| Chicken coccidiosis is an intestinal intracellular parasitic protozoan disease caused by one or more species of Eimeria,which is a serious hazard to the poultry industry.At present,the main means of prevention and control of the disease are vaccination and antibiotics,but vaccination has the problem of“spreading poison”,while the long-term widespread use of antibiotics can cause the emergence of drug-resistant strains of parasites,resulting in drug residues and other food safety problems,seriously affecting human health.Increasing the development of drug-resistant coccidial species has stimulated the search for alternative control methods or new drugs,and this has become a top priority for the poultry industry.Specific strains of lactic acid bacteria,in particular some of the genera Lactobacillus,are extensively used as probiotics since their ability to modulate the immune system has been demonstrated as well as their capacity to inhibit the growth or invasion of pathogenic bacteria and Eimeria.In this experiment,lactic acid bacteria were isolated from the cecum of white feather broiler chickens,and intestines were selected for potential probiotics based on their bile-salt resistance,low p H tolerance,potential adhesion to epithelial cells and the compound ratio of lactic acid bacteria was determined based on the number of viable bacteria.The anticoccidial effect of the compound lactobacilli was evaluated by Eimeria tenella(E.tenella)chick infection model,and the main research contents and results are as follows.In this study,the strains were isolated from the cecum contents of adult white-feather broiler under anaerobic conditions.The agar plate streaking method,morphological observation and 16S r RNA gene sequence analysis were used to identify and screen the strains,and the antibacterial ability,adhesion and tolerance of the strains were evaluated.The compound proportion of lactic acid bacteria was determined according to the number of viable bacteria.The results showed that three strains of lactic acid bacteria were screened as(Lactobacillus salivarius HO 66,L.salivarius HO 66),(Lactobacillus reuteri,L.reuteri),and(Lactobacillus brevis ATCC 14869,L.brevis ATCC 14869)respectively.L.reuteri,L.salivarius HO 66 and L.brevis 14869 showed that the number of viable cells was 1×10~3,1×10~4 and 1×10~3 CFU/m L after p H 1.5–5.The three strains of lactic acid bacteria showed good acid tolerance.The viable counts of L.reuteri,L.salivarius HO 66 and L.brevis 14869 reached 1×10~6,1×10~7and 1×10~6CFU/m L after 6 h culture in gastric juice.After 6 h of culture in artificial intestinal fluid,the viable count was 1×10~8,1×10~7 and 1×10~5CFU/m L respectively,showing strong activity,indicating that the three lactic acid bacteria could survive in intestinal fluid and gastric fluid.In the bile salt tolerance experiment,the viable counts of L.reuteri,L.salivarius HO 66 and L.brevis 14869 at bile salt concentration of 0.5 were 1×10~8,1×10~5 and 1×10~5 CFU/m L,respectively indicating that the three lactic acid bacteria were highly tolerant to bile salt.The results showed that the antibacterial diameters of the three strains against Escherichia coli(E.coli)were 12 mm,17 mm and 12 mm,respectively,which were moderately sensitive.The inhibition diameters of Salmonella were 17 mm,22 mm and 11 mm,respectively,which were all above moderate or highly sensitive levels.The diameters of inhibiting Staphylococcus aureus(S.aureus)were 13.4 mm,18 mm and 11.7 mm,respectively,reaching moderate or highly sensitive levels.According to the above results,three strains of lactic acid bacteria have the potential to inhibit S.aureus,Salmonella and S.aureus infection.The adhesion rates of L.reuteri,L.salivarius HO 66 and L.brevis 14869 to DF-1 were all above 15%after adhesion test.According to the above test results,the strains of L.reuteri,L.salivarius HO 66 and L.brevis14869 were mixed and cultured in different proportions(2:2:1,1:1:1,3:1:1).After counting count was 1×10~8,1×10~7 and 1×10~6 CFU/m L,respectively.According to the results,the viable count was the highest when the proportion of composite bacteria was 2:2:1.Forty-five 1-day-old chickens were randomly divided into PBS group,challenged group and compound lactobacillus group.From 3 days of age,each chick in the PBS group and the challenged group was fed with 1 m L PBS every other day,and each chick in the compound lactic acid bacteria group was fed with 1 m L compound lactic acid bacteria(1×10~9 CFU/m L).At the age of 14 days,5×10~4 eggs of E.tenella were orally inoculated in the challenge group and the compound lactobacillus group.The peripheral blood of chickens at 14 and 21 days of age was collected for flow cytometry detection,and the contents of CD4~+T cells and CD8~+T cells in each group before and after insect attack were detected.The body weight of chickens in each group was monitored every day after insect attack,and the relative weight gain rate,caecum pathological score,caecum pathological change,and anticoccidial index(ACI).The results of flow cytometry showed that oral administration of compound lactic acid bacteria could increase the number of CD4~+T cells and CD8~+T cells in the peripheral blood of healthy chickens,but there was no significant difference compared with PBS group and challenge group(p>0.05).The level of CD8~+T cells was significantly increased in the compound lactobacillus group compared with the challenged group(p<0.001).The average weight gain rate and relative weight gain rate of chickens were calculated,and the average weight gain rate and relative weight gain rate of chickens in the compound lactobacillus group were 34±14.92(95%)and18.9±8.33(52%),respectively,which were significantly higher than those in the insect attack group(p<0.01).At the same time,the insect reduction rate of compound lactobacillus group was significantly reduced by 38%compared with the attack group(p<0.0001).The cecal lesion score showed that the lesion value of 20 in the compound probiotics group was significantly reduced compared with the attack group,and the difference was significant(p<0.05).H&E staining and pathological sections showed that the number of coccidia oocysts was reduced and the inflammatory infiltration symptoms were significantly alleviated in the composite lactic acid bacteria group.ACI index determined that the composite lactic acid bacteria group had a good index of 165.The compound lactic acid bacteria developed in this study can significantly enhance the level of anti-coccidial immune cells,improve the weight gain of chickens,reduce the cecum lesions and reduce the ovulation of chickens after oral immunization,which has good anti-coccidial effect and lays a foundation for the application of anti-coccidial probiotics. |
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