Isolation And Identification Of Salmonella Isolates From Gestational Mink And Their Pathogenicity In Mice

In recent years,the mink-breeding industry due to intensive methods of farming is burdened with losses from numerous infectious agents,of which one is the Salmonella.Salmonella species are the most important zoonotic pathogens worldwide and cause considerable harm to mink.This disease causes serious complications during mink pregnancy,such as fever,diarrhea and the weight quickly decreases.Similarly,salmonella infection during pregnancy can lead to miscarriage and has been associated with huge losses to mink farming.This study aimed to survey the serotypes,antimicrobial resistance,and virulence-associated genes of Salmonella recovered from different mink farms.Meanwhile,the pathogenicity of the representative strains to mice has been assessed in this study.From March to May 2019,liver samples,intestinal and intestinal contents of the mink showing gastroenteritis were collected in Weifang,Shandong Province.The samples were kept insterile centrifuge tube and transferred to the laboratory for further bacteriological analysis.Presumptive Salmonella colonies were identified by biochemical methods,gram-stain reaction and polymerase chain reaction?PCR?amplification of the inherent gene invA.And all Salmonella isolates were serotyped according to the Kauffmann-White scheme by slide agglutination with O and H antigen-specific sera.In this study,thirty-five Salmonella isolates were collected from mink farms in China.Thirty-four of the Salmonella isolates were Salmonella Enteritidis and the other 1 belonged to Salmonella Typhimurium,named as S.Enteritidis-SD-1 to S.Enteritidis-SD-34,and S.Typhimurium-SD-1,respectively.PCR targeting 10 virulence genes were performed in this study to detect the identified Salmonella serovars virulence.The virulence genes analysis showed that all Salmonella isolates were positive for invA gene and mgtC gene,100%,followed by sopE gene 97.14%,tolC gene 97.14%,stn 94.29%,avrA gene 88.57%,spiA gene 82.86%,hilC gene 68.57%,spvB gene 62.86%,and pefA gene 42.86%.The susceptibility of Salmonella to 13 antimicrobials was tested using the disc-diffusion method.For the Salmonella isolates,the most resistance was to Neomycin?42.86%?,followed by Kanamycin?34.29%?,Tetracycline?31.43%?,Ampicillin?28.57%?,Gentamicin?25.71%?,Streptomycin?25.71%?,Amoxicillin?22.86%?,Ciprofloxacin?22.86%?,co-trimoxazole?17.14%?,Norfloxacin?11.43%?,Ofloxacin?11.43%?,Cefoperazone?5.71%?.And less resistance was detected to Cephazolin?2.86%?.In addition,12 isolates exhibited multidrug resistance.All 35 Salmonella isolates were also assessed for the presence of 12 antibiotic resistance genes.The prevalence of int1 gene for the isolates were 100%,aadA1 gene 51.43%,blaTEM-1 gene 42.86%,blaPSE-1 gene 37.14%,aadA gene 34.29%,Cat3 gene 31.43%,tetB gene 31.43%,gyrB gene 28.57%,sul?gene 28.57%,tetA gene 25.71%,parE gene 22.86%,and cat2 gene 17.14%.To clarify the pathogenicity of the isolates in mice,the experiments were performed on66 healthy Kun-ming mice,being divided into 11 groups on average.According to serotypes,virulence gene content of the isolates,S.Enteritidis-SD-1 and S.Typhimurium-SD-1 were selected for animal experiments.As a result,the mice infected with Salmonella showed obvious clinical symptoms,such as loss of appetite,sluggish action,vomiting and diarrhea,and the feces were liquid or watery.At the time of autopsy,it was found that the internal organs were yellow stained in varying degrees,irregular thickening of gastric folds,punctate hemorrhage of intestine,enlargement of spleen,hemorrhage and necrosis of liver,and hemorrhage of lung.Histopathological study showed that S.Enteritidis-SD-1 infected mice had alveolar hemorrhage and edema,intestinal mucosa and villi shedding,hepatocyte necrosis,spleen edema and macrophage increase.In addition,S.Typhimurium-SD-1 infected mice had a large number of cellulose like exudates in the alveoli,intestinal villi shedding,liver necrosis and spleen macrophage increase.The median lethal dose of S.Enteritidis-SD-1 and S.Typhimurium-SD-1 were 1.02×10^6.0CFU and 2.14×10^6.0CFU,respectively.This study proves that Salmonella Enteritidis is widespread in mink.Salmonella isolates were commonly resistant to antimicrobial drugs and multiple drug resistances were very serious.It was shown that the representative strains has strong pathogenicity in mice.This study provided a theoretical basis for effective prevention and treatment of salmonella in mink.Our study also provides experimental evidence for the clinical treatment of salmonella.These data have significant clinical and public health import.