Biological Characteristics Of Erysipelothrix Rhusiopathiae Isolated From Anhui And Estabishment Of A Real-Time PCR Method

Swine erysipelas is a time-honored bacterial infectious disease,and it was rarely seen more than 20 years in China.However,Guangdong,Jiangsu,Fujian,Yunnan,Hunan,Hubei,Anhui and other area were reported increased the incidence of Swine erysipelas in recent years.At the same time,the disease in the United States,Japan,Brazil and other countries also increased significantly,which caused cause great economic loss.This study collected clinical information form anhui province swine erysipelas popular characteristic;Agar gel precipitation tests were used to invistigate serotype;PCR and sequence analysis methods were used to study phylogeny of spa A gene;PFGE technique was used to identify genetic typing of the advantage bacteria;Broth microdilution method was used to detect the drug sensitivity;Perceptual experiments using acriflavine isolates of acriflavine resistance;.At the same time,We established SYBR Green I Real-Time PCR first time in China,which provided the necessary technical support for the research of molecular biology and epidemiology of swine erysipelas in Anhui area.The results show that Swine erysipelas were occurde in 2-10 months in Anhui,the peak period were 7-9 months,the incidence of pigs are mainly concentrated in the fattening stage(52.4%),acute septicemia(92.9%)were the most clinical symptoms;All42 strains belonged to serotype 1a.spa A gene was 98.5% to 100% homologous to the reference strains.All strains were Met-203 and Ile-257-type strains;PFGE revealed 8different genotypes,with 88.8% to 100% similarity.ER2(54.8%)was the dominant PFGE genotype,and erysipelas attenuated vaccine strains(G4T10 and GC42)were the same genotype strains;The sensitive rate of isolated strains for Penicillins,Quinolones and Chloramphenicols were over 80%,and Aminoglycosides and Lincosamides were above60%.100% strains resisted 4 or more drugs,and 95.2% strains resisted of 5 or more drugs.GEN+KAN+STR+CLI+LIN consists of the maximum ratio of 76.2%;4 strains(9.5%)isolates resisted acriflavin;The specificity experiment of Real-Time PCR show that there is no specific amplification in commom bacteria of pigs.It has good specificity,and the minimum 20 copies of nucleic acid were detected.It can be detected more than 100 times higher than common PCR,the repeat between within groups and between groups were between 0.033-0.17,the variation coefficient were between 0.22-2.09,which has a very good stability.The model test in mice showed that Real-Time The detection rate(71.7%)was significantly higher than that of the bacterial isolates(41.7%)and common PCR(50%)(P < 0.05),and the detection rate of Real-Time PCR was significantly higher than that of common PCR.The results show that Erysipelothrix rhusiopathiae serotype 1a was the prevalent serotypes in Anhui area.The homology of the spa A was high and had small variations.All the isolates came from one clone strain.Met-203-type and Ile-257-type strains had high pathogenicity and were the most prevalent pathogens type in Anhui area.The strains in Anhui showed closely related PFGE gene type,there was no correlation between PFGE genotype and location separation and separation between isolates derived from the same clone,but there is variation in the dissemination of in the process,the outbreak of swine erysipelas in Anhui has nothing to do with the attenuated virulence;Anhui swine erysipelas Bacillus strains of multi drug resistance is strong,the proportion of GEN+KAN+STR+CLI +LIN were 76.2%,penicillins and quinolones were the first choice to treat swine erysipelas in Anhui;established swine erysipelas bacillus by Real-time PCR detection method for the first time in China,the specificity,sensitivity high,than that of conventional PCR sensitivity is more than 100 times,with good repeatability can be used for the qualitative and quantitative detection of clinical samples.