Etiological Study On Pneumonia Infectious In Yak Farm

In a medium sized yak farm,there were a lot of yaks which had a loss of appetite,depressed depression,dried nose,high body temperatureand died in large numbers.After dissection of dead yaks,pleural effusion,lung marble-like lesions,rumen full of food were observed,and clinicallydiagnosedpneumonia.In order to know the cause of this severe disease,organ samples of heart,liver,spleen,lung and kidney of dead yaks were collected to isolate the pathogen and conduct related study.1.Isolation and identification of pathogenic bacteria of yak pneumonia,pathogenicity of isolated strains and determination of drug resistance spectrum were carried out.The pathogenic bacteria were isolated and purified from yak disease materials.Gram staining microscopy and morphological observation were carried out.The pathogenic bacteria were identified by VITEK and 16S rRNA PCR amplification and sequencing.The growth curve and biofilm formation ability of the three strains were determined by UV spectrophotometry combined with OD value and viable bacteria counting and crystal violet staining.K-B susceptibility paper method was used to test the susceptibility of 20 commonly used drugs in clinical isolation,and 28drug-resistant genes were detected by PCR combined with drug-resistant phenotype.The results showed that the isolated strains were classified into three types:Gram-negative bacteria,Proteusmirabilis,Escherichia coli and Klebsiella pneumoniae.The three strains were identified as Proteus mirabilis,Escherichia coli and Klebsiella pneumoniae.The known sequence homology of the three strains with the related strains was more than 99%.They were temporarily named GY1,GY2 and GY3.The three strains were delayed at 0-4 h,logarithmic at 5-10 h,and logarithmic at 12 h-Among the three strains,Proteus mirabilis and Klebsiella pneumoniae were moderately biofilm-forming strains and Escherichia coli were weak biofilm-forming strains.All strains were resistant to six drugs,including compound neotamine,oxacillin,erythromycin,vancomycin,clindamycin and tetracycline,and were resistant to ciprofloxacin,norfloxacin,and tetracycline.Ofloxacin,meropenem,florfenicol and polymyxin were susceptible to six drugs.The resistance genes of Proteus mirabilis were ermE and aac.The resistance genes of Escherichia coli were sul2,tetB,vanA and vanB.The resistance genes of Klebsiella pneumoniae were vanA,sul1,sul2,aac,SHV and fexA.Combining the results of drug resistance phenotype and drug resistance gene,Klebsiella pneumoniae has the broadest drug resistance spectrum.2.The pathogenicity and virulence gene detection of the isolated strains were completed.Animal regression test was carried out in mice.LD₅₀0 of the strain was determined by Koch's improved method.Animal regression test was carried out.Bacteria were isolated and identified from the organs of dead mice.Pathological histology of organs was observed.Virulence genes carried by pathogenic strains were detected by PCR.The results showed that LD₅₀0 of Proteus mirabilis was 1.12*10⁸ CFU,E.coli LD₅₀0 was 1.11*10⁸ CFU and Klebsiella pneumoniae LD₅₀was 1.094*10⁸ CFU.The corresponding pathogens were isolated from the organs of dead mice.Histological observation showed that there were different pathological changes in the organs of mice,and the mixed infection of Proteus mirabilis and Klebsiella pneumoniae was the most serious.The virulence genes of Proteus mirabilis are hpmA,hpmB,Fli,zapA,rpoA;E.coli is ompT,phoA,ompA;Klebsiella pneumoniae is kfuBc,arr,wabG,ompT,fimH.According to the above tests,Proteus mirabilis and Klebsiella pneumoniae had the strongest pathogenicity among the three isolates.