Study On The Therapeutic Effect Of Chinese Medicine Compound "Sishen Wan Plus And Minus" On Chickens Infected With Salmonella Pullorum

Chicken pullorum is an infectious disease caused by Salmonella pullorum that can affect poultry of all ages.Especially in chicks,chicken white dysentery can easily cause acute sepsis in chicks,resulting in higher morbidity and mortality.Although traditional antibiotics are effective in the treatment of chicken dysentery,prolonged use may lead to resistance of Salmonella dysentery,thus accelerating the emergence of resistant strains in the environment,which poses a potential risk to food safety.Traditional Chinese medicine compound"Sishen pills"from the Ming Dynasty Wang Kantang’s"syndrome and treatment criteria",containing psoralea,nutmeg,Wu Shu,schisandra and other herbs,mainly used for warming the kidney to dispel cold,shibing the intestine to prevent diarrhea.And this study according to the clinical symptoms of pullorosis,loose stool,such as white ash discharge water present evil cold,feather shock characteristics,traditional Chinese medicine under the guidance of the teacher to join in changchun Chinese bulbul,herba agrimoniae,radix bupleuri,cassia twig,rhizoma atractylodis,dried tangerine or orange peel,magnolia bark,liquorice,Sishen Wan plus and minus（SSJ）was prepared and applied in the clinical study of chicken white dysentery.The objective of this study was to investigate the therapeutic effect of SSJ on chicken pullorosalmonellosis.Therefore,the inhibitory effect of SSJ on Salmonella pullorum was investigated in vitro.Secondly,the therapeutic effect of SSJ treatment on infected chicks and its effects on intestinal flora and related metabolites were investigated.Finally,fecal microbiota transplantation（FMT）was used to explore whether the therapeutic effect of SSJ on chicken salmonella was realized by regulating intestinal flora and its metabolites.These results will provide a theoretical basis for SSJ in the clinical treatment of chicken salmonella pullorum.The specific research results are as follows:（1）minimal inhibit concentration（MIC）of SSJ in salmonella pullorum was detected by microdilution method,and the results showed that the MIC of SSJ in S.pullorum was 250 mg/m L.The results of growth curve measurement showed that the growth of bacteria was significantly inhibited under 1/2 MIC and 1/4 MIC conditions,while the growth and reproduction of bacteria were not affected at 1/16 MIC and below,and the growth curve was close to coincidence with that of the control group.The effect of SSJ on biofilm formation of experimental bacteria was studied by crystal violet staining.The results showed that SSJ could effectively inhibit biofilm formation at 1/2MIC and 1/4 MIC,but had no significant inhibitory effect at 1/16 MIC and below.Finally,the ability of SSJ to adhere to and invade DF-1 of chicken embryo fibroblasts was measured.The results showed that SSJ could significantly inhibit the adhesion and invasion ability of salmonella pullorum to DF-1 of chicken embryo fibroblasts without affecting the growth concentration of salmonella pullorum.（2）In vivo experiments,different concentrations of Salmonella pulsentery were used to infect 3-day-old chickens,and the median lethal dose(LD₅₀)was calculated to be 2.2×10⁶ CFU/m L,and the chickens were infected with LD₅₀ in subsequent experiments.Then,the infected chicks were treated with 0.4 g/kg,0.8 g/kg,1.6 g/kg SSJ and flufenicol,respectively,for 14 days.Mortality,barrier associated tight junction proteins（ZO-1,Occludin,Claudin-2,and Claudin-1）in the ileum,histopathology（duodenum,ileum,and liver）,bacterial load（spleen,liver,and cecum）,serum immunoglobulins（Ig M,Ig G,and Ig A）,and inflammatory causes were measured to evaluate the therapeutic effect of SSJ（IL-10,IL-8,IL-1β,TNF-α,and IL-6）on chicken pullorosalmonellosis.The results showed that SSJ treatment significantly decreased the mortality of chicken salmonella pullorum,inhibited the expression of proinflammatory factors in serum,alleviated histopathological injury,increased the expression of intestinal barrier protein,inhibited bacterial colonization of peripheral organs,and increased the serum immunoglobulin.（3）Then,16S r RNA sequencing and metabolomics techniques were used to explore the effects of SSJ on the intestinal flora and metabolites of chicks.The results showed that after SSJ treatment,the intestinal flora richness and diversity were significantly increased compared with the model group,and the number of beneficial bacteria was increased while the number of harmful bacteria was decreased.Fecal metabolomics results showed that compared with the control group,the bile acid metabolic pathway was disturbed in the model group,but after 1.6 g/kg SSJ treatment,the bile acid metabolic pathway and metabolites were restored,and the related metabolites in the glycine,serine and threonine metabolic pathways were significantly increased.（4）Finally,we transplanted the faeces of 1.6 g/kg SSJ recipient chicks into chickens infected with Salmonella pullorum.FMT-SSJ was used to detect the mortality,histopathology and proinflammatory cytokine expression of chicks,so as to explore whether SSJ can play an anti-infection role through intestinal flora.The results showed that FMT-SSJ could significantly reduce the mortality,reduce the histopathological injury,and inhibit the expression of proinflammatory factors and immunoglobulin in serum.In summary,this study explored the therapeutic effect of SSJ on chicken white dysentery salmonellosis,looking for traditional Chinese medicine treatment of chicken white dysentery.These findings will provide clinical data for SSJ treatment of chicken salmonella pullorum.