Study On The Biological Features Of Salmonella Infantis From Different Hosts And The Effect Of SPI/SPI2 On The Pathogenicity Of Salmonella Infantis

Salmonella Infantis(SIn)is a pan-host zoonotic pathogen.At present,the morbidity rate of SIn in poultry are increasing year by year,which brings serious harm to poultry breeding industry,and it is also easy to infect people and cause public health issues.However,the study on the biological features and pathogenic mechanism of SIn is relatively few,which restricts the effective prevention and control of SIn.And so,SIn isolates from different host were collected,their biological features were analyzed,including antibiotic resistance and disinfectant tolerance,as well as the genetic diversity and pathogenicity.To explore the effect of SPI1 and SPI2 on the pathogenicity of SIn,SIn212ΔSPI1 and SIn212ΔSPI2 were constructed and analyzed their biological features and the dynamics in the hosts.The preliminary study on biological features and pathogenicity of SIn will promote to be further understood.1 Analysis of biological features of SIn from different hostsA total of 56 SIn isolates were collected,There are 2 strains of SIn isolated from chickens,5 strains from pigs,4 strains from humans,2 strains from cattle,42 strains from the environment,and one strain from the UK.Antibiotic resistance assay showed that there were different degrees of antibiotic resistance in all SIn isolates.Among them,the drug resistance rates of Olaquindox was 37.5%,Ampicillin was 30.0%,Chloramphenicol was 26.8%,Tetracycline was 20.0%,Flufenicol was 16.0%,and other antibiotics were below 5.4%.Multiple drug resistant(MDR)of SIn isoaltes were 21.4%.The analysis of disinfectant tolerance showed that SIn isolates were sensitive to 5 kinds of disinfectants.The MIC of Salmonella were 156.25 μg/mL for Sodium Hypochlorite,312.5 μg/mL for 10%Povidone Iodine,6.25 μg/mL for Glutaraldehyde,62.5 μg/mL for Compound Phenol.However,the MIC of SIn2 and SInDup strains were 312.5 μg/mL for 25%Glutaraldehyde,and the MIC of SIncdml,SIn83,SIn308 and SIn212 were 156.25 μg/mL for 25%Glutaraldehyde.The results of the MBC were consistent with that of the MIC.The detection of virulence gene showed that all Salmonella isolates carried virulence genes of sipA,sopB,sopD,sifA,sitC,spvC,sopE,sipD,and pefA,but no sopE2 gene,and the positive rate of ssaR gene was 94.6%.PFGE analysis showed that there were some difference among SIn isolates,but genetic homology of SIn isolates from same host was very high,even up to 100%.In in vitro cellular assay,After SIn212 infected RAW264.7 cells,the invasion rate was higher than that of SIn308 and SIncdm1,while the invasion rate of SIn212 infected HD-11 cells was significantly higher than that of SIn83 and SInDup.In in vivo pathogenicity assay,in mice,the loads of 6 SIn isolates(different host sources)in the cecum showed a gradual decline,and the loads of SIn212 in the cecum was higher than that of the other 5 strains,but all 6 strains could not been isolated in the liver,only SIn212,SIn83 and SIn308 strains can not be isolated in the spleen until the 14th day;while in chicks,6 SIn strains can be isolated in the liver.It colonizes the cecum on the 1st day post infection and can spread from the intestine for systemic infection.The pathogenicity of 6 SIn strains to chicks of different ages was different,after 3-day-old and 14-day-old chicks being orally administrated with SIn strains,all 3-day-old chicks died on the 4th day,while the 14-day-old chicks had been no deaths,so it was speculated that these studied SIn strains were more susceptible to young chickens and stronger pathogenicity.2 Preliminary study on the effect of SPI1 and SPI2 on the pathogenicity of Salmonella InfantisBased on homologous recombination,SIn212ΔSPI1 and SIn212ΔSPI2 were constructed and their biological features were preliminarily analyzed.after SIn212ΔSPI1 and SIn212ΔSPI2 being successfully constructed,their biochemical features were same as those of wild strains;Growth features were slightly slow.In vitro assay on HD-11 cells showed that the adhesion rate of SIn212ΔSPI1 and SIn212ΔSPI2 to HD-11 cells had no significant difference compared to wild strain,and the their invasion rate were significantly decreased.Intracellular proliferation assay showed that the 2 mutants and the wild strains proliferated during 1-24 h,and showed a downward trend during 24-48 h.The proliferation rate of the 2 mutants was lower than that of the wild strains.In vitro assay on RAW264.7 showed that there was no significant change in the adhesion rate of the 2 mutants,but the invasion rate of SIn212ΔSPI1 was increased significantly,that of the SIn212ΔSPI2 decreased significantly.In the intracellular proliferation assay,both the mutants and the wild strains proliferated during 1-24 h,the proliferation of SIn212ΔSPI2 began to decrease at 24 h,while the SIn212ΔSPI1 and the wild strains continued to proliferate.In vivo pathogenicity assay in mice showed SIn212ΔSPI1 and SIn212ΔSPI2 in the cecum were lower than those of the wild strains,and the two mutants did not appear in the liver and spleen;But the same assay in poultry showed that the two mutants and the wild strains could still be isolated from the liver,spleen and cecum in chicks up to 21 days,and there were extrenteric infections.In conclusion,the genetic differences of the isolates collected from different host were small,but their pathogenicity to different hosts was significantly different,and the pathogenicity to chicken was stronger than that to mice.Although SIn212ΔSPI1 and SIn212ΔSPI2 are easier to be eliminated in mice and chicks than parent strains,a special phenomenon was found that the invasion rate of SIn212ΔSPI1 to RAW264.7 cell was significantly increased.These results not only expanded the theory about Salmonella pathogenic mechanisms,but also offered a base for the development of vaccines and drugs against SIn in the future.