Epide Miological Investigation Of Riemirella Anatipestifer In Guangdong Province And Research Plasmid Carrying FloR Gene

Riemirella anatipestifer is a gram-negative pathogen belonging to the Flavobacteriumfamily in the rRNA superfamily V that can easily infect young ducks,geese and other waterfowl animals.It has been widely spread all over the world,especially Asian contries.Compared with Salmonella and Escherichia coli,R.anatipestiferhave numerous serotypes,25 types has been found so far.It is difficult to develop corresponding inactivated vaccines based on serotypes,and isolated strains are highly resistant to antibiotics,thus causing significant economic losses to the waterfowl breeding industry.At present,although there are many reports on the drug resistance of R.anatipestifer,there are few studies on plasmid-mediated molecular resistance mechanisms.The purpose of this study wasto understand the epidemiology of R.anatipestifer isolates in Guangdong provinceinrecent years,and continuously monitor the drug resistance.Furthermore,the molecular mechanism of drug resistance was also studied.It can not only guide the clinical use ofR.anatipestiferinfectionin Guangdong province,but also trace to its source from the molecular perspective and control the epidemic of animal-derived diseases caused by R.anatipestifer.From August 2015 to June 2017,115 samples of typical infectious serositis of ducks and gooses in Guangdong were collected and R.anatipestifer isolates were cultured.The outer membrance protein gene was used to identify isolates by specific PCR.A total of 93 strains of R anatipestifer were obtained,among which 63 were from ducks and 30 from geese.Rabbits were immunized with I serotype strain-RA1 to prepare type I antiserum.Serotype I was identified by plate agglutination method for the 220 strains from 2007 to 2017 years in our laboratory,and 119 strains were serotype I.The proportion of serotype I was 54.09%,which was the dominant serotype in Guangdongrecently years.The drug sensitivity test was carried out 93 strains of R anatipestifer by agar dilution method.The results showed that isolates had higher resistance to clindamycin and enrofloxacin,and there wereseriousphenomenon of multiple drug resistance.Theresistance rates of isolates to cefoxitin,streptomycin,amikacin,doxycycline,tetracycline,quinolol and other drugs were low.The strainswere sensitive or intermediateto ampicillin,amoxicillin and cefotifuroxil.The resistance genes were detected by PCR,A total of 17 resistant geneswere detected in 93 isolates.The aph(3’)-Ⅰa and ermA genes were first detected for R.anatipestiferin Guangdong area.The PFGE were carried out by SmaI-digested chromosomal DNA from 119 isolates.In total,33 different Sma I-PFGE types(with similarity percentage of ≥85%)were detected among 110 available PFGE profiles in R.anatipestifer isolates,including 19 clusters and 14 single types.Resultshowed diverse genotyping band patterns,Low degree of homology was observed among these isolatesand there was a clonal spread on a small scale.The plasmid carrying floR gene was located by S1-PFGE and Southern blot.The plasmid was extracted by alkaline lysis and sequenced.The results showed that pRAGD185 contained 18982 bp and had an overall C+G content of 38.61%.The plasmid contained 16 putative protein-coding regions.pRAGD185 included 2 catB genes,2 tetX genes,moveable protein genes,multiple ABC transporter genes,The synthesis enzyme gene,floR gen,lysR gene,ISCR2,IS4351,dhfr gene and three unknown protein genes were identified.