Study On Isolation,Identification And Application Of Salmonella Phage From Chicken

Chicken is a natural host of Salmonella,vulnerable to Salmonella invasion.At present,antibiotic therapy is the main measure for the prevention and control of Salmonella.However,with the deepening understanding of the negative effects of antibiotics,the research and development of high-efficiency,low-cost and environmentally friendly alternative products have attracted great attention from the society.Phages widely present in nature have the advantages of high bactericidal efficiency,low application cost,and no obvious side effects,so they have become a research hotspot as candidates for alternative products.The purpose of this study was to screen Salmonella phage strains with excellent biological characteristics from nature,and to study their therapeutic effects on chickens infected with Salmonella by using the experimental model of Salmonella infection in chickens,so as to provide theoretical support for the prevention and control of Salmonella diseases and the purification of Salmonella pathogens by using phages.The main test results are as follows:1.Isolation,identification and biological characteristics of phages.A strain of Salmonella streptitidis bacteriophage(SJT-90)was isolated and purified from the wastewater of laying hens breeding enterprises by double-layer plate method.Its morphology was observed by transmission electron microscopy,and its split spectrum,optimal infection complex,one-step growth curve,pH stability and thermal stability were determined by conventional methods.The results showed that SJT-90 was long-tailed and belonged to the tailed bacteriophage.Phage SJT-90 could lyse 27Salmonella strains with a lysis rate of 75%(27/36).The titer was 2.5?5.9×10⁹ PFU/mL and the optimal multiplicity of infection was 0.01.The incubation period was 20 min,the burst period was 40 min,and the cleavage amount was about 141 PFU/cell.The phage had good tolerance to temperature,and the titer was still 1×10⁵ PFU/mL after culturing at 70?for 60 min.The phage had a certain tolerance to strong acid and alkali,and could grow in the pH range of 2.0?12.0.2.Establishment of Salmonella infection model.Three-day-old chickens were fed with 3×10¹⁰ CFU/mL Salmonella enteritidis once.The clinical symptoms and histopathological results of infected chickens were consistent with the pathogenesis of Salmonella enteritidis.3.Effects of SJT-90 on the expression of intestinal villi,inflammatory factors and tight junction proteins in chicks.The phage therapy experiment was designed by orthogonal test.The results showed that after 1 h of challenge,0.5 mL of 3×10¹⁰PFU/mL phage suspension was fed to the chicks at an interval of 1 h,and then fed again according to the same method for three consecutive times.The incidence of chicks was the lowest.After the treatment of artificially infected chickens with the above methods,compared with the infection group,the duodenum and jejunum of the SJT-90 group were intact,and the villus height and villus-crypt ratio were significantly increased(P<0.05).SJT-90 could promote the expression levels of ZO-1 and Claudin-1 in duodenum and jejunum,but had no significant effect on the expression levels of ZO-1in jejunum and no significant effect on the expression levels of ZO-1 and Claudin-1 in ileum(P>0.05).In summary,the phages isolated from the sewage of laying hens breeding enterprises were used to establish the model by chicks.After 1 h interval,the 3×10¹⁰PFU/mL phage suspension was continuously fed for three times,which could prevent the normal structural damage of Salmonella on chick intestinal villi,promote the expression of tight junction proteins in duodenum and jejunum,and inhibit the expression of inflammatory factor genes in duodenum and jejunum.