen(5.3%). The serotyping of57H. parasuis strains showed serovar4(22.8%) and serovar5(21.1%) were the most prevalent, followed by serovar12(10.5%), serovar13(10.5%)and serovar14(8.8%), whereas17.5%of the isolates were non-typeable by agar geldiffusion (GD) test. Serovars1,2,6, and10were only represented by a small number ofisolates. A mice infection experiment indicated that serovar12and serovar14weremost virulent, followed by serovar5and serovar4, serovar13was weakly virulence.We selected16strongest virulence strains in serovar4(4),5(4),12(3),13(2) and14(3) for the further research.In a guinea pigs infection experiment of the16representative strains, the resultwas compliant with previous research in mice. Compared with mice, guinea pigs hadlonger morbidity period, and more similar pathological changes with pigs, so they aremore suitable to be infection models of H. parasuis than mice. A pig infectionexperiment with different infection dose showed virulence of different strains wassimilar in same serovars. The absolute lethal dose (LD100) were6.8×109CFU (S4S1),7.2×109CFU (S4S2),4.4×109CFU (S5S2),3.7×109CFU (S5S3),3.9×109CFU(S12S2), and3.0×109CFU (S12S3), respectively. In growth curve, peak growth of H.parasuis was detected after10h to14h culturing in Tryptic Soy Broth (TSB) mediumwith amount of S4S1, S5S3, S12S3were2.5×109CFU/mL,1.6×109CFU/mL and1.1×109CFU/mL, respectively. The sequence determination of16S rRNA PCRproduction showed that5highest homology (over99%) sequences with the test strains were all16S rRNA sequences of H. parasuis published by NCBI databanks. Keepingtime of H. parasuis in different conditions was10d (Tryptic Soy Agar, TSA and TSBmedium,2–8℃),1year (TSB medium with30%glycerine,-20℃),3years (TSBmedium with30%glycerine,-80℃.15%Freeze-dried skim milk,-20℃or-80℃),respectively. The selection of strong virulence strains and research of their biologicalcharacterization made foundation to vaccine development of H. parasuis.A microagglutination test (MAT) in serovars4,5and12of H. parasuis wasestablished. Through the optimization of the reaction conditions, the optimalconcentration of agglutination antigen was1.6×109CFU/mL (S4S1),1.7×109CFU/mL(S5S3) and1.3×109CFU/mL (S12S3), respectively. The optimal reaction temperatureand time were37℃,6–7h. Specificity and sensitivity test showed the results werenegative after detected positive serum of5common bacterial diseases (SwinePasteurellosis, oedema disease, Swine paratyphoid, Swine Streptococcosis, Bordetelladisease) and5common viral diseases (Classical Swine Fever, Aujesky’s Disease,Porcine Circovirus Disease, Porcine Reproductive and Respiratory Syndrome, PorcineEpidemic Encephalitis B). The method was2–4times sensitive than corresponding tubeagglutination test, and serum antibody could detected14d after immunization. Thedetection results of clinical samples indicated combination of conservation periodpiglets may lead seriously H. parasuis infection, and the results of immunized pigletsserum samples demonstrated MAT can used to detect the antibody level afterimmunization.