Pathogenic Study Of Four Major Sequence Types Of Porcine-derived Salmonella Species In Mice

Salmonella,as an important foodborne zoonotic pathogen,has important public health significance in China and even the world.China is the largest country in pork production and consumption,and pork products are prone to contaminate during the whole pork production chain including pig breeding,slaughtering,and processing,posing a serious threat to human health.Previous studies by our laboratory showed that the lineages ST19,ST34,ST40 and ST469 were the dominant sequence types(STs)of Salmonella strains isolated from the whole pork production chain.However,data on the pathogenicity in vivo of the four STs of Salmonella are lacking.Therefore,this study was conducted to evaluate the pathogenicity in vivo of the four major STs of porcine-derived Salmonella strains.The main results are shown as follows:Firstly,G.mellonella larvae model were used to preminarily evaluate the pathogenicity of 12 porcine-derived Salmonella strains belonging to four STs.The optimum infection condition for Salmonella was 2.5×10~3CFU of bacterial count for each animal and 24 hours of infection period.The results showed that three Salmonella strains with the same ST exhibited the consistent pathogenicity to G.mellonella larvae.Next,four porcine-derived Salmonella strains were selected to be representative of the four STs(ST19 strain 11207,ST34 strain 03130,ST40 strain 04017,ST469 strain03202)and the pathgenicity in vivo was conducted in mouse model.Each strain was infected at a dose of 10~7CFU by gavage administration in BALB/c female mice.The results showed that,after 7 days of infection,the body weight of mice in the ST19 group and the ST34 group was reduced by 24.8%and 31.0%,respectively.In contrast,the body weight of mice in the ST40 group only decreased by 6.2%,and mice in the ST469 group increased body weight by 3.0%.Clinical symptom results.Mice in the ST19 group exhibited symptoms such as depression,shaggy hair,moderate or slight appetite loss,and loose stools.Mice in the ST34 group exhibited severe symptoms including seriously shaggy hair,watery diarrhea,moderate or severe anorexia,depression,tremors,thin,and in some cases,hemiplegia,ataxia,and coma.Mice in the ST40 group showed slight appetite loss,slightly shaggy hair,and fecal impaction.Mice in the ST469 group exhibited mild symptoms of depression and anorexia.Autopsy finding results.Mice in the ST19 and ST34 groups showed hepatomegaly with white spots,congestion and white spots on the spleen,overall thinning of intestinal wall with gas accumulation and yellow fluid,and cecal atrophy.However,mice in the ST40 group had a few pinpoint-sized white spots on liver surface,slightly enlarged spleen and mild intestinal wall edema;and for the mice in the ST469 group,there was a small amount of yellow fluid in the intestine,and no other obviously pathological change was observed.Observation under microscope revealed that in the ST19-infected mice,the liver plate structure was disrupted,hepatocyte underwent degeneration and necrosis,and plenty of inflammatory cells were clustered in the liver sinusoids;in the spleen,the number of splenic nodules decreased,the relative area of red pulp increased,and lymphocytes in the white pulp underwent necrosis;the villous structure of the cecum was destroyed,and plenty of inflammatory cells were present in the interstitium;the epithelial cells of small intestine underwent degeneration,necrosis,and shedding.In the ST34-infected mice,there was infiltration of a quantity number of red blood cells and inflammatory cells in the hepatic tissue,and a quantity number of red blood cells infiltrating in the red pulp of the spleen with neutrophils clustering in the white pulp;the epithelial cells of the cecum underwent necrosis and dissolution,and plenty of necrosis and inflammatory cells were observed in the epithelial cells of small intestine.In the ST40-infected mice,there was a slight degeneration in the hepatocytes and an accumulation of inflammatory cells in the hepatic tissue;a small amount of lymphocytes in the spleen underwent coagulation necrosis;a small amount of inflammatory cells were sparsely distributed in the cecum and small intestine tissues.In the ST469-infected mice,the pathological changes in the liver,spleen,cecum,and small intestine were mild,and a small number of inflammatory cells were distributed in the tissues.The bacterial load in the liver,spleen,and cecum of mouse model showed that as early as day 1 post infection,bacteria were detected for the three organs in the ST19,ST34,and ST40-infected mice,while bacteria were detected only in the cecum in the ST469-infected mice.The bacterial load in the ST40-infected mice reached its peak at day 1,whereas in the mice infected by the other three STs bacterial load continuously and slowly proliferated and reached the peak at day 3,and then slowly decreased.The results of fluorescence quantitative RT-PCR showed that the expression levels of TNF-αand IL-1βin the liver,spleen,and cecum of mice infected with each of the four STs of Salmonella strains were significantly upregulated,whereas IFN-γwas significantly upregulated in the liver and spleen but significantly inhibited in the cecum.In summary,our study indicates that among the four Salmonella strains with different STs,the ST34 strain 03130 exhibited the strongest virulence,followed by ST19strain 11207,ST40 strain 04017,and ST469 strain 03202.These results provide data support for conducting Salmonella risk assessments and developing source control strategies.