Screening And Preliminary Application Of The Bacillus Velezensis C205 For Prevention And Treatment Of Necrotizing Enteritis In Chickens

Clostridium perfringens is a gram-positive anaerobic bacterium that resides in the intestines of humans and animals.When pathogenic C.perfringens reaches a certain concentration in the gut,it can cause necrotic enteritis.For many years,antibiotics have been used as growth promoters and to control necrotic enteritis in poultry.However,the improper use of antibiotics in livestock production has led to various negative effects,including decreased animal immunity,increased bacterial resistance,and the presence of drug residues in animal-derived food,which has attracted significant attention from society.Therefore,the development of antibiotic alternatives has become an urgent matter.Bacillus velezensis is highly resistant and produces abundant antimicrobial metabolites such as lipopeptides,polyketides,and bacteriocins.It also regulates intestinal microflora balance,enhances immunity,and exhibits anti-stress effects.As a substitute for feed antibiotics,B.velezensis has received considerable attention.In this study,a strain of B.velezensis C205,which exhibited antagonistic effects on the growth of C.perfringens,was selected from a collection of laboratory-preserved probiotic strains using anaerobic co-cultivation and in vitro antibacterial assays.The feasibility of this strain in preventing and treating necrotic enteritis caused by C.perfringens was evaluated through animal experiments.The research results are as follows:1.Screening and identification of B.velezensis C205The laboratory-preserved six probiotic strains and C.perfringens ML2 were revived,and antagonistic spore-forming bacteria capable of inhibiting the growth of ML2 were screened through anaerobic co-culture.The results showed that the strain with the number C205 could significantly inhibit the growth of ML2 under initial inoculation ratios of 1:1and 10:1.In the in vitro Oxford cup antibacterial test,the supernatant of the fermentation broth of C205 strain had a good inhibitory effect on ML2,with an inhibition zone of about 20 mm.The Gram staining of the C205 strain showed a blue-purple rod shape.16S r RNA sequencing results showed that the C205 strain belonged to Bacillus velezensis,and was therefore named B.velezensis C205.The growth curve results showed that C205grew well in RCM medium,and was in the logarithmic growth phase between 2-8 hours,and entered the stable period after 8 hours.2.Establishment of a quantitative real-time PCR detection method for C.perfringensA pair of specific primers were designed based on the cpa gene of C.perfringens and quantitative real-time PCR detection method was established to detect C.perfringens.After optimizing the reaction conditions,the method showed good linear relationship,with a standard curve of Y=-3.410*X+40.48,a regression coefficient of R~2=0.9986.Compared to conventional polymerase chain reaction(PCR),this real-time quantitative PCR method displayed a much higher sensitivity of 3.67×10~2copies/μL,which demonstrated its superior capability for detecting low abundance targets.Furthermore,the coefficient of variation(CV%)for this method was observed to be consistently below5%,which is a testament to its outstanding reproducibility and reliability.3.Evaluation of the preventive and therapeutic effects of B.velezensis C205 on necrotic enteritisThe C.perfringens ML2 infection model was established by oral administration.The preventive group was given feed containing C205 strain(4×10~6CFU/g)one week before ML2 infection,while the treatment group was given feed containing C205 strain(4×10~7CFU/g)one day after ML2 infection.Enramycin was used as a drug control.The results showed that C205 could effectively reduce the intestinal bleeding and distension caused by ML2 infection and protect the health of chickens.Compared with the group infected with C.perfringens group,C205 could alleviate the damage to intestinal villi caused by ML2 infection.Quantitative rael-time PCR detection showed that the load of bacteria detected was 10~6CFU/g in the C.perfringens infection group,while in the preventive group it was 10~4-10~5CFU/g(P<0.05),in the C205 treatment group it was 10~4CFU/g,and in the enramycin treatment group it was 10~3CFU/g(P<0.05),indicating that both C205 and enramycin had good preventive and therapeutic effects on C.perfringens infection.In conclusion,B.velezensis C205 selected in this study has a strong inhibitory effect on C.perfringens and can improve the damage to intestinal epithelium caused by C.perfringens infection,showing potential for developing probiotic products.