| Clostridium perfringens is an important zoonotic pathogen,which has seriously affected the healthy breeding of livestock and poultry in veterinary clinic.However,there is a lack of systematic research on the epidemiological and virulence characteristics of the bacterium in sheep and waterfowl,which restricts its clinical prevention and control to a certain extent.Based on the molecular epidemiological investigation of Clostridium perfringens in eastern China,the genotype and drug sensitivity of clinical isolates of Clostridium perfringens were further analyzed.Finally,the virulence of Clostridium perfringens from different sources and toxins was tested in mice to provide basis for precise prevention and control of Clostridium perfringens.1.Molecular Epidemiological Analysis of Clostridium perfringens from Sheep and Waterfowl in Eastern ChinaSamples were collected and DNA was extracted for PCR detection.The results showed that Clostridium perfringens were detected in 175 fecal samples(18.74%)and 4 tissue samples(20%)of 934 clinical fecal samples and 20 tissue samples of sheep,respectively.Among them,the positive detection rate of healthy stool samples was 19.51%,the positive detection rate of morbidity stool samples was 2.38%,and the positive tissue samples were all dead samples.Clostridium perfringens were detected from 202 stool samples(43.72%)and 3 tissue samples(3.45%)of 462 clinical stool samples and 87 tissue samples of waterfowl,respectively.Among them,the positive rate of healthy stool samples was 45.79%,the positive rate of morbidity stool samples was 4.35%,and the positive tissue samples were all healthy tissues.The results showed that the detection rates of Clostridium perfringens in faeces and tissues of sheep were not significantly different.The detection rates of faeces in waterfowl samples were significantly higher than those in tissues.The detection rates in healthy faeces of sheep and waterfowl samples were significantly higher than those in pathogenic faeces.It suggested that Clostridium perfringens is widely carried in clinical healthy sheep and waterfowl,and the mortality of sheep and waterfowl is not necessarily due to Clostridium perfringens infection;the total detection rate of waterfowl samples is higher than that of sheep samples,which may be related to the humidity of waterfowl living environment.The highest positive rate of Clostridium perfringens from sheep was Yangzhou,reaching 21.45%.The highest positive rate of Clostridium perfringens from waterfowl was Jiangsu Province,reaching 68.75%.There was no significant difference in the positive rate of Clostridium perfringens from all parts of Jiangsu Province.It is suggested that Clostridium perfringens from sheep is more prevalent in Yangzhou and Clostridium perfringens from waterfowl is more prevalent in Jiangsu Province.2.Type analysis of the Clostridium perfringens isolatesClostridium perfringens were isolated from the above positive bacterial liquid.The single colonies were identified by PCR,Gram staining,methylene blue staining,biochemical and milk fermentation tests.Using published primers,the types of toxins contained in the isolated strains were detected by PCR,and the toxin types of each strain were determined.The results showed that 63.24%(43/68)of 68 strains of Clostridium perfringens from sheep were genotype A(a toxin),36.76%(25/68)were genotype D(a toxin and ? toxin),and 9 strains of Clostridium perfringens from waterfowl were genotype A(9/9).No B(?,?,? toxins),C(?,?toxins),E(?,? toxins)were found.It is suggested that most of the sheep in this area carry type A bacterium,a few carry type D bacterium and waterfowl carry type A bacterium.3.Drug sensitivity analysis of Clostridium perfringens isolatesThe paper diffusion method was used to test the susceptibility of selected strains and cultured strains from different regions and animal species,and the susceptibility of strains to antibiotics was judged by the size of the bacteriostatic zone.50 isolates from 77 isolates were selected according to the region for the experiment.Among them,45 were sheep-derived bacteria and 5 were waterfowl-derived bacteria.The results showed that the proportion of resistant strains to Lincomycin and Gentamicin was relatively high,and the drug resistance rates were 34%(17/50)and 32%(16/50),respectively.Most strains were double-resistant,of which 56%(28/50)were resistant to two or more antibiotics,and only a few were single or triple-resistant.In Nantong,Suqian and Xuzhou,the Sheep-derived strains were more resistant to gentamicin and lincomycin,while in Lianyungang,the Sheep-derived strains were more resistant to doxycycline,and the resistance rate was more than 50%.The resistance rate of waterfowl-derived strains to tetracycline was higher in Xuzhou and Jiujiang areas,and that of Enrofloxacin was higher in Nanchang area.The resistance rate of waterfowl-derived strains to Compound trimethoprim was higher in Jiujiang and Nanchang areas,with 100%resistance rate.The resistance of strain type A to tetracycline,enrofloxacin and compound trimethoprim was slightly higher than that of strain type D,but there was no significant difference in other strains.The results indicated that there was abuse of antibiotics in farms,which led to the emergence of multi-drug resistant strains4.Toxicity comparison of different toxin-producing Clostridium perfringens isolates and pathogenicity test in miceMice were randomly divided into four groups.After plate colony counting,Clostridium perfringens culture medium(group 1),Clostridium perfringens type A strain BC51(group 2),Clostridium perfringens type D strain HC40(group 3)and Clostridium perfringens type A strain IY1-2(group 4)were injected intraperitoneally.The LD50 of bacteria was calculated by Reed-Muench methodThe results showed that LD50 was 5.6×10757 CFU/mL in group 2,5.7×105.5 CFU/mL in group 3 and 3×106 375 CFU/mL in group 4.It can be seen that the virulence of type D strain HC40 from sheep was the strongest,followed by strain IY1-2 from waterfowl,and strain BC51 from sheep was the weakest.The virulence of different toxin-type Clostridium perfringens of the same species is different,and the virulence of different species of the same toxin-type is different,which may be related to the synergistic effect of a and ? toxins carried by type D bacteria,the time of colonization of strains in the gut of different species of animals,and the differences in other virulence factors carried by the same toxin-type strains of different animal sources.The results of this experiment provide some ideas and directions for the follow-up studyAfter infection with Clostridium perfringens,mice developed rapidly with abdominal enlargement and obvious intestinal blockage;their skin was bronze or even black;the odor of hydrogen sulfide could be detected after dissection;and pathological changes such as hemorrhage,congestion,necrosis and ulceration were found in liver,stomach and small intestine It is suggested that the bacteria multiply in large quantities in animals,and the toxins produced reach the whole body along with the blood circulation system,leading to multiple organ damage The data obtained in this experiment provide a reference for the establishment of a stable model of Clostridium perfringens infection in mice,and the establishment of an infection model can also provide a suitable animal model for the in-depth study of Clostridium perfringens in various disciplines.The results of this study suggest that Clostridium perfringens are widely carried in clinically healthy sheep and waterfowl,mainly type A,and there is still abuse of antibiotics in farms.This study provides a basis for the precise prevention and control of the disease,and also provides ideas and directions for further research. |
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