Epidemiological Characteristics And Pathogenicity Analysis Of Salmonella Isolated From Meat Duck Farms In Shandong Province And Surrounding Areas

Salmonella is generally recognized as an important zoonotic and foodborne pathogen,which seriously endangers the development of breeding industry and public health due not only to its pathogenicity,but also to its ability to adapt to different environment and a broad range of transmission routes.In recent years,with the intensive and large-scale development of duck farming,the prevalence and spread of Salmonella in duck farms have shown diversity and complexity.This study aims to evaluate the contamination status of Salmonella in different breeding procedure systematically and transmission lines between isolates obtained from living ducks and duck farm environmental samples.Then,we can reveal the main ways and critical control points of Salmonella transmission links during duck breeding.The above researches are vital prerequisites for effectively preventing and controling Salmonella infection in ducks.In this study,we isolated and identified Salmonella from duck embryos,cloacal swab samples from ducks at different ages,and rearing environment samples in large-scale breeding farms and hatchery.In order to clarify the epidemic characteristics among isolates and explore the potential sources of Salmonella infection in the duck’s body,we performed serotype and molecular typing of isolates from diverse environmental sources and ducks at different ages.In addition,we conducted pathogenicity tests in ducklings to assess duck-origin Salmonella virulence and track the dynamics in response to Salmonella infection in vivo.The above research results will provide theoretical basis and data support to prevent and control duck-derived Salmonella infection.1.Contamination status and serotype distribution of Salmonella from ducks in breeding processIn present study,736 samples collected from cloacal swabs,environment（fece,water,feed,soil and air）and healthy duck embryos from large-scale meat duck farms in Heze,Jining,Liaocheng and Guantao.Through conventional culturing methods,double PCR amplification and microbial mass spectrometry identification,a total of 160 Salmonella strains were isolated,with a total isolation rate of 21.74%;the detection rates of Salmonella from all samples in the breeding chain were as follows:duck embryo（40%）>air（35%）>feces（25%）>duck bodies（22.71%）>feeds（17.46%）>drinking waters（13%）>soils（6.25%）.The positive rate of Salmonella carried in ducklings at 1-day-old（44%）and 15-day-old（37.97%）was significantly higher than that at 25-day-old（4.42%-4.49%）.This result indicated that Salmonella infection tends to decrease with increasing age.24 serotypes were identified from 160 Salmonella strains.The dominant serotypes were S.Typhimurium（30.15%）,S.Kottbus（13.97%）and S.Enteritidis（10.29%）.S.Typhimurium were mainly isolated from ducks of different ages and healthy embryos.S.Kottbus mainly existed in duck’s cloacae and environmental samples（fece,water,soil and feed）.A majority of S.Enteritidis was isolated from ducks,environmental samples and embryos.We preliminarily speculated that S.Typhimurium was mainly transmitted vertically,depended on horizontal transmission for S.Kottbus and possessed the above two transmission ways for S.Enteritidis.From the perspective of geographical distribution,Salmonella from various geographical sources had their own predominant serotypes,which indicated that the prevalence of Salmonella had even been limited to a specific region.2.Molecular typing and traceability analysis of Salmonella isolates belonged to dominant serotypesUsing the Clustered Regularly Interspaced Short Palindromic Repeats（CRISPR）typing to identify genotypic relationships among isolates of S.Typhimurium and S.Enteritidis,13CRISPR types were detected in S.Typhimurium isolates,and a total of 3 different S.Enteritidis CRISPR were identified,which named using a number suffix to TCT and ECT,respectively.The common CRISPR type TCT3 among S.Typhimurium strains was mainly derived from duck embryos and duck’s bodies,indicating might have the potential to vertically transmit between duck embryos and ducks.Duck embryos were the main source of infection:TCT strain was the most abundant strain isolated from the embryos,and the genetic relationship of TCT strains was 95.8%,which fully reflected that Salmonella strains from duck embryos were rich in evolutionary sources,but had genetic stability.Therefore,duck embryos became the gene storage pool of different strains.The dominant type of S.Enteritidis isolated from duck’s bodies,environment（feces,waters and feeds）and duck embryos was ECT1,indicating that S.Enteritidis isolates were spread by horizontal and vertical transfer.The genetic kinship of S.Enteritidis isolates from different sources was more than 90%,revealed a high degree of genetic stability.S.Kottbus were identified 4 ST types by multilocus sequence typing（MLST）.ST1546was common among strains isolated from Liaocheng,which showed clonal transmission among different duck farms in this region.However,ST198 were detected in isolates from different areas.Isolates collected from feces,feeds and soils in different areas belonged to ST1690.ST321 isolates were observed from duck’s bodies and feeds in different areas.This revealed that S.Kottbus had horizontal spread among strains from same or different regions,because S.Kottbus were isolated primarily from environmental samples.The above traceability analysis at the molecular level was consistent with the epidemic characteristics of Salmonella serotypes during breeding.3.Pathogenicity test of Salmonella isolates from ducksThe recombinant strains of S.Kottbus-pFCcGi and S.Enteritidis-pFCcGi were successfully constructed by electroporation with fluorescent plasmid pFCcGi.The LD₅₀of origin strains（S.Kottbus and S.Enteritidis）by infecting ducklings were 1.23×10¹⁰,8.77×10~5CFU/m L,respectively.LD₅₀values were found to be 1.71×10¹⁰（S.Kottbus-pFCcGi）and5.99×10~6（S.Enteritidis-pFCcGi）CFU/m L for recombinant strains in ducklings.The above results showed that the pathogenicity of S.Enteritidis-pFCcGi was higher than that of S.Kottbus-pFCcGi,which was cosistent with pathogenic differences between S.Kottbus and S.Enteritidis from origin strains.The heart,liver,spleen,lung,kidney,duodenum,caecum,thymus and bursa of fabricius were collected at 2h,6h,12h,24h,48h and 120h after the infection to prepare paraffin sections for HE staining,immunofluorescence and q PCR.The results confirmed that the ducklings with infecting S.Enteritidis-pFCcGi had a longer duration of disease than with infecting S.Kottbus-pFCcGi.The degree of inflammation,hemorrhage and steatosis of all organs in ducklings from S.Enteritidis-pFCcGi group was more serious than that in S.Kottbus-pFCcGi group.Bacterial colonization signals were observed in both groups at 2h after infection.Meanwhile,the cecal contents were collected and significantly higher bacterial cfu was found.Salmonella load reached its maximum at 24h after infection.Meanwhile,the bacterial load of different tissues in S.Enteritidis-pFCcGi group was higher compared with investigated tissues in S.Kottbus-pFCcGi group.The colonizing bacteria were observed in both groups at 120h after inoculation.