Isolation And Identifition Of Goat Klebsiella Pneumoniae And Its Establishment Of Methods For Detection

Klebsiella pneumoniae is a conditional pathogen that easily develops drug resistance and causes disease in humans and animals.It can cause meningitis,pneumonia,inflammation of urinary system,wound infections,and even sepsis.However,Klebsiella pneumoniae has not been valued in the goat industry.Due to the widely used antibiotics,high-density breeding and rapid fattening breeding models in the rapid development of the goat industry,the harm of the bacteria to the goat industry has increased.Goat-derived Klebsiella pneumoniae does not have a systematic clinical detection technique.Therefore,the prerequisite for controlling the disease is to establish clinical detection technology for Klebsiella pneumoniae.In the present study,Klebsiella pneumoniae was isolated from the lungs of infected-goats.Indirect enzyme linked immunosorbent assay(ELISA)and indirect immunofluorescence(IFA)detection techniques were established,which laid the foundation for Klebsiella pneumoniae in the development of a rapid diagnostic kit.Two bacterial strains were successfully isolated from goat lungs,forming off-white,round bumps,neat edges,moist,and sticky colonies on the LB medium,which can be picked up and easily drawn wire.Gram staining confirmed that the isolates were both Gram-negative bacteria.All Kunming mice died within 8 hours after inoculating 0.2 mL 2×10~9 CFU/mL cultures of two isolated bacteria and the inoculated bacteria were isolated again from the lungs of the dead mice.The biochemical test results were consistent with the biochemical characteristics of Klebsiella pneumoniae.Drug sensitivity tests found that bacteria are sensitive to drugs including cephalosporins and aminoglycosides.The 16s rRNA test results proved that the isolates were Klebsiella pneumoniae,and the specific bands were obtained by specific primer detection.Collectively,the isolate was Klebsiella pneumoniae.Homology analysis of Klebsiella pneumoniae from other animals was performed,and the result indicated that the evolutionary relationship was similar to that of Klebsiella pneumoniae from fur animals,with the highest homology of 99.5%.Klebsiella pneumoniae was cultured at a concentration of 2.0x10~100 CFU/mL and then inactivated to prepare a white oil adjuvant antigen,and finally a Klebsiella pneumoniae goat polyclonal antibody was produced by immunizing Laiwu black goats using inactivated bacteria.An indirect ELISA method for detecting Klebsiella pneumoniae was established based on this polyclonal antibody,and the detection conditions of the detection method were screened and optimized.According to the test results,the optimal working concentration of the indirect ELSIA bacterial antigen is 2.99×10~7 CFU/mL,the optimal working concentration of the polyclonal antibody is 1:6 400,and the optimal working concentration of the enzyme-labeled secondary antibody is 1:5 000.The antibody dilution is 1%BSA phosphate buffer.The best working conditions was determined include coating with carbonate buffer for 12 h at 4°C,blocking with carbonate buffer containing 1.5%BSA for12 h at 4°C,incubation of primary antibody for 60 min,secondary antibody incubation for45 min,and substrate reaction for 30 min.The titer of the polyclonal antibody measured by the established indirect ELISA method was 1:12 800.The coated antigen could not cross-react with other bacterial serum,and the intra-assay and intra-assay variation coefficients of the indirect ELISA were below 7.20%.Through clinical application,the detection rate of 1 320 goat serums was 6.74%higher than that of the agglutination test.The test results showed that the indirect ELISA method has the advantages of simple operation,strong specificity,and high sensitivity.Furthermore,the prepared Klebsiella pneumoniae white oil adjuvant antigen was used to immunize healthy rabbits to obtain a goat Klebsiella pneumoniae rabbit polyclonal antibody,and to establish an IFA detection method for detecting Klebsiella pneumoniae.This polyclonal antibody was screened and optimized for detection conditions.The test results showed that the dilution of polyclonal antibody is 1:80,the incubation time is 30 minutes,the dilution of FITC-labeled goat anti-rabbit IgG is 1:400,and the incubation time is 30minutes.The specific yellow-green fluorescence is the clearest and the non-specific fluorescence is the weakest on smears of goat Klebsiella pneumoniae.There was no cross-reaction between the prepared polyclonal antibodies and other bacteria.IFA detection was performed on 124 clinical materials,and the coincidence rate of comparison with the results of bacterial isolation and identification was 98.4%.The experimental results proved that the IFA method has the advantages of simple operation and strong specificity.In the study,the two detection methods established provide theoretical basis for epidemiological investigation,clinical diagnosis and detection of Klebsiella pneumoniae in goats from antibody detection and clinical antigen detection.They also provide technical support for better control in goats,which has important public health significance.