| Colibacillosis is one of the common bacterial infectious diseases that endanger the chicken industry.The infection rate in chicken flocks in my country can reach 5%-30%,and the fatality rate is as high as 90%,ranking first among chicken diseases.At present,the clinical prevention and treatment of Escherichia coli infection in chicken mainly rely on antibiotics.Due to the large amount and irregular use of antibiotics,drug-resistant strains appear,resulting in drug residues and other problems,which endanger public health safety.One of the most effective ways to solve the above problems is to screen alternative antibacterial products for the prevention and treatment of chicken colibacillosis.In this study,the strains with strong pathogenicity and serious drug resistance were selected from Escherichia coli strains as host bacteria,and the corresponding lytic phages were isolated,and the biological characteristics and antibacterial effects of phages were evaluated in vitro.,to provide a theoretical basis for the prevention and treatment of chicken colibacillosis,and to pave the way for subsequent research on phage preparations.The main research contents are as follows:1.By collecting 430 samples of heart,brain,cecum,etc.of sick and dead chickens,anal swabs of diarrhea chickens,sewage and feces in the environment from some largescale chicken farms in Henan Province,374 positive strains were isolated,with a positive rate of 86.98%.Virulence gene identification,drug resistance detection and serotype identification were performed on the isolates.The results showed that among the 17 virulence genes related to Escherichia coli,136 strains of Escherichia coli contained 10 or more virulence genes in 374 strains.These Escherichia coli carried tsh,cva C,iss,iro N,iuc D,and irp2,there are 108 strains with 3 or more genes among the 6 genes,The 108 strains of Escherichia coli were tested for drug susceptibility,and the results showed that among the 21 drugs,8 strains of Escherichia coli were resistant to 20 or more strains.The serotypes of these 8 strains of Escherichia coli are 2 strains O82 strains O35,2 strains O76 and 1 strain O145,and 1 strain has not be determined.2.Phages were isolated by using the screened 8 strains of Escherichia coli as host bacteria.Sewage and fecal samples were treated,cocultured with 8 strains of Escherichia coli,and verified by doublelayer plate and spot method.The isolated phages were purified,and their titers and host profiles were determined.The results showed that only with O145 Escherichia coli as the host,two phages with different plaque sizes were isolated,and no corresponding phages were isolated from other strains;the titers of the two phages were 3×109 PFU/m L,8.5 ×108 PFU/m L,the host profiles of the two phages were determined respectively,and it was found that both phages had strong specificity,and were named as Bp1 and Bp2.3.Both phages are of narrow spectrum.Because the plaques formed by phage Bp1 are relatively large,only the biological identification of phage Bp1 was carried out.From the observation of transmission electron microscope,it can be seen that the head of phage Bp1 is hexagonal and the tail is longer;the MOI of phage Bp1 is 0.001;by measuring the growth curve of phage Bp1,it is found that the incubation period of phage Bp1 is about 20 minutes,and the lysis period is 50 minutes;Phage Bp1 was incubated at 40°C for 30 min,60 min and 50°C for 30 min,and still maintained a high biological activity,and its stable biological activity could be maintained when the p H was close to 7;Phage Bp1 has a good inhibitory effect on its host bacteria in vitro.In a word,the screened chicken-derived Escherichia coli is pathogenic and the drug resistance is serious;The isolated bacteriophage Bp1 has good lytic property,It also has strong inhibitory and elimination effects on host bacteria in vitro,This will pave the way for the evaluation of the antibacterial effect of phage in animal s and the study of phage preparations. |
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