| Clostridium perfringens is resident in the gastrointestinal tract of humans and animals.Necrotic enteritis occurs when the concentration of the pathogenic clostridium perfringens in digestive juices exceeds a certain level.For many years,antibiotics have been used as growth promoters and to control necrotic enteritis in poultry,but the emergence of antibiotic resistance has led to a worldwide ban on their use as feed additives.With the trend of limiting and forbidding antibodies,the incidence of necrotic enteritis of poultry increased gradually,resulting in increased loss of poultry industry.We need to find alternatives to antibiotics to control necrotic enteritis in poultry,and phages are one of them.This study aims to isolate clostridium perfringens from poultry and study its drug resistance,and use the isolated clostridium perfringens to model necrotizing enteritis in poultry.The therapeutic effect of phage was compared with that of antibiotics,which provided a theoretical basis for phage to replace antibiotics to control necrotic enteritis in poultry and a new idea for green breeding.The main research contents and results are as follows:1 Isolation and identification of Clostridium perfringens(1)Isolation and purification of Clostridium perfringensIn this study,a total of 500 cecum samples were collected from chicken farms in Hunan,Hubei,Shandong and Shanxi provinces,and 139 clostridium perfringens strains were isolated and purified,with a separation rate of 27.8%.(2)PCR method was used to detect the virulence-associated genes and serotype of the isolated clostridium perfringens.The results showed that among the 139 isolated clostridium perfringens,113 were type A,accounting for 81.29%,and 26 were type G,accounting for18.71%.Among them,65 were cpb2 toxin genes,accounting for 60.75%.No C type and CPE toxin producing strain has been identified.(3)Antimicrobial resistance of Clostridium perfringensThe minimum inhibitory concentration(MIC)method was selected to detect antimicrobial resistance of clostridium perfringens.The results showed that 139 strains of clostridium perfringens showed resistance to most antibiotics,including 100%to chloramphenicol,75.35%to amoxicillin,60.56%to ciprofloxacin,and high sensitivity to penicillin,enrofloxacin,cephalosporins and other antibiotics.The MIC values of 139 strains of clostridium perfringens against neomycin sulfate and erythromycin were 92.25%and 31.70%higher than the clinically recommended dose.The MIC values of amprimycin and pomomycin were within the clinically recommended dose range,but the ratios of specific MIC values≥128μg/m L were 85.42%and 56.25%,respectively.2 Lysis test of phageThe lysis rate of phage was measured by double-layer plate drop method.The bacteriophage was provided by Wuhan Green-AGR Co.,LTD.It is a bacteriophage cocktail preparation,the main component is clostridium perfringens phage.The results showed that the bacteriophage could lyse 134 isolates of clostridium perfringens,with the lyse rate up to96.4%.3 Animal testing(1)Infection with Clostridium perfringensIn this experiment,5 strains of G type and sixty 40-day-old Roman grey laying hens were selected,intragastric administration 1×10~9 cfu/m L mixed bacterial solution was continued for 1m L.After 9 days,random examination was performed.The results showed that except the control group,all groups had pathological changes,severe bleeding in duodenal intestinal wall and mesentery,obvious dilation in jejunum and caecum,thinning of intestinal wall,dense hemorrhagic spots in duodenal inner wall.These symptoms indicate clostridium perfringens infection was successful.(2)Intestinal morphology and lesion scoreAfter the treatment experiment,the chickens in each group were dissected and the intestinal lesions were observed.The results showed that the intestinal condition of the control group was normal.In the challenge group,there was a large amount of gas in most of the intestinal lumen,and a large amount of bleeding and a small amount of necrosis in the intestinal wall.In the high-dose phage treatment group,low-dose phage treatment group and antibiotic treatment group,there were a few bleeding spots in the intestinal lumen,and most of them were less than 5 bleeding spots.Each group was scored according to the scoring criteria for intestinal lesions.The results showed that the control group had the lowest score,and the lesion score was significantly increased after the challenge of clostridium perfringens(P<0.01),while the lesion score was significantly decreased after the treatment of high dose phage,low dose phage and antibiotics,and was significantly lower than that of the challenge group(P<0.01).There was no significant difference between the high dose phage group,the low dose phage group and the antibiotic group.(3)Intestinal histological observationDuodenum and jejunum were sectioned and pathological changes were observed.The results showed that in the challenge group,there were obvious lesions,necrosis and shedding of villous epithelial cells of duodenum and intestines,disappearance of large crypt and infiltration of inflammatory cells in jejunum,and no obvious lesions in the sections of other groups.Villus length and crypt depth were measured in each group.The data showed that villus length in both duodenum and jejunum was significantly reduced in the challenge group and basically recovered after treatment,while crypt depth in the challenge group was significantly shallower and recovered after treatment.(4)Intestinal tight junction detectionThe intestinal tight junction proteins claudin-1,Claudin-2 and Occludin were detected by fluorescence quantitative PCR.The results showed that the m RNA level of claudin-1 in duodenum was down-regulated in challenge group compared with control group,and significantly up-regulated in high-dose phage treatment group(P<0.01),and significantly up-regulated in high-dose phage treatment group and antibiotic treatment group compared with challenge group(P<0.01).The low dose phage treatment group was significantly up-regulated(P<0.05).The m RNA level of claudin-2 in duodenum was significantly down-regulated in antibiotic treatment group compared with control group(P<0.01),and up-regulated in challenge group.Compared with challenge group,the m RNA level of claudin-2in high-dose phage treatment group and antibiotic treatment group was significantly down-regulated(P<0.01),and that in low-dose treatment group was significantly down-regulated(P<0.05).There was no significant difference in m RNA level of occludin in duodenum among all groups.The m RNA level of claudin-1 in jejunum was significantly up-regulated in the low-dose phage treatment group compared with the control group(P<0.05),but there was no significant difference among other groups.There was no significant difference in the m RNA level of claudin-2 in jejunum among all groups.The m RNA level of occludin in jejunum in the low-dose phage treatment group was significantly up-regulated compared with that in the challenge group(P<0.05),but there was no significant difference among the other groups.(5)Intestinal clostridium perfringens detectionFluorescence quantitative PCR was used to detect clostridium perfringens in intestinal contents of each group.The results showed that the number of clostridium perfringens in the challenge group was the highest,up to 10~5 cfu/g,which was significantly higher than that in the control group and the low-dose phage treatment group(P<0.01),and significantly higher than that in the high-dose phage combination treatment group(P<0.05).The number of clostridium perfringens decreased significantly in both the high-dose and low-dose phage treatment groups.In conclusion,phage has a good killing effect on clostridium perfringens both in vivo and in vitro.Animal experimental results showed that both high dose and low dose phage can effectively relieve intestinal injury caused by clostridium perfringens,and its therapeutic effect is comparable to that of antibiotics.Therefore,phage is a good substitute for antibiotics. |
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