Isolation And Identification Of Pathogen Of Subskin Abscess And Control Of Cecropin AD

Subcutaneous abscess is one of the common skin diseases of sheep,which is widely prevalent in China.It has a long course of disease and strong infectivity.The disease can lead to emaciation and decrease of resistance of sheep,which seriously affects the benefit of sheep breeding and production.The presence of pathogenic bacteria in the environment and the broken skin integrity of sheep were the main causes of this disease,which caused great difficulties in the prevention and treatment of sheep subskin abscess.In recent years,research reports on the pathogenic bacteria of subskin abscess showed that the main pathogenic bacteria of subskin abscess were Corynebacterium pseudotuberulosis(C.pseudotuberulosis)and Staphylococcus aureus(S.aureus).After the formation of subskin abscess,it will continue to increase.Conventional antibacterial drugs are not effective in the treatment of this disease,and it is easy to produce drug resistance.Since 2020,China has fully entered the era of "forbidden resistance in feed end,reduced resistance and limited resistance in breeding end".Therefore,the research and application of antibiotic substitutes in breeding industry has become a hot spot.At present,antibiotic substitutes on the market mainly include antimicrobial peptides,Chinese herbal medicine and plant essential oils,etc.among which antimicrobial peptides have been studied and applied for more than 40 years,and are known for their strong thermal stability,wide antimicrobial spectrum,difficulty in developing drug resistance and ability to improve body immunity.In this study,samples of subskin abscess were collected from some large-scale Hu sheep farms in Qingyang City,Gansu Province and Yulin City,Shaanxi Province.Isolation,purification,identification and antimicrobial susceptibility tests were conducted successively.Then,the antimicrobial activity of antimicrobial peptides against the isolated strains was determined by AGAR diffusion method and microdilution method.In addition,the isolated pathogenic bacteria were used to infect rabbits to form subcutaneous abscess.At the same time,antimicrobial peptide(cecropin AD)was used to prevent and treat subcutaneous abscess in rabbits.Finally,the antimicrobial peptides were used in the prevention and treatment of subskin abscess in Hu sheep farm to verify the prevention and treatment effects of antimicrobial peptides on subskin abscess.The experimental results are as follows:(1)Sampling of 26 representative cases of subskin abscess from some large-scale Hu sheep farms in Qingyang City,Gansu Province and Yulin City,Shaanxi Province was conducted.After laboratory isolation and purification,PCR identification and sequencing,it was found that only 10 samples of Staphylococcus aureus and 7samples of Corynebacterium pseudotuberulosis were isolated.There were 8 cases of mixed infection of S.aureus and C.pseudotuberulosis)(including 1 case of secondary infection of Staphylococcus epidermidis,1 case of mixed infection of S.aureus and Corynebacterium mucosa,and1 case of mixed infection of C.pseudotuberulosis and Staphylococcus epidermidis.The isolation rates of S.aureus and C.pseudotuberculosis were 69.2%(18/26)and57.7%(15/26),respectively.The gene sequences of the 18 strains of S.aureus and the standard sequence of S.aureus in the gene bank(KX456106.1)were all more than98.04% homology.The genetic sequence of 15 isolates of C.pseudo tuberculous was more than 99.31% with the standard sequence of C.pseudo tuberculous in the gene library(KX580966.1),so S.aureus and C.pseudotuberulosis were the main pathogens of subcutaneous abscess.(2)Antibiotic susceptibility test results showed that all 18 strains of S.aureus were resistant to penicillin,phenoxacillin,cefalexin and ampicillin,and the drug resistance rate was more than 44%.The drug resistance rates of the 15 strains to penicillin and ampicillin were 100%,and the drug resistance rates to neomycin,kanamycin,amikacin,ceftriaxone and levofloxacin were over 45%.(3)By agar diffusion diffusion test,it was found that the diameters of antimicrobial zone of S.aureus isolated by cecropin AD were 40 mm,which was significantly larger than the maximum diameters of antimicrobial tablets tested(33.5mm).The minimum inhibitory concentration was 3906 μg/m L.Because the in vitro culture medium of pseudo C.pseudotuberulosis contains phospholipids which inhibits the function of antimicrobial peptides,the in vitro antibacterial effect of antimicrobial peptides on C.pseudotuberulosis by agar diffusion diffusion method is not obvious.(4)The use of cecropin AD for the prevention and treatment test found that cecropin AD can completely prevent(incidence rate of 0)and cure(cure rate of 100%)experimental rabbit subcutaneous abscess caused by S.aureus.However,it could not prevent and treat subcutaneous abscess caused by C.pseudotuberulosis(5)The discovery of using cecropin AD to prevent and treat subcutaneous abscess disease in sheep.The incidence of abscess in the prevention group was 0.6%(n= 834),which was significantly lower than that in the control group(12.4%).The cure rate was 90.0%(n=110)by surgical drainage combined with cecropin AD,while the incidence rate was still 100% in the control group.Therefore,subedermal abscess is mainly caused by S.aureus and/or C.pseudotuberculosis,and cecropin AD has a significant effect on prevention and treatment.