| Salmonella is one of the most common foodborne pathogens,with a wide range of hazards that can cause pollution of various agricultural products.At present,the food contamination caused by Salmonella is a great international concern.It is estimated that there are approximately 1.4 million cases of lmonella bacterial infection in the United States each year.In China,70%to 80%of bacterial food poisoning is caused by Salmonella.Research on the status of contamination and distribution of Salmonella in food animals is important public health implications.This study isolated and identified Salmonella from swine and sheep.Drug susceptibility testing and drug resistance genes were adopted to preliminary grasp the distribution and resistance phenotype of Salmonella in Jiangsu Province.Also,the relationship between the drug resistance phenotypes and the resistance genes of Salmonella isolates were explored.The results of the study are conducive to guiding the rational application of antibiotics in production practice,as well as providing a basis for research on public health security and agricultural product safety caused by Salmonella.As an invasive intracellular bacteria,Salmonella has a strong invasive potential after being attenuated which grows slowly in the host and can induce powerful cellular,humoral and local mucosal immune responses effectively and continuously.One strain of S.Typhimurium virulent strains rSC0032 was screened out from our 86 isolates.Three mutations(APcrp::TT araC PBAD crp,?sopB and ?pmi)were introduced into rSC0032 to construct regulated delayed attenuated Salmonella vaccine vectors,named as rSC0117.The attenuation efficiency of rSC0117 was evaluated in BALB/c mice.1 Isolation and identification of Salmonella from swines and sheepTo investigate the distribution and the dominant serotype of Salmonella from swines in Jiangsu Province.Samples mainly from a pig slaughterhouse in Jiangsu Province from October 2016 to October 2017 were detected with enrichment,purification,PCR,serotype identification and 16S rRNA cluster analysis.The results showed that 80 isolates were identified as Salmonella from 459 samples in the slaughterhouseand of Jiangsu,the serotype of isolates included S.Derby(35/80),S.Rissen(16/80),S.Newlands(11/80),S.Typhimurium(9/80),S.Nchanga(3/80),and S.Chester(2/80),and 1 strain could not be identified.64 Salmonella were isolated among 230 intestine samples with a detection rate of 27.83%;The postive rate of liver and feces samples were 8.43%(14/144)and 3.17%(2/63),respectively.2 strains of S.Typhimurium and 4 strains of S.Choleraesuis were isolated form the rest samples.Based on the 16S rRNA gene sequence of Salmonella,129 Salmonella strains of 32 serotypes were analyzed by phylogenetic phylogenetic tree.The 16S rRNA phylogenetic analysis organized the strains into three clusters and the same serotype could be divided into different clusters.The homology within each branche ranged from 99.6%-100%with the genetic distance ranged from 0-0.002.The maximum genetic distance of all strains was 0.003 as the 16S rRNA gene homology was 98.8%-100%.Since most of the serotypes of Salmonella were not able to be distinguished as unique by 16S rRNA analysis,it can be indicated that the 16S rRNA gene sequence is not the most appropriate locus to definitively identify Salmonella serotypes,or to deduce phylogenetic relationships among bacteria in the same genus.2 Antibiotic resistance,resistance genes and virulence genes analysis of Salmonella isolatesAntibiotic resistance of 86 Salmonella isolates was determined by antibiotic susceptibility test(K-B method)with 22 antibacterials.The results showed that,the highest incidence resistance in Salmonella isolates was against tetracycline(76.74%),then followed by ampicillin(70.93%),cotrimoxazole(67.44%),sulfisoxazole(67.44%),and chloramphenicol(44.19%).Although the isolates did not develop resistance to drugs such as polymyxin B,they had extremely high mediation rates to them;while they were completely sensitive to cefoxitin and amikacin.Among the 86 strains of Salmonella isolates,9 strains were non-resistance.Of the other drug-resistant strains,57(66.28%)strains displayed multidrug resistance.The percentage of 4 and 5 kinds of antibiotics resistance were 19.77%(17/86)and 17.44%(15/86)respectively,which account for the highest rate among the resistant strains.22 resistance genes were detected by PCR in Salmonella isolates.The analysis showed that 19 resistant genes were detected in the isolates,of which tetA gene(60.47%)displayed the highest positive rate of detection,followed by catAl and aadAl with 45.35%(39/86)and 39.53%(34/86),respectively.No qnrA,qnrC,and qnrD genes were detected,and no resistance gene was not detected in 12 isolates.In order to recognize the virulence genes of Salmonella isolates,the virulence genes of mogA,spvB and spvC were detected by PCR skill.As a result,71(82.56%)isolates carried at least the mogA gene,of which only 3(3.49%)strains carried the mogA,spvB and spvC virulence genes at the same time,and all were Salmonella typhimurium;another 4 strains of S.Choleraesuis which carried both mogA and spvC genes were isolated from illness swine samples.The results showed that swine products in the slaughterhouse were contaminated with multi-drug resistance Salmonella commonly,even a small fraction of them might carry the spv virulence plasmid,which might facilitate the dissemination of the resistance genes to consumers along the production chain,which suggests importance of controlling Salmonella during slaughter for public health,underlying strict hygiene method and HACCP management to reduce cross-contamination.3 Construction and evaluating the characteristic of delayed attenuated Salmonella vectorTo screen Salmonella strains with strong immunogenicity and broad host range as attenuated vaccine vectors,we determined the virulence of 7 strains of S.Typhimurium isolates in this section.The results showed that,all 7 strains of S.Typhimurium were pathogenic to mice,and the lethal time and dose were different.Among the strains,4 isolates had relatively weak pathogenicity,their median lethal dose(LD50)ranged from 103 to 104 CFU,the remaining 3 strains with stronger pathogenicity which LD50 were about 100 CFU,which had the virulence plasmids spvB and spvC.The results of LD50 showed that,Salmonella contamination in pig and sheep slaughterhouse was not only serious,but also was induced the isolates with strong virulence.In the three strains with strong virulence,two of them were isolated from swine samples and one from sheep.Since Salmonella with high virulence could induce good immunogenicity,one isolate derived from pig with high level of LD50 was chosen to construct the delayed attenuated Salmonella vaccine vector,named rSC0032.Three mutations ?Pcrp::TT araC PBAD crpy,?sopB and Apmi were introduced into rSC0032,and which became new live attenuated Salmonella typhimurium rSC0117(rSC0032 APcrp::TT araC PBAD crp,?sopB,?pmi)by suicide vector-mediated homologous recombination technology.Subsequently,the colonization ability of the attenuated strains were evaluated in BALB/c mice.With the passage of time,the amount of bacteria in organs gradually decreased.At the 3d,the two attenuated strains in liver and spleen were significantly lower than that of the wild strains(P<0.001),indicating that its virulence decreased significantly.The colonization ability of the two attenuated strains in the Peyer's patches was strong,which means that attenuated strains likely retained excellent immunogenicity.At the 7d,bacterial count in Peyer's patches of rSC0117 took a slight decrease,indicating that the attenuated bacteria can induce the immune response of the host safely and effectively. |
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