Establishment Of Rapid Detection Method For Main Enterococcus From Chicken

As an important opportunistic pathogen,enterococcus can cause many kinds of animal and human infections,especially chickens,which are most sensitive to enterococcus.It can cause retarding growth,endocarditis,septicemia,encephalomalacia,osteomyelitis and other diseases of chicks,and bring serious losses to the poultry industry.Therefore,it is of great significance to explore the species and virulence of enterococci in chickens and establish a rapid detection method for main enterococci in chickens,for the prevention and treatment of enterococci in chickens and early infection.In order to investigate the infection of enterococcus gallinarum and enterococcus hirae in chicken source,1-day-old sick and weak chicks were collected for gross autopsy and observation of microscopic pathological changes.The bacteria were isolated from chicken the liver and morphological observation and biochemical identification were carried out.The 16S rDNA gene sequence was amplified by PCR.The isolates strains were identified by colony morphology and sequencing comparison.The results indicate that the belly of the sick,weak chick was larger,the umbilical hemorrhage,autopsy showed that the liver was earth yellow,the yolk malabsorption;The microscopic examination showed pulmonary hemorrhage;Liver tissue hyperemia,congestion,full of vacuoles.The isolated strains could grow on nutrient agar medium,and the colony morphology was slight uplift.On bile salt and aesculin agar,the medium was brown and black.Gram staining is positive cocci;Biochemical identification results were consistent with the characteristics of enterococcus.Comparison of 16S rDNA sequences showed that EH-1 strain was identified as enterococcus hirae,and EG-1 and EG-2 strain was identified as enterococcus gallinarum.This study was the first to identify enterococcus gallinarum and enterococcus hirae infection in chickens in China.In order to understand the virulence of different enterococcus species and virulence gene carrying status.Six virulence genes of enterococcus were detected by PCR,and the virulence of 3 strains of enterococcus was analyzed by chicken embryo pathogenicity test.The results showed that only ace gene was detected in EG-1.Six virulence genes were not detected in EH-1 and EG-2 isolates.Four virulence genes including efa A,asal,ace and gel E were detected in enterococcus faecium Efm-1.After inoculation in allantoic cavity of chicken embryo,no obvious lesions were found in viable chicken embryo,while obvious hyperemia and bleeding lesions were found in dead chicken embryo.The median lethal dose of enterococcus gallinarum,enterococcus hirae and enterococcus faecium were 9.33×10~4CFU,6.10×10~6CFU and 1.31×10~6CFU,Respectively.It is suggested that the number and species of virulence genes carried by different enterococcus species were different,and the pathogenicity of the three enterococcus species in this study is weak and there is no significant correlation between the virulence and the carrying status of different virulence genes.In order to establish a high-speed detection method for the major enterococcus from chickens,specific primers for enterococcus gallinarum and enterococcous hirae were designed.SYBR Green I fluorescence quantitative PCR method was established for the two enterococcus species.Based on SYBR Green I single fluorescence quantitative PCR method for the four enterococcus species.Double fluorescence quantitative PCR of enterococcus gallinarum and enterococcus faecalis,enterococcus hirae and enterococcus faecium was established.The results indicate that its high specificity and sensitivity,the lowest concentration of enterococcus gallinarum was 8.9×10~1CFU/m L and the lowest concentration of enterococcus hirae was 6.4×10~1CFU/m L.The repeatability was good and the coefficient of variation of CT values ranged from 0.78%to 2.9%and from 2.9%to 3.3%.The established double quantitative PCR method for detection of main enterococcus from chicken can effectively distinguish the four enterococcus species.The lowest concentration of the detected strain could reach 10~1CFU/m L.The coefficient of variation between CT value and Tm value was less than 4%.It can be used for the rapid detection of four kinds of enterococcus mixed infection,improve the detection efficiency and more economic benefits.