Characterization Of Haemophilus Parasuis From Central Vietnam And Protection Efficacy Assessment Of A Chinese Commercial Vaccine Against The Vietnamese Strains

H.parasuis?HPS?is a gram-negative bacterium dependent on the growth of nicotinamide adenine dinucleotide?NAD?.It is causing Gl?sser's disease in swine,which is characterized by serofibrinous polyserositis,meningitis,and arthritis.Vietnam has the fifth-highest pig production in the world.Until now,there is still a lack of data about the epidemiological characteristics and the prevalence of antimicrobial resistance?AMR?and antimicrobial resistance genes?ARGs?of HPS in Vietnam.Also,to the best of our knowledge,the vaccines for prevention and control of Gl?sser's disease have still not been developed and used in Vietnam.In the first study,the prevalence of serotypes and distribution of virulence-associated genes?VGs?in HPS strains isolated from swine in Quang Binh and Thua Thien Hue provinces,Central Vietnam was studied.A total of 56 HPS strains were isolated from814 lungs and swab nasal samples?56/814,6.9%?.The isolation rate was significantly different in different regions,ages,and origins of specimens?p<0.05?.Serotypes of these strains were identified by using one-step multiplex PCR?m PCR?.The results have shown that the most prevalent serotypes were serotype 5?15/56,26.8%?,followed by serotype 2?13/56,23.2%?and serotype 4?10/56,17.9%?.Nine virulence-associated genes?VGs?were then analyzed in the HPS strains.The vta1 gene?35/56,62.5%?was most frequently detected,followed by vta3 gene?24/56,42.9%?,vta2?22/56,39.3%?,HPM-1371?20/56,35.7%?,cap D?17/56,30.4%?,HPM-1372?7/56.12.5%?,and lsg B and HPM-1373?both shared 5/56,8.9%?.Seven out of 56 strains?12.5%?did not carry any VGs tested in this study.Strong associations between some serotypes and VGs were detected,in which virulence-associated genes HMP-1371,HMP-1372,vta3,vta2,cap D were mainly clustered in serotype 5/12,and vta2 clustered in serotype 2.The results in this study are to present the first baseline information in the epidemiological characteristics of HPS isolates from swine in Central Vietnam.In the second study,the AMR prevalence,as well as their association with ARGs in the above isolated HPS strains was investigated.All the strains were tested for the antimicrobial resistance?AMR?against 25 antibacterial agents by using a broth microdilution method and for the presence of the antimicrobial resistance genes?ARGs?by PCR method.These strains were shown to have a high rate of resistant to trimethoprim/sulfamethoxazole?94.6%?,followed by resistant to colistin?91.1%?,chloramphenicol?91.1%?,penicillin?85.7%?,gentamicin?83.9%?,lincomycin?82.1%?,and amoxicillin?78.6%?.All of the strains were multiple drug-resistant?MDR?to at least three to eighteen antimicrobial agents.The phenotypes resistance to each antimicrobial agent was significantly associated with at least one other;all of them are positive associations excepting between amoxicillin and tobramycin.The most frequencies ARGs in the strains recovered were bla TEM-1?94.6%?,int?76.8%?,gyr A?58.9%?,and rmt D?50.0%?.The strongest associations were observed between tet B and tet H?OR=153.0,p=0.001?,tet A and tet B?OR=75.0,p=0.001?,tet H and aac?6'?-Ib?OR=49.0,p=0.003?,aad A1 and tet B?OR=23,p<0.01?,aac?6'?-Ib and sul II,par C and erm?A??OR=23.0,p<0.01?.The phenotype resistance to cefuroxime,chloramphenicol and tobramycin were strongly relationship with blarob-1?OR=26.3,p=0.002?,catl?OR=25.1,p=0.004?,and str B?OR=23.5,p=0.001?,respectively.The results of this study showed that the prevalence of AMR in the HPS isolates from pigs is slightly high in Central Vietnam.A high correlation between some AMR phenotypes and ARGs was observed.The present study is the first report about H.parasuis AMR in Central Vietnam,which is helpful for the clinical control of HPS infection,as well as the development of policies and clinical practice guidelines to reduce AMR in swine production in Vietnam.In the third work,a highly virulent HPS strain SH0165 serotype 5 recovered from hog pen in North China was serially passaged in vitro for 250 times at 37?(H₃₇)and 40?(H₄₀),respectively.The growth and their virulence of the sub-strains were assessed by using the Bio-screen C system and animal?guinea pig as well as piglet?infection models,respectively.The results showed that the parental strain was growing significantly slower than the sub-strains?p<0.01?.In the guinea pig infection assay,all of the animals infected with parental and H_37-100?the strain serially passaged for 100 times at 37??strains died between 36 to 48 h post-infection,which showed gross necropsy,including fibrinous layers on serosal surfaces in enterocoeliam thorax,pericardium,articular cavity,meningitis,and polyserositis.In contrast,the sub-strains H_37-150,H_37-200,H_37-250,H_40-150,H_40-200 and H_40-250 revealed significantly attenuated virulence,showing a cumulative percent survival at 72 h post-infection of 20.0%,60%,80%,20.0%,40%,and 80%,respectively.In the piglet infection assay,only one out of three piglets?20%?infected with parental,H_37-150,and H_37-200 strains died with visible clinical signs and lesion.The number of HPS recovered from lung and brain specimens of piglets infected with H_37-200,H_37-250,H_40-150,H_40-200,and H_40-250 sub-strains was significantly lower than that from piglets infected with the parental strain?p<0.01?.This study is the first report that the in vitro serially passaged HPS strains were significantly virulence attenuated,providing an alternative approach to develop attenuated HPS vaccine.In the fourth study,we evaluated the efficacy of a commercial HPS vaccine in protection against the challenged with HPS serotypes 4 and 5 isolates from Vietnam using a guinea pig infection model.A significantly high level of antibodies,IFN-g,and IL-4 in sera from vaccinated animals was observed.After challenged with the Vietnamese strains,a significantly milder lesion score and lower bacterial loads in the brain,lung,and liver tissues were observed in the vaccinated guinea pigs when compared with the non-vaccinated animals.The typical symptom of Gl?sser's disease was observed in the non-vaccinated guinea pigs.Taken together,the results in this study suggest that the bivalent inactivated vaccine made from Chinese strains of HPS serotypes 4 and 5 can provide excellent protection against the infection of the homologous strains from Vietnam.