| Klebsiella pneumoniae is an important conditional pathogen,which can cause infection in pigs,cattle,sheep,rabbits,other economic animals and humans,and leads to bronchitis,pneumonia,urinary system inflammation,mammary system inflammation and traumatic infection.In serious conditions,this bacterium even can cause sepsis,meningitis and pleurisy.In recent years,the drug resistance of Klebsiella pneumonia has a significant increase due to the extensive use of antibiotics.However,there is few reports on the identification of this bacterium and its prevalence in large-scale pig farms at home and abroad.In this research,samples were collected from some intensive pig farms in Jilin Province,which were suspected to be infected with Klebsiella pneumonia.And the isolated bacteria were identified by morphological analysis,16 s r DNA sequence analysis,physiology and biochemistry test.The screened strains were further tested by drug resistance,pathogenicity,virulence genes and capsule type.In order to provide some theoretical support for the infection,prevention and control of Klebsiella pneumoniae,the whole bacterial antigen was prepared by three different methods,and the indirect ELISA was established to investigate the prevalence of Klebsiella pneumoniae of intensive pig farms in Jilin Province.1.Isolation and identification of Klebsiella pneumoniae from pigs.In order to understand the infection of Klebsiella pneumoniae of some intensive pig farms in Jilin province,the samples suspected to be infected with Klebsiella pneumoniae were collected.Fourteen strains of Klebsiella pneumoniae were isolated by 16 s r DNA sequence analysis,morphological analysis,physiological and biochemical tests.Through the detection of antibiotic resistance,the result showed that most strains were highly resistant to ?-lactam,aminoglycoside and tetracycline.Through the detection of pathogenicity and pathogenetic genes,all strains carried Khe gene(100%),among which seven stains carried ramp gene(50%).The detection rate of wac G gene was 0%.The results of Capsular classification showed that the capsule types of isolated strains were K20(12/14,85.71%)and K1(2/14 14.29%)and the capsular type of the most virulent strain was K20.2.Establishment of indirect ELISA methodThe antigens were prepared from the most virulent strain by three different methods.Firstly,the optimal concentrations of antigens and serum were determined by square matrix titration.Secondly,the dilution ratio,action time and substrate action time of enzyme-labeled antibody were optimized and its critical value was determined.Finally,sensitivity,specificity and stability of ELISA were tested.The results showed the protein concentration of ELISA based on heat-resistant antigen was 14.820 mg / m L and the best dilution multiple of antigen was 160-fold.When the serum was exposed to serum for 1.5 h,the dilution multiple of enzyme labeled antibody was 500 times and the action time was 1 h,Which was in the best reaction condition.The critical value calculated by the formula was 0.610.This method was sensitive,specific and stable.So that it could be used to detect the serum antibody of Klebsiella pneumoniae.3.Seroepidemiological survey of Klebsiella pneumoniae in some intensive pig farms in Jilin ProvinceFive hundred and fifty-eight serum samples were detected by ELISA.The prevalence of Klebsiella pneumoniae was serious in large-scale pig farms in Jilin Province and the total positive rate was 30.46%(170/558).From the location of sample collection,the positive rate of large scale pig farms in Changchun was highest,at 8.60%(48/558),following Yanji 6.09%(34/558)and Gonzhuling was the lowest with 1.61%(9/558).According to the age stages of the samples,the positive rates of antibodies against Klebsiella pneumoniae in newborn piglets and sows were the highest 12.19%(68/558)and 14.70%(85/558),respectively,while the positive rates of fattening pigs and sows were lower at 1.97%(11/558)and 1.61%(9/558). |
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