Study On The Mechanism Of Alfalfa Polysaccharide Manipulating Gut Microbiome Of Salmonella-challenged Broilers

Alfalfa polysaccharide(APS)has anti-inflammatory and antibacterial capacities;however,the effects of APS on intestinal health and the microbiota in Salmonella-challenged broilers remain unclear.Salmonella is a zoonotic pathogen that harms human and animal health,and is also considered to be a major foodborne pathogen that seriously threatens food safety.In this experiment,a broiler chicken infected with Salmonella was used as a model to study the effect of adding alfalfa polysaccharide to the diet on the intestinal microbiome of Salmonella infected broilers.The alfalfa provided by the experimental farm of Shandong Agricultural University was used for the experiment.The alfalfa polysaccharide was extracted by water extraction and alcohol precipitation.For the breeding test,240 1-day-old broilers(mixed sex)were selected,and the broilers were randomly divided into 24 pens of 4treatments(10 birds per pen and 6 pens per treatment).The experiment was conducted with a two-factor factorial design,and the pens were considered replicate units.The 4 treatment groups were as follows:(1)The CON group(A basal diet-fed group),(2)The CON+SA group(A basal diet-fed group challenged with 3 mL of Salmonella enteritidis by oral gavage at 11and 18 days of age),(3)The APS group(An APS-supplemented basal diet-fed group,dose:500 mg/kg diet;),and(4)The APS+SA group(An APS-supplemented basal diet-fed group challenged with Salmonella enteritidis).During the experiment,broilers were free to eat and drink.The main findings are as follows:(1)Alfalfa polysaccharide extracted by water extraction and alcohol precipitation method is composed of fucose,arabinose,galactose,glucose,xylose,mannose,galacturonic acid and glucuronic acid with a molar ratio of 2.02:6.25:3.66:16.46:2.36:0.77:57.13:11.36 and molar weight of 3.30×10~6 Da.(2)During the 42-day growth period,the two groups with alfalfa polysaccharides significantly increased(P<0.05)intestinal immunoglobulin G and A.(3)At 21 and 42 days,the length of duodenum and jejunum in the APS group was significantly longer than that in the CON group(P<0.05),and the crypt depth in the APS+SA group was significantly smaller than in the CON+SA group.These changes were significantly increased(P<0.05)Vill length/crypt depth(V/C)ratio.At 42 days,the activity of intestinal diamine oxidase(DAO)and the relative expression of tight junction proteins(claudin-1,occludin and MUC-2)in the two groups supplemented with alfalfa polysaccharide were higher than those of the control group(P<0.05).(4)The average OTU numbers were 325,405,and 647 in the CON group at 14,21 and42 days of age,respectively.Similarly,there were 309,456 and 434 OTUs in the APS group;361,482 and 431 OTUs in the CON+SA group;and 419,408 and 699 OTUs in the APS+SA group at 14,21 and 42 days of age,respectively.At 14 and 42 days,the number of OUTs in the APS+SA group was significantly greater than that in the APS group(P<0.05).It can be seen that the addition of alfalfa polysaccharide to the diet increased the number of OTUs in the cecum of broilers infected with Salmonella.(5)At the gate level,the caecum microorganisms of broiler chickens of different ages were dominated by Firmicutes and Bacteroides.As the age of broiler chickens increased,the abundance of Bacteroides continued to increase,while the abundance of Firmicutes and the ratio of Firmicutes to Bacteroides(F/B)continued to decrease.(6)Odoribacter and Bacteroides are core microorganisms in broiler cecum flora that are directly related to broiler weight.In summary,dietary APS supplementation improved intestinal barrier functions and immunity in broilers by modulating the morphology of the gut lining and the composition and structure of the intestinal microbiota regardless of whether Salmonella infection was present.Salmonella infection increased the abundance of facultative anaerobic bacteria or potential pathogens in the gut microbiota,whereas inclusion of APS in broiler feed augmented beneficial bacterial proliferation.Alfalfa polysaccharide may be a potential immune enhancer,enhance the intestinal health of broilers,improve immunity,and promote rapid growth of broilers.