| Swine erysipelas that is also called diamond skin disease or red fever is an acute,fever infectious disease caused by Erysipelothrix rhusiopathiae.In recent years,there are more and more reports on the outbreaks of Erysipelothrix rhusiopathiae,which resulting in significant economic losses to the swine industry.To date,there are mainly two methods to be used to control swine erysipelas in the pig production by using vaccination and drug therapy.As is shown in previous reports,Erysipelothrix rhusiopathiae is resistant to a large number of antibiotics.Once the drug tolerance is forming for them,which will cause a major outbreak of swine erysipelas.Bacteriophage is a bacterial virus that can adsorb onto the surface of the host and propagate in it,which will causes the bacteria lysis eventually.So,phage plays a specific role in killing the host bacteria.In our study,one phage against Erysipelothrix rhusiopathiae was isolated and purified.The host spectrum for the phage of Erysipelothrix rhusiopathiae were also investigated,which indicating that the phage can kill most of Erysipelothrix rhusiopathiae specifically.Therapy of this phage in mice were also researched and a series of studies have been done.Lyase,the key protein for the lysis of phage,was screened through genome information analysis and a series of optimizations for its lytic conditions have been done.The contents of this research contain seven parts as following:1.Isolation and characterization of bacteriophage of Erysipelothrix rhusiopathiaeA bacteriophage infecting pathogenic Erysipelothrix rhusiopathiae was isolated from a swine farm in Yancheng city of Jiangsu province experiencing an outbreak of acute swine erysipelas in 2014 and designated as SE-I.SE-I has an icosahedral head,long tail and a double-stranded DNA genome.The 34,997 bp genome has a GC content of 34%and contains 43 open reading frames(ORFs)encoding packaging,structural,lysin-holin,and hypothetical proteins.Components of purified SE-I were separated using SDS-PAGE electrophoresis and analyzed using liquid chromatograph mass spectrometry.9 proteins were identified,encoded by ORF9,ORF15,ORF23,ORF30,ORF31,ORF33,ORF39,ORF40 and ORF 42.A phylogenetic tree constructed with the large terminase subunit reveals that SE-I is closely related to staphylococcus phages P954 and phi3396.SE-I can infect 7 of 13 E.rhusiopathiae strains,but is unable to infect Salmonella,Streptococcus suis,Staphylococcus aureus.Our study is the first report of the isolation,characterization,and genomic and proteomic analysis of phage infecting E.rhusiopathiae,and may lead to the development of new anti-E.rhusiopathiae agents.2.Animal experiment of phageBeing antibiotics that can affect normal flora in human and animals.In recent years,phages in the treatment of bacterial diseases of human and animals has been widely concerned.A bacteriophage infecting pathogenic E.rhusiopathiae was successfully isolated in the research work by our laboratory.In order to verify whether SE-1 has the function of killing bacteria in vivo.Median lethal dose of E.rhusiopathiae was determined.The injection sites,time and doses were optimized to make SE-1 more suitable for medical treatment.The loads of.rhusiopathiae in blood were calculated by colony forming unit to detect the effect that phage SE-1 played in mice.The results showed that the quantity of bacteria in the experimental group was decreased by 10 folds at 72h after the injection of the phage in the comparison with the negative control without any phage SE-1 injection.our results will provide a new way for the subsequent development of new drugs treating erysipelas infection.3.Expression and application of Erysipelothrix rhusiopathiae phage lysinLysin of bacteriophage,which can hydrolyze cell walls efficiently,is a kind of protein expressed by DNA of phage to release offspring phages and kill specific bacteria in vitro in the last phase of infection.The aim of this experiment is to clone and express lysin of bacteriophage of Erysipelothrix rhusiopathiae(Ely)and identify its biological activity.After sequencing the genome of phage of Erysipelothrix rhusiopathiae and analyzing its biological characteristic of ORFs,we amplified the gene of predicted lysine by polymerase chain reaction(PCR).the products were cloned into pET-28a to establish a recombined vector pET-28a(+)-Ely and transferred into BL21(Rosetta).After 8 hours induced by IPTG(1mmol/1)in 28℃.When SDS-PAGE performed,the objective protein existences in cell supernatant and correspond to the anticipated molecular weight and the purity of the protein which purified by Ni2+-NTA is over 90%.The Erysipelothrix rhusiopathiae strain was exposed to this protein with final concentration of 100 μg/ml,10 μg/ml,1μg/ml and cultured in BHI medium.Colony count was performed every 1h until 7h,the untreated group was exposed to PBS of same volume only.This study is to identify whether this protein can produce the effect for lysis of Erysipelothrix rhusiopathiae strain or not,and which will provide a new idea for treatment of drug-resistance Erysipelothrix rhusiopathiae. |
