Epidemiological Investigation Of Clostridium Perfringens From Yak And Study On Biological Characteristics Of Qinghai-1 Strain

Clostridium perfringens（C.perfringens,Cp）,formerly known as Clostridium welchii,is a zoonotic opportunistic pathogen that can infect both humans and animals.It can usually cause human and animal gas gangrene,food poisoning,lamb dysentery,sheep gangrene,bovine enterotoxemia,necrotizing enteritis and other diseases.Yaks are mainly grazed,and it is difficult to implement disinfection and epidemic prevention measures.Clostridium perfringens disease of yaks occurs frequently in spring and autumn,which is one of the common diseases of yaks.In this study,744 yak fecal samples were collected from Tibet and Qinghai provinces,and Clostridium perfringens was isolated.The isolated strains were analyzed by drug sensitivity test.Then the toxin gene and drug resistance gene of the isolated strains were detected by PCR method.Finally,their diversity and affinity were analyzed by MLST.After that,the biological characteristics of Clostridium perfringens Qinhai-1 strain from yak were studied,and the pathogenicity of mice was studied using mice as model.Subsequently,bioinformatics technology was used to further understand the Clostridium perfringens from yak at the molecular level.The purpose is to investigate the prevalence of Clostridium perfringens in yaks,provide basis for the prevention and treatment of Clostridium perfringens disease in local yaks,and provide reference data for understanding the pathogenic mechanism of Clostridium perfringens from yaks and the prevention and control of related diseases in the future.The following results are obtained from this study:1.In this study,744 yak dung samples were collected from the Qinghai-Tibet Plateau.Through isolation,culture and identification,144of them were confirmed to be positive,with an overall positive rate of19.35%.According to MLST analysis,the housekeeper genes of 144strains of Clostridium perfringens from yak have 15～50（12.95 on average）alleles,89 STs and 11 CCs.2.Among 144 strains of Clostridium perfringens from yak,108strains（75%,108/144）are Clostridium perfringens type A,25 strains（17.36%,25/144）are Clostridium perfringens type C,4 strains（2.78%,4/144）are Clostridium perfringens type D,and 7 strains（4.86%,7/144）are Clostridium perfringens type F.No iap and net B genes were detected in this isolated strain.3.The drug sensitivity test showed that 144 strains of Clostridium perfringens isolated from yak feces had extensive resistance to 26antibiotics.The antibiotic drugs with severe drug resistance of isolated bacteria include streptomycin（93.75%）,sulfamethoxazole（86.81%）,kanamycin（81.25%）,erythromycin（81.25%）,polymyxin B（75%）,gentamicin（69.44%）,etc;The sensitive antibiotics include vancomycin,minocycline,cefotaxime,florfenicol and doxycycline.4.All isolates were resistant to 3 to 16 antibiotics,at least 3 drugs,and at most 16 drugs.None of the isolates were resistant to 26 antibiotics used in this study.More than half of the strains were resistant to 5～7drugs,with a total of 77 strains,accounting for 53.47%.98.61%of the isolates were multidrug resistant,and the most resistant mode was MAC-AGA-POL-SAs,with 24 strains（16.67%）.5.The isolated strains carried 14 drug resistance genes,the detection rate was 42.43%（14/33）,and the detection rate of the drug resistance gene erm（B）was the highest,with the detection rate of 74.31%.The detection rate of drug resistance genes erm（B）,mcr-2 and sul2 was more than 50%;The detection rate of drug resistance genes erm（X）and sul1was 30%～50%;The detection rate of drug resistance genes bla SHV,bla TEM,aph（3’）-III-F,tet A,tet B,tet M,Flor,qnr S,aac（6’）-Ib-cr was less than 15%;The other 19 drug resistance genes were not detected.6.The strain Qinghai-1 of Clostridium perfringens from yak is Clostridium perfringens type A;MLST typing confirmed that the strain was ST-414,which was a new ST-type strain.The growth curve showed that the strain had a slow period of 2 hours,a logarithmic growth period of 6 hours,a stable period of 8 hours,and then entered the decay period.The best time of producing toxin is 12 hours,and its content is 3.480mg/m L.LD₅₀is 5.955×10⁸CFU/m L。Animal experiments show that it can make mice have different degrees of clinical symptoms in spirit,appetite,action and neurological symptoms.Abdominal distension and vaginal bleeding were found in the infected mice.The autopsy revealed red fluid accumulation in the abdominal cavity and bleeding in small intestine and other tissues.There were different degrees of cell lysis and erosion in the intestine of the dead mice;The liver cells showed vacuolar degeneration;Renal tubules showed degeneration with hyaline tubules,and no hyperemia,edema and inflammatory cell infiltration were found in the interstitium;Histocyte proliferation can be seen in the white pulp of spleen,and congestion can be seen in the red pulp;Congestion of cerebral interstitial blood vessels;No pathological changes were found in the heart,lungs and stomach.The main pathogenic organs are intestine,liver,spleen,kidney and brain.7.Bioinformatics analysis showed that the total length of the chromosome genome of the strain of Clostridium perfringens Qinhai-1from yak was 3 042 374 bp,and the GC content was 28.58%;The total length of the plasmid was 38 989 bp,and the GC content was 26.22%.There are 2 906 genes（including 2 721 coding genes）,94 t RNAs,30r RNAs（10 for 5S r RNA,16S r RNA and 23S r RNA respectively）,60misc RNAs,1 tm RNA and 65 pseudogenes.The functional element analysis showed that the strain contained 1 gene island;968 repetitive sequences;It does not contain CRISPR（clustered regular interval short palindrome repeat sequence）;No prophage.The proprietary database annotation shows that there is 1 resistance gene;6 carbohydrate enzymes;1 drug resistance related gene;188 cytochrome P450 proteins;237virulence related factors;And 828 transmembrane proteins（162 of which are signal peptide proteins）.To sum up,the infection rate and toxin type of Clostridium perfringens in yaks were obtained through the epidemiological investigation of Clostridium perfringens from yaks;The drug resistance of Clostridium perfringens from yak and the types of drug resistance genes were determined by drug sensitivity test;Through biological characteristics,the biochemical characteristics,growth characteristics and pathogenicity of the strain of Clostridium perfringens Qinhai-1 from yak were understood;Through bioinformatics analysis,the structure and function of the strain of Clostridium perfringens Qinhai-1 from yak were recognized at the molecular level.