Protective Mechanism Of Copper/Zinc-modified Palygorskite On Chicks Against S.Typhimurium Infection

Salmonella,widely distributed in nature,is a common pathogen of human and animal,which could cause typhoid,paratyphoid,food poisoning,gastroenteritis,sepsis and many other diseases.Salmonella can cause pullorosis,typhoid and other diseases in poultry.Food poisoning caused by Salmonella is the first bacterial food poisoning,which is harmful to human and animal health.Palygorskite is a kind of natural mineral clay,which can absorb toxic and harmful substances and protect the intestinal mucosal barrier.Therefore,palygorskite is often used as a green,safe and efficient feed additive.In this paper,copper/zinc-modified palygorskite was prepared to study the antibacterial activity of copper/zinc-modified palygorskite against Salmonella Typhimurium in vitro,and the protective effect of copper/zinc-modified palygorskite against Salmonella Typhimurium infection in chickens was studied through infection protection experiment.The protective mechanisms of intestinal integrity,bacterial load,intestinal stem cell proliferation and apoptosis,and the regulation of microbiota were studied.1.Preparation and antibacterial activity of modified palygorskite in vitroIn this study,the anti-S.Typhimurium activity of palygorskite modified with copper and zinc ions was studied in vitro.A series of modified palygorskites（Pal）with different concentrations of copper and zinc ions were prepared by ion exchange method,and the concentrations of copper and zinc ions were determined by inductively coupled plasma spectrometer.Agar drilling method and plate counting method were used to determine the in vitro antibacterial activity and minimum bactericidal concentration of the modified palygorskite,and the effects of the modified palygorskite on the growth curve of Salmonella Typhimurium.The results showed that the content of copper ions in Cu-Pal,Zn-Pal,Cu/Zn-Pal-1,Cu/Zn-Pal-2 and Cu/Zn-Pal-3 was 51.21±2.81 mg/g,0 mg/g,48.23±0.77 mg/g,36.51±1.60 mg/g,15.78±0.71 mg/g respectively;zinc ion content of 0 mg/g,16.23±0.48 mg/g,3.14±0.76 mg/g,8.83±0.45 mg/g,14.26±0.35 mg/g respectively.In vitro agar diffusion assay results showed that Pal itself had no bacteriostatic effect,but Cu/Zn-Pal-1 had better bacteriostatic effect,and the diameter of inhibition zone was 15.33±0.58 mm,which was significantly higher than that of other groups（P<0.05）.The minimum bactericidal concentration of Cu/Zn-Pal-1 was 5 mg/m L,which was lower than that of other modified palygorskites.The results of the growth curve of Salmonella Typhimurium showed that the simple mixture of Cu²⁺,Zn²⁺and Pal could not inhibit the growth of Salmonella Typhimurium,while Cu/Zn-Pal-1 could effectively inhibit the growth of Salmonella Typhimurium in the form of complex.These results indicated that Cu/Zn-Pal-1 had good anti-S.Typhimurium activity.2.Protective effect of copper/zinc-modified palygorskite on chicks against S.Typhimurium infectionIn this study,the protective effect of Cu/Zn-modified palygorskite（Cu/Zn-Pal-1）against S.Typhimurium infection in chicks was investigated.The protective rate of Cu/Zn-modified palygorskite（Cu/Zn-Pal-1）against S.Typhimurium infection was evaluated by recording the morbidity and mortality of chicks.The performance of chicks was evaluated by weighing their weight and weight before and after feeding.The load of S.Typhimurium in the tissues and feces of infected chicks was determined by plate counting method.The contents of copper and zinc in tissues and feces were determined by inductively coupled plasma spectrometer.The results showed that the incidence of S.Typhimurium infection in chickens was up to 100%,and 40%of the chicks died.Meanwhile,the average daily feed intake（21.65±0.81 g）,average daily gain（10.52±0.60g）and average final weight（232.97±27.63 g）of infected chicks were significantly lower than those of the control group.In CZP+SAL group,the morbidity,mortality and protection rate were 20%,0%and 80%,respectively.Cu/Zn-Pal-1 supplementation increased the protection rate of infected chicks.The average daily feed intake was 26.57±0.95 g,the average daily gain was 12.97±0.13 g and the average final weight was 280.41±54.53 g.Cu/Zn-Pal-1 supplementation significantly improved the performance of the infected chicks.After feeding S.Typhimurium to chicks,the Salmonella loads in liver,spleen,small intestine,cecum and feces were 10^3.7 CFU/g,10^4.2 CFU/g,10^5.0 CFU/g,10^5.9CFU/g and 10^5.7 CFU/g,respectively.In addition,the liver and spleen were significantly swollen,and the heart had yellow and white,rice-sized,necrotic nodules on the surface.There are numerous needle-like hemorrhages on the surface of the small intestine.In CZP+SAL group,the loads of Salmonella in liver,spleen,small intestine,cecum and feces were10^2.6 CFU/g,10^2.8 CFU/g,10^3.8 CFU/g,10^3.9 CFU/g and 10^3.2 CFU/g,respectively.Dietary Cu/Zn-Pal-1 supplementation significantly reduced the load of S.Typhimurium in tissues and feces of chickens,and alleviated the pathological changes of various organs induced by S.Typhimurium.After feeding chicks fed Cu/Zn-Pal-1 diet,the copper ion content in liver and feces was 164.063±10.670μg/g and 168.011±11.747μg/g,respectively,which was significantly higher than that in the control group,but there was no significant difference in copper ion content in other tissues and organs.These results indicate that dietary Cu/Zn-Pal-1 has a good protective effect on S.Typhimurium infection in chickens.3.Effects of Cu/Zn-modified palygorskite on intestinal barrier,inflammatory response and oxidative stress response in infected chicksIn this study,the effects of Cu/Zn-modified palygorskite（Cu/Zn-Pal-1）on intestinal mucosal barrier,inflammatory response and oxidative stress response of chicks infected with S.Typhimurium were studied.The morphological structure of villi and crypt in small intestine was observed by HE staining,and then the villi height and crypt depth were measured.The gene transcriptional levels of tight junction protein and inflammatory factors in intestine were detected by real-time quantitative PCR.The protein expression levels of tight junction protein were detected by Western Blot assay.The contents of immunoglobulin and pro-inflammatory factors in small intestine were detected by ELISA.The contents of diamine oxidase（DAO）,D-lactate（D-Lac）,malondialdehyde（MDA）and total superoxide dismutase（SOD）in chicks were detected by kits.The results showed that S.Typhimurium infection caused severe atrophy of villi in small intestine,decreased villi height and crypt depth,decreased the expression levels of tight junction proteins（Claudin-1,Occludin and ZO-1）,and increased the contents of DAO and D-Lac in serum.Dietary Cu/Zn-Pal-1 can restore the normal physiological morphology of intestinal wool,significantly increase the expression levels of tight junction protein,and significantly reduce the contents of DAO and D-Lac in serum,suggesting that Cu/Zn-Pal-1 can reduce the permeability of intestinal mucosal barrier in infected chicks.The transcriptional levels of NF-κB signaling pathway related genes and pro-inflammatory factors（IL-6,TNF-α,IFN-γ,IL-1 and IL-8）in small intestine of chicks infected with S.Typhimurium were significantly increased,resulting in severe inflammatory reactions in intestine.In addition,the contents of MDA and SOD in small intestine of infected chicks increased significantly,and the oxidative stress reaction occurred in small intestine.Cu/Zn-Pal-1 supplementation significantly decreased the transcriptional levels of NF-κB signaling pathway related genes and pro-inflammatory factors,and significantly increased the content of immunoglobulin and decreased the contents of pro-inflammatory factors IL-6 and TNF-αin intestine of infected chicks.In addition,dietary Cu/Zn-Pal-1 significantly reduced the content of MDA and increased the content of SOD in intestine of infected chicks.In conclusion,Cu/Zn-Pal-1 can indirectly maintain the structural and functional integrity of the intestinal mucosal barrier by reducing the load of S.typhimurium,thereby alleviating the intestinal inflammatory response and oxidative stress response induced by S.Typhimurium.4.Effects of copper/zinc-modified palygorskite on proliferation,apoptosis and differentiation of intestinal epithelial cells in infected chicksThe effect of Cu/Zn-modified palygorskite（Cu/Zn-Pal-1）on the proliferation,apoptosis and differentiation of intestinal epithelial cells in chickens infected with Salmonella Typhimurium were explored in this study.The transcriptional levels of proliferation related genes,Wnt/β-catenin signaling pathway related genes,apoptosis related genes and Notch signaling pathway related genes in small intestinal tissue were detected by quantitative real-time PCR assay.The expression levels of PCNA protein andβ-catenin protein in small intestinal crypts were detected by immunohistochemistry.The expression levels of proliferation proteins and apoptosis proteins in intestinal epithelial cells were detect by Western Blot.The goblet cells in small intestinal tissue were detected by PAS staining.The results showed that the S.Typhimurium infection significantly reduced the expression levels of PCNA protein andβ-catenin protein in the small intestinal crypts of chicks,and decreased the transcriptional levels of Wnt/β-catenin signaling pathway-related genes Wnt3,Lrp5 and Axin2,and inhibited the Wnt/β-catenin signaling pathway in intestinal stem cells.The protein expression levels of PCNA andβ-catenin in the small intestine of infected chicks were increased by the Cu/Zn-Pal-1,and the Wnt/β-catenin signaling pathway in intestinal stem cells were activated and the proliferation of intestinal stem cells were promoted by adding the Cu/Zn-Pal-1.In addition,S.Typhimurium infection increased the transcriptional levels of apoptosis genes Caspase3,Caspase8 and Bax,while decreased the transcriptional level of anti-apoptosis gene Bcl-2,which induced the apoptosis of intestinal epithelial cells in chicks.The transcriptional levels of above genes were significantly reversed by adding the Cu/Zn-Pal-1 to the diet.Intestinal stem cells undergo differentiation as well as proliferation and apoptosis.S.Typhimurium challenge increased the transcriptional levels of Notch signaling pathway related genes DLL1 and Hes1,decreased the number of goblet cells and the transcriptional levels of Muc2 and Tff2in the intestine of chicks.The expression levels of DLL1 and Hes1 genes were significantly reduced by adding the Cu/Zn-Pal-1,and the the number of goblet cells and the expressionlevel of Muc2 and Tff2 genes were increased by adding the Cu/Zn-Pal-1,the results indicated that Cu/Zn-Pal-1 inhibited the Notch signaling pathway and promoted the differentiation of intestinal proliferating cells into goblet cells,thereby increasing the secretion of mucin 2（Muc2）.In conclusion,Cu/Zn-Pal-1 repaired the damage of the intestinal mucosal barrier induced by Salmonella Typhimurium in chicks and maintained intestinal homeostasis by regulating the proliferation,apoptosis and differentiation of intestinal epithelial cells.5.Effects of copper/zinc-modified palygorskite on intestinal microbiota of infected chicksThe effect of copper/zinc-modified palygorskite（Cu/Zn-Pal-1）on the intestinal flora of chickens infected with S.Typhimurium was explored in this study.Optimized sequence numbers and OTU numbers for high-throughput sequencing were obtained using data optimization and OTU clustering.The sequencing depth was detected by Alpha diversity analysis.The species and relative abundance of microorganisms in the samples were obtained using species composition analysis.The differences between samples in each group were detected using Beta diversity analysis,and the effect of each species abundance on the difference effect were evaluated using species difference analysis.The results showed that 1110881 optimized sequences,465057648 base pairs and 9216 OTU were obtained based on 16S r RNA high-throughput sequencing,and the length of 99.67%optimized sequences was between 401bp and 450bp.Alpha diversity analysis showed that the sequencing depth covered all species of the 24 intestinal contents samples,which was sufficient to reflect the abundance and diversity of intestinal microbiota of chicks.At the phylum level,Cu/Zn-Pal-1 could significantly reduce the relative abundance of Firmicutes and increase the relative proportion of Proteobacteria and Bacteroidetes.At the genus level,Cu/Zn-Pal-1 supplementation significantly increased the relative abundance of Ligillactobacillus in the gut of infected chicks,and also enhanced the relative proportions of Vibrionimonas,Rhodanobacter and Bradyrhizobium.Beta diversity analysis showed that there wasa significant difference between groups overall.In conclusion,Cu/Zn-Pal-1 can regulate the intestinal microbiota of infected chicks and alleviate the damage of S.typhimurium to intestinal epithelial cells.