The Effect Evaluation Of Phage Therapy In Chickens Infected Salmonella Pullorum

Salmonella Pullorum causes acute systemic diseases,leading to severe gastrointestinal symptoms and high mortality rates in young chicks.In adult chickens,Salmonella Pullorum can be infected for a long time without causing clinical diseases,but it is transmitted vertically or horizontally.Some developed countries in the aquaculture industry have eliminated chicken dysentery through strict purification measures,but in countries where the intensification of poultry farming is still in its early stages,the disease remains a major threat.In recent years,due to the abuse of antibiotics,the problem of bacterial resistance has become increasingly severe,and people have begun to search for new antibiotic substitutes.Phages,as a virus hosted by bacteria,can specifically lysize host bacteria and are one of the potential alternative solutions to antibiotic abuse.Currently,Salmonella Pullorum is still prevalent in chicken flocks in China,and there are few reports of natural infection cases of Salmonella Pullorum treated with phages.In addition,chronic infection of adult chickens makes it difficult to completely eliminate long-term vertical transmission of Salmonella Pullorum.Therefore,this study screened a strain of phage CKT1 with excellent characteristics through isolation and identification,evaluated the therapeutic effect of phage CKT1 on chicks naturally infected with Salmonella Pullorum through clinical trials,and evaluated the effect of phage therapy on controlling vertical transmission of Salmonella Pullorum by constructing a vertical transmission model of Salmonella Pullorum.1.Biological characteristics and genome analysis of Salmonella phage CKT1This study isolated and identified Salmonella phages from sewage samples from different cities in Shandong Province,in order to screen phage strains with excellent characteristics.7strains of phage were isolated in this study,of which the phage CKT1 produced the highest viral titer（P<0.0001）and the lowest resistant mutation rate（P>0.05）.The research results indicate that the seven phage strains have the same host spectrum and have the ability to lysize Salmonella Pullorum,Salmonella Typhimurium,and Salmonella Enteritidis.Among them,the phage CKT1 has the highest efficiency in lysizing the three serotypes of Salmonella.Under electron microscopy,the phage CKT1 exhibits a symmetrical polyhedral head and a long tail,with a diameter of approximately 60 nm and a tail length of approximately 120 nm.The results of its infection characteristics against the host Salmonella strain CVCC526showed that the adsorption rate was about 92%at 9 min,the incubation period was 10 min,the outbreak period lasted for about 50 min,and the average outbreak volume was 147PFU/cell.When the multiple infection index（MOI）is 0.001,the highest titer of phages can be reached 4 h after infection,approximately 2.3×10¹⁰PFU/m L。Phage CKT1 can significantly inhibit the growth of Salmonella Pullorum CVCC526 in vitro（P<0.0001）.Phage CKT1 exhibited stable activity within 2 h at 37-60°C,but rapidly decreased activity at70°C and lost activity within 30 min at 80°C.In addition,the phage remained stable in p H 4-11 environment for 2 h.The phage CKT1 genome does not contain integrase genes,does not carry resistance or virulence genes,and is a safe phage.2.Evaluation of the therapeutic effect of phages on naturally infected chickens with Salmonella PullorumThis study evaluated the therapeutic effect of phage CKT1 on natural infection of Salmonella Pullorum in chicks by gavaging 1-day old chicks of a local breed naturally infected with Salmonella Pullorum.The results showed that phage CKT1 reduced the mortality rate of chicks,reduced the isolation rate of Salmonella Pullorum in cloacal swabs,and reduced the positive rate of Salmonella Pullorum antibody.In addition,the administration of phage CKT1 significantly reduced the liver index（P<0.01）of chicks,improved liver enlargement,and significantly reduced the Salmonella loads in the liver（P<0.01）and spleen（P<0.0001）.However,phage therapy resulted in an increase in non-Salmonella such as Escherichia coli colonization in the liver and spleen（P<0.05）.Therefore,in this study,Escherichia coli phages were isolated,and three treatment groups（Salmonella phage group,Escherichia coli phage group and mixed phage group）and the control group were designed for three consecutive days of oral administration.The results showed that the mortality rate of all treatment groups decreased,and there was a significant improvement in chick weight（P<0.01）.The liver index of all treatment groups decreased,with significant differences in the mixed phage group（P<0.01）.In addition,the spleen index of chicks in all phage groups increased,with significant differences in the Salmonella phage group（P>0.05）.All treatment groups could reduce the bacterial loads in the liver and spleen（P<0.01）.The bacterial loads in the liver and spleen of chicks in the mixed phage group was lower than the detection line,while the bacterial loads in the spleen of chicks in the E.coli phage group was not significantly reduced（P>0.05）.3.Evaluation of phage control effect on vertical transmission of Salmonella PullorumIn this study,22 week-old white feathered broilers were selected to construct an adult chicken vertical transmission model by injecting Salmonella Pullorum CVCC526 into their pectoral muscles.The effect of phage CKT1 on controlling vertical transmission of Salmonella Pullorum was evaluated through two consecutive days of oral administration.The experimental results showed that phage CKT1 significantly reduced the isolation rate of Salmonella Pullorum in eggs（P<0.05）.After phage administration,the colonization of Salmonella Pullorum in the cecal contents significantly decreased（P<0.05）,and the Salmonella loads in organs such as the liver,spleen,and heart significantly decreased（P<0.01）,but had no significant effect on organ index（P>0.05）.In the phage treatment group,Salmonella in the oviducts and ovaries decreased below the detection level（P<0.01）.In addition,the administration of phage CKT1 significantly reduced air pollution,and the total number of bacteria in the air of the phage treatment group significantly decreased（P<0.0001）.Phage CKT1 reduced significantly reduced the level of group D Salmonella specific antibody Ig G in the serum（P<0.01）.In summary,this study screened a Salmonella phage CKT1 with excellent characteristics,which has good lysis ability,proliferation ability,and safety;The phage CKT1 effectively reduces the mortality rate of naturally infected chickens with Salmonella Pullorum,reduces the colonization of Salmonella Pullorum in organs,but leads to an increase in non-Salmonella colonization.Continuous administration of drugs effectively reduces the colonization of Salmonella and non-Salmonella in organs;The phage CKT1 effectively reduces the colonization of Salmonella Pullorum in the liver,spleen,heart,oviduct,ovaries,and cecal contents of adult chickens,reduces the isolation rate of Salmonella Pullorum in egg laying,and also reduces the level of bacterial contamination in the ambient air and the level of Salmonella specific antibody Ig G in serum.