| Salmonella,being a pathogen of zoonotic disease and having a wide range of hosts,is an important food-borne pathogenic bacteria;and that has the characteristic of vertical propagation bringing the most threatening to the breeding industry,especially in the breeding duck farms.Since 2014 to 2016,the rate of embryonic mortality and weak hatching of the large-scale duck hatchery in Shandong was significantly higher,and the major dead duck embryo was bacon,which was suspected to be caused by large area bacterial infection.The resistance rate of Salmonella to antibiotics is increasing as the extensive use of antimicrobial agents in the prevention and treatment of livestock and poultry diseases,especially the emergence of multiple drug resistance,which brings difficulties to the effective prevention and cure of salmonellosis.This study was aimed to investigate the infection status of pathogenic bacteria in dead duck embryos,and further to explore the reasons of death during hatching.We preliminarily understand the drug resistance situation of Salmonella typhimurium in Shandong and explored the interaction between resistance and antibiotics by using the experiment of drug sensitivity test and drug resistance gene detection,providing a theoretical basis for the rational drug use in the production.In addition,we analyzed the virulence genes of ducks Salmonella,which is significant for the development of safe and effective vaccine.The study is divided into four parts:1.Isolation and identification of bacteria from dead duck embryos in Shandong provinceA total of 282 strains were isolated from 315 dead duck embryos of 12 batches in Shandong province and was divided into 26 species,by isolating and culturing the bacterial and observing their morphological staining and 16 SrDNA in the non-damaged dead duckembryos,among which 24 s were infected by bacterial with the infection rate being 77.8%.The percentage of Escherichia coli,Salmonella and Proteus mirabilis in all the bacteria isolates was 38.30%(108/282),26.24%(74/282)and 8.16%(23/282)and they were the main flora in dead duck embryos.The results showed that bacterial infection is the main reason for the increasing of mortality and weak hatching,and Salmonella was account for a major role.2.The investigation of resistance of Salmonella from dead duck embryosThe resistance of of 17 kinds of antibiotics(aminoglycosides,quinolones,?-lactam drugs,etc.)was tested in the 74 Salmonella strains by KB method.The results showed that the 74 Salmonella strains have resistance to 17 kinds of antibiotics.The resistance of ?-lactam drug is high: the resistance rate of ampicillin and cefotaxime was 31.08% and 29.73%,respectively.In terms of aminoglycoside,the highest resistance rate was gentamicin(22.97%),followed by streptomycin(9.46%)and kanamycin(2.70%);while in the quinolone drug,the resistance rate was lower,and the resistant drug rate of ciprofloxacin and norfloxacin was 4.05%,and the resistance rate of ofloxacin was only 2.70%.Similarly,the drug resistance rates tetracycline and chloramphenicol were still low with 5.41% and 4.05%,respectively.The study showed that the quinolones and kanamycin can be selectively used in the treatment and prevention of pathogenic bacterial infections in the duck hatching field in Shandong.The drug instructions strictly control the dose when quinolones are selected.3.The detection and identification of resistance gene from the Salmonella isolatesUsing multiplex PCR method,the aminoglycoside modifying enzyme(AME)genes of the 74 Salmonella strains were tested.A total of 13 strains(17.56%,13/74)were identified as positive for ant(3'')-Ia,aac(6')-Ib and aph(3')-IIa,while no strain was positive for 16 S methylase gene.The quinolone-resistant genes,qnrS,oqxA and oqxB were detected positive as 14.85%,9.46% and 2.70%,respectively,while qnrA,qnrB,qnrC and qnrD were not detected in all the 74 Salmonella strains.TEM gene,as a ?-lactam resistance gene,was detected from 27.03% of the 74 Salmonella strains.The results showed that AME gene,quinolone-resistant gene and ?-lactam resistance gene were widely existed in the Salmonella isolates from duck embryos,and the drug resistance of the Salmonella strains is not closelyrelated to the drug resistance genes.4.The detection and identification of virulence genes in Salmonella isolates from dead duck embryosEight kinds of virulence genes were detected in the 74 Salmonella strains of using PCR.The detection rates of SPI-1,SPI-3,SPI-4,SPI-5,stn and invJ was 81.19%,94.59%,94.59%,79.73,100% and 66.22% respectively,while the SPI-2 and fim were not detected.The percent of strains with 2,5 and 6 kinds of virulent was 2.7%,31.06% and 56.76% in 74 Salmonella strains,respectively.The results showed that multiple virulence island core protein genes were existed in in the Salmonella isolates from dead duck embryos in Shandong province,suggesting the toxicity of Salmonella being strong in contaminated duck embryos and that may be the major reason for the death in the hatchery process. |
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