Isolation And Identification Of Bacteriophage ?DS8 From Chicken Origin And Its Preliminary Application

Bacteriophage is a general term for a virus that infects microorganisms such as bacteria,fungi,algae,actinomycetes,or spirochaete.It has some characteristics of the virus:individuals are small,do not have a complete cell structure,and contain only a single nucleic acid.Because it can cause the host bacteria to lyse,it is called phage.Traditional antibiotic therapies have had little effect in combating resistant bacteria,and phage as a new type of biological antibacterial agent is expected to replace antibiotics in the treatment of bacterial infections.In this article,26 strains of Shigella were first isolated and identified from susceptible broilers in the Fufeng chicken farm in Guangxi.By measuring the pathogenicity of Yunnan broilers as experimental animals at the same concentration,it was found that broilers infected with BDS8 has more severe diarrhea than other isolates.After broilers infected with isolate BDS8 were fed normally for 7 days,15days later,the inflammatory lesions in the internal organs of the broiler chickens were clearly found by dissection.The heart was enlarged and covered with yellow pericardium,and the mesenteric hemorrhage was obvious.The drug resistance of the isolate BDS8 was tested by the drug-sensitive paper method,and it was found that the Shigella isolate BDS8 had multi-drug resistance.BDS8 was used as the host,and healthy breeder manure was used as a sample.The host was isolated from breeder manure samples using the Coomassie brilliant blue method and named DS1-DS26.?DS8 bacteriophage was selected and concentrated by PEG(Polyethylene glycol)precipitation method.The final titer reached 10¹¹ PFU/m L.Transmission electron microscopy revealed that the head of bacteriophage?DS8 was icosahedral,with a diameter of 61±2 nm,a long tail,and the tail length was 165±2 nm,belonging to the family Phaephidae,long-tailed phage order.The biological characteristics of bacteriophage?DS8 were identified with strict host specificity.The infection cycle was 120 minutes,including a 75-minute incubation period,followed by a 45-minute outbreak period,with an outbreak of 52±5 PFU/cell per infected cell.The optimal multiplicity of infection was 0.1,and the increase in phage reached its peak at this ratio,which was 2.8×10⁹PFU/m L.The p H/thermal stability test found that the phage maintained high activity in the range of p H 4-10 and 4-50?.The genome of bacteriophage?DS8 was extracted,and the genome of?DS8 was found to be double-stranded circular DNA by gene sequencing analysis.Genetic analysis showed that the genome was 41,088 bp in length and GC content was 46.6%.A total of 79 ORFs were estimated by Blast alig nment,ranging in size from 96 bp to 2910 bp.The component analysis of phage?DS8 and genomic function prediction revealed that it did not have known related virulence genes.Through animal experiments,treatment and prevention of phage were used to treat Shigella infected Yunnan broiler chickens.It was found that whether it was the change of Shigella content in broiler manure,the content of phage in broiler manure,or the survival rate After 15 days of breeding,the anatomy of broilers was very effective,which obviously prevented the lesions of broilers.Biodiversity analysis of broiler manure from the phage-treated group revealed that at the Phylum,the Firmicutes accounted for 98.6%and 86.3%of samples of the treatment group before0 h infection,respectively.It decreased rapidly at 2 h after infection,to 21.3%and12.5%,respectively,and gradually rose to 42.2%and 71%at 12 h after treatment by phage,respectively.At 48 h after treatment,it finally rose to 94.2%.In contrast to this,the relative abundance of Proteobacteria was 1.2%and 2.1%at 0 h without bacterial infection.After being infected by bacteria,the relative abundance of Proteobacteria rose to 70.6%and 85.3%.At 12 h after phage treatment,the relative abundance of Proteobacteria decreased to 56.1%and 20.2%.After 48 h it fell back to 0.4%and32.4%.Genus analysis found that probiotics in the intestines of broilers,including Lactobacillus,Enterococcus and Sphingomonas,all showed a downward trend after bacterial infections,but eventually returned to pre-treatment levels after phage treatment.The harmful bacteria in the intestines of broilers,including Pseudomonas,Shigella,and Escherichia,are just the opposite.After the bacterial infection,the relative abundance has greatly increased,but after phage treatment,it finally fell back.normal level.In summary,bacteriophage?DS8 has a very good effect in treating broilers infected with Shigella BDS8 and has a faster response.It has less effect on the intestinal flora and recovers quickly.This is also the actual use of bacteriophage for production.Broilers provide a solid theoretical foundation currently.