Evaluation Of Therapeutic Effect Of Phage On Salmonella Pullorum And Its Effect On Cecal Microflora

Salmonella is an important zoonotic pathogen,which not only endangers the health of Chinese people,but also seriously restricts the development of poultry industry in China.The characteristics of Salmonella can evade chicken immunity and spread vertically through parent-child generation determine that population purification is the best way to solve avian salmonellosis.However,in view of the low level of intensification,uneven level of feeding management and frequent outbreaks of Salmonella in poultry industry,the use of antibiotics in aquaculture has always been the main way to prevent and treat Salmonella.In recent years,due to the rapid growth of Salmonella resistance and the national policy to reduce the use of veterinary antibiotics,we plan to expand the application of phages to poultry production.In the early growth of chicks,the immune system was immature and the intestinal flora was not fully developed,so it was susceptible to bacterial diseases such as Salmonella.The mechanism of antibiotic prevention or treatment of bacterial infection was to inhibit the growth of pathogenic bacteria and improve intestinal flora.Bacteriophages were green drug to replace antibiotics,but few studies have been conducted on their effect on early cecum flora development in chicks.The normal establishment of cecal microbe in chicks is related to promoting intestinal development,enhancing digestive function and increasing host resistance to environment.In this study,we evaluated the therapeutic effect of phages on chicks infected with Salmonella pullorum,focusing on how phages affect the cecal flora to regulate intestinal homeostasis.1 Isolation and Identification of PhageWe collected sewage and feces from poultry farms in different regions and isolated and purified three strains of Salmonella phage SM5,SM6 and SM8 with titers of 10⁷,10¹⁰and 10⁸PFU/m L,respectively.Among them,SM6 had the highest titer,and the phage plaque was clear and bright,which met the standard of virulent phage.The results of Transmission electron microscopy showed SM6 belonged to the Siphoviridae family.104strains of avian Salmonella kept in the laboratory were used to determine the lysis spectrum and the cleavage rate reached 84.6%.Other biological characteristics of SM6 were examined.The optimal multiplicity of infection was 0.001,and phage titer was 10¹¹PFU/m L.The resistance of phage to external environment was strong,and the titer was unchanged when incubated at 60℃or p H 5-11 for 1 h.2 Evaluation of therapeutic effect of phage on Salmonella pullorum in chicksOral administration of phage did not cause changes in body weight and serum endotoxin levels.We also observed significant increases in the jejunal villus height,V/C ratio and m RNA expression levels of occludin and ZO-1 and a significant decrease in the m RNA expression level of TNF-αin P2 group,suggesting that rational use of phage can enhance intestinal barrier function.Chicks of M group showed typical symptoms of Salmonella infection:listless,disordered feathers,accompanied by diarrhea,and significantly lower body weight than other groups.The results of both real-time fluorescence quantitative PCR and 16S r RNA high-throughput sequencing showed that the number of Salmonella in the cecum of chicks in M group was much higher than that in NC group,indicating Salmonella colonization was successful.In addition,we observed that the incidence rate of chicks decreased,symptoms were significantly reduced,the body weight was recovered,and the number of Salmonella in cecum was significantly decreased in PT1,PT2,and AMXT groups.The PCR results showed that the bacteriophage reduced the positive rate of Salmonella in chicks over time,suggesting that the phage can resist the invasion of Salmonella pullorum to the host by directly reducing the number of Salmonella in the intestine.In addition,Salmonella pullorum infection damaged the tissue morphology of duodenum and jejunum,down-regulated the m RNA expression of claudin-1,occludin and ZO-1 in jejunum,increased the level of endotoxin in serum and up-regulated m RNA expression of IL-1βand TNF-α.Phage therapy can alleviate the changes of small intestinal tissue morphology,down-regulation gene expression of tight junction protein,elevation of serum endotoxin and up-regulation of inflammatory factors levels caused by Salmonella pullorum in chicks.3 Effects of Salmonella pullorum and bacteriophage on cecal microflora of chicksThe 16S r RNA sequencing results of PBS treatment group showed that although phage did not significantly alter the community richness and diversity.However,the magnitude of changes in the composition of the cecal microbiota correlated with the time of phage use,the earlier the use of phages,the lower the diversity of the cecum flora.The community composition analysis showed that the Firmicutes was predominant in the cecum of 14-day-old chicks,and the abundance of Lachnospiraceae and Ruminococcaceae ranked top two at family level.Further analysis of genus-level differential groups revealed that oral phages significantly increased the relative abundance of Enterococcus,with a tendency to decrease the abundance of Ruminococcus＿torques＿group.Combined with the results of LEf Se analysis,the P1 group had the most differential flora.In addition,we also observed an increase in the abundance of Bifidobacterium and Subdoligranulum in the P2 group.This may be the reason for the enhanced intestinal barrier function of chicks.The results of 16S r RNA sequencing in the Salmonella treatment group showed that oral administration of phage during 3-5d significantly affected the Shannon and Simpson index,leading to a reduction in community diversity.NMDS analysis and community composition analysis also showed that among the three treatment groups,the PT1 group had the greatest changes in community structure,and Enterococcus and Lactobacillus were the dominant genera in the cecum.The community structure of the PT2 group was the closest to that of the NC group.Further analysis of the differential bacterial groups at the genus level revealed that the relative abundance Salmonella,Methylophilus,Harryflintia,and Halomonas were increased in the M group,while the use of phages significantly reduced the relative abundance of Gammaproteobacteria such as Salmonella.Combined with LEf Se results,the PT1 group had the most differential flora.Among the three treatment groups,the PT2 group showed the minimal changes in intestinal flora,avoiding the use of phages in the first stage of cecal microbiota development would greatly reduce the impact on gut flora while ensuring the therapeutic effect.In summary,this study drew the following conclusions:（1）In this study,a strain of phage targeting Salmonella was isolated which has a wide lytic spectrum,high proliferation titer,good tolerance to high temperature and acid and bases,and potential for application in aquaculture.（2）Phage can effectively resist Salmonella pullorum infection in chicks by reducing morbidity,alleviating weight loss and intestinal barrier function destruction caused by Salmonella,significantly reducing the number of Salmonella in the intestinal tract,and not affecting the normal flora.（3）Oral phage-induced changes in intestinal barrier function may be related to changes in the intestinal flora induced by phages.The magnitude of the changes in the composition of the intestinal flora correlates with the duration of phage use.Additionally,the mechanism of Salmonella phage resistance against Salmonella typhimurium may be related to:direct lysis of Salmonella pullorum in chicks and regulation of intestinal flora.