Isolation,Identification And Virulence Genes Detection And Analysis Of Salmonella From Duck Embryos

Salmonella is generally recognized an important foodborne pathogen in the research fields of zoonotic diseases and public health,with serious harm to breeding industry and public health.At present,more than 2600 serotypes have been discovered.Many serotypes can cause various illnesses in animals and humans and even death.Salmonella can both transmit horizontally or vertically.Breeding eggs play an important role in the spread of Salmonella and food contamination.Shandong is a major duck farming province.Studies have suggested that duck embryos mortality has increased significantly in recent years.Salmonella infection is an important cause.This study aimed to further evaluate the infection status of Salmonella in duck embryos through the isolation and identification of Salmonella derived from duck embryos,and to clarify the infection status of Salmonella in vertical transmission by eggs;the serotype distribution and predominant serotypes of the isolates were determined in local duck farms;through the serotype classification;explored the genetic evolutionary relationship among isolates through genotyping of isolates;through detecting the virulence genes from the isolates,the detection rate of the virulence genes and the numbers of virulence genes carried by each isolate are clarified;in order to clarify the pathogenicity of isolates selected from dominant serotype,measured LD₅₀and observed the clinical symptoms and histopathological changes in animal pathogenicity tests.The above research results laid the foundation and provide experimental materials for the epidemiological study of Salmonella from ducks,and made recommendations for the standardization of biological safety in duck farms and duck breeding hatcheries.1.Isolation and identification of Salmonella from dead duck embryosIn the present study,conventional culturing methods,morphological staining,biochemical test,PCR identification and serotyping were used to isolate and identify Salmonella from 335 undamaged dead duck embryos collected from multiple large-scale breeding duck farms in Shandong.The results showed that 60 Salmonella strains were divided with 17.9%infection rate.Their dominant serotypes were Salmonella Cottbus and Salmonella Typhimurium.Genotyping results showed that the similarity coefficient of each strain was41%-100%,and there were 7 genotypes in total.2.Detection and identification of virulence genes of Salmonella derived from duck embryosThe 23 virulence genes of 60 Salmonella strains were detected by PCR.The results showed that detection rates of invA,sipC,sipA,sopA,ssaB,orf319,pipC,misL,stn were100%,the detection rates of sseL,mgtC,sopB,spvB,sopE,pef,spvC,siiE,spvD were 60%;the average detection rates of spvR,spvA,ssaR,rck,and sefA were 40%.The detection rate of virulence genes tested in 60 strains was 100%.The statistical results about the detection rate of virulence genes showed that a single strain carried at least 14 kinds of virulence genes and a maximum of 23 virulence genes,and all isolates generally carried multiple virulence genes.3.Pathogenicity test of Salmonella derived from duck embryosA lethality test was carried out using 10 Salmonella isolates selected at random.The results showed that the lethality rates were 33%to 100%.The pathogenicity test in mice was used to analyze the correlation between the pathogenicity and the numbers of virulence genes carried by the isolates.Salmonella typhimurium SD04 strain and Salmonella Cottbus SD20strain used to detect LD₅₀.SPF mice were injected intraperitoneally with 0.2mL/mouse at a concentration of 10⁴?10⁹CFU/mL.Meanwhile,the control group was given the same dose PBS.After inoculation,heart,liver,spleen,lung,kidney and small intestine tissues were collected at 6h,12h,24h,48h,72h and 96h,respectively,which made HE pathological sections and immunofluorescence detection.The results showed that the LD₅₀of the SD04and SD20 strain were 1.26×10⁵and 1.86×10⁸CFU/mL,respectively.The infected mice showed messy fur and diarrhea at the initial stage.Autopsy and pathological sections showed that various tissues and organs of the mice had different degrees of bleeding and necrosis.Salmonella was colonized in the tissues and organs collected in the test group,and there were no obvious clinical symptoms and histological changes in the control group,and no Salmonella was found.For Salmonella strains isolated and identified in dead duck embryos from local hatchery,we detected the distribution of serotypes and molecular typing,and carried out virulence-associated genes screening and pathogenicity analyses,which indicated that Salmonella infection had 17.9%positive ratio in large scale duck hatchery in Shandong Province.The dominant serotypes were Salmonella Cottbus and Salmonella Typhimurium.Salmonella isolates carried generally virulence genes,which were mainly harbored in Salmonella pathogenicity island(SPI).Pathogenicity studies preliminarily indicated that pathogenicity was positive correlation with the numbers of virulence genes carried.