Isolation,Identification And PCR Detection Of A New Strain Of Duck-Derived Aerococcus Viridans

Aerococcus viridans belongs to the genus Aerococcus and is an opportunistic pathogen that is prone to drug resistance and can infect humans and animals,causing endocarditis,urinary tract infections,bacteremia,septicemia,pulmonary External infection,eye infection,postoperative infection,meningitis and arthritis.At present,the disease has been found in aquatic animals,pigs,sheep,etc.,but rarely reported in ducks.In July 2020,a pathogenic bacteria Y4 was isolated from the liver and spleen of dead ducks in a duck farm in Henan.The morphological,physiological and biochemical and 16 S r RNA identification of the bacteria was carried out,and the bacteria were determined to be green balloon bacteria.Through the animal regression pathogenicity test,it was determined that the bacteria had certain pathogenicity.Based on the whole gene sequencing technology and drug sensitivity test,the drug resistance and drug resistance mechanism of Aerococcus viridans Y4 were analyzed.Genomes were compared,and the unique genes of Aerococcus viridans were screened out,and then specific primers were designed to establish a rapid PCR diagnostic method,which provided a quick and easy detection method for the monitoring of Aerococcus viridans of duck origin.The result is as follows:1.In this study,a pathogen Y4 was isolated from the liver and spleen of dead ducks in a duck farm in Henan.It was characterized by a smooth surface,raised,milky white,and α-hemolytic Gram-positive cocci on the blood agar plate.Microscopic examination showed double and quadruple cocci.The results of biochemical identification showed that the isolate Y4 was positive for maltose,sucrose,raffinose,nitrate reduction,MP-VP test,urea,lysine decarboxylase,ornithine decarboxylase,esculin,etc.,and was positive for xylose.,lactose,glucose,sorbitol,hydrogen sulfide,citrate,peptone,mannitol,phenylalanine,methyl red tests were all negative.The 16 S r RNA gene sequence and Blast alignment of the isolated strain Y4 showed that the homology between the 16 S r RNA gene sequence and the published Aerococcus viridans on NCBI was between 94.8% and 99.9%.Based on the above identification,it is indicated that the bacteria is a duck-derived Aerococcus viridans.2.In this study,an animal regression test was performed on the isolate Y4,and the results showed that the bacteria had pathogenicity,but the pathogenicity was weak.After the challenge,the sick ducklings were depressed,lost their appetite or died,and were afraid of the cold.The necropsy of the dead ducklings showed that the liver and spleen were enlarged,there was a capsule outside the heart,and there was bleeding in the intestinal tract.The histopathological section showed that there were red blood cells in the liver,the liver cells were disorderly arranged,some were necrotic,and there were inflammatory cells,and there was steatosis.The boundary between the red and white pulp areas of the spleen was unclear,and the lymphocytes in the germinal center were decreased,and the cells were necrotic.The intestinal villi epithelial cells in the intestine lyse,die,and the intestinal villi become shorter.3.In this study,the whole genome of Aerococcus viridans Y4 was sequenced,and the CARD database was used for antibiotic resistance(CARD)analysis.The RGI analysis parameters were set,the data format was selected from the protein sequence,and the strict alignment mode was selected as the alignment standard.The results showed that Aerococcus viridans Y4 was resistant to antibiotics such as aminoglycosides,chloramphenicol,macrolides,tetracyclines,diaminopyrimidines,lincosamides,and streptomycin,and the resistance mechanism was antibiotic inactivation.and antibiotic target site variation.Combined with the drug susceptibility test,the resistance of the bacteria to lincomycin,streptomycin,erythromycin and kanamycin was consistent with the gene sequencing results.4.In this study,a pair of specific primers were designed by identifying the template sequence with PCR amplification specificity in the bacterial genome DNA sequence,and using BLAST primer to compare the primers online.The annealing temperature of primers was optimized.Based on the evaluation of sensitivity and specificity,a PCR detection method of Aerococcus viridans with good accuracy and specificity was established.In this study,the pathogenicity and drug resistance of Aerococcus viridans Y4 were studied,and a PCR detection method was established to provide technical support for clinical prevention and control of Aerococcus viridans disease in ducks.