MOLECULAR CHARACTERIZATION AND IMMUNOPROTECTIVE ACTIVITY OF CAPSULAR POLYSACCHARIDE OF KLEBSIELLA PNEUMONIAE ISOLATED FROM FARM ANIMALS AT TAIF GOVERNORATE

摘要

Klebsiella pneumoniae is a Gram-negative enterobacterium that has historically been and currently remains, a significant cause of human disease and several kinds of infections in animals. In the present work, trials for the isolation of Klebsiella pneumoniae from diseased and apparently healthy farm animals (cows, sheep, goats and camels) were done for recognition of Klebsiella pneumoniae subspecies. It was noticed that there was a marked variation between incidences of Klebsiella pneumoniae subspecies in examined animals as regards to health condition. The frequency was greater among samples collected from diseased animals 25.2% as compared with apparently healthy one 5.5%. It was found that there was great difference between the prevalence of Klebsiella isolated from various animal origins. On biochemical identification Klebsiella pneumoniae subsp. pneumoniae was the most prevalent followed by Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. Rhinoscleromatis. Klebsiella pneumoniae subsp. rhinoscleromatis was not isolated from apparently healthy animals. The in vitro sensitivity of isolates of Klebsiella pneumoniae subspecies recovered from different animal species to 23 antimicrobial agents was tested. It was found that were resistance to cefoxitin, cefotaxime, cefoperazone, ceftazidime, ceftriaxone, aztreonam, amoxicillin and ampicillin. The most potent antibiotics showing 100% activity against Klebsiella pneumoniae subsp. isolated in this study were imipenem, ciprofloxacin, norfloxacin, gentamicin and kanamycin. While 96.2% of all examined isolates were sensitive to amoxicillin/clavulanic acid and ticarcillin/clavulanic acid. SDS-PAGE analysis showed that CPSs of Klebsiella pneumoniae subspecies contained wide variety of different molecular weights which ranged from 15.52 kDa to106.29 kDa and gave 10-13 bands. Evaluation of humoral immune response of mice immunized with CPSs was done using ELISA. It was found that the highest immune response was obtained 14 days post 1st dose of immunization with extracted CPSs of the three Klebsiella pneumoniae subspecies. When mice were immunized with CPSs of Klebsiella pneumoniae subspecies, they were protected against virulent challenge with homologous or heterologous strains, as a result the mortality rates were reduced from 80, 75 and 65 to 5 to 15% of Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis, respectively. Determination of the reisolation rates of Klebsiella pneumoniae subspecies from lung, liver and spleen were done on both immunized mice and challenged and on control mice. Histopathological studies have been done on both dead infected non-immunized and immunized mice. Lungs were the main organs that showed macroscopic and microscopic pathological changes. Finally, ELISA has been used for detection of CPSs of Klebsiella pneumoniae subspecies antibodies in sera of examined animals. The sensitivity of ELISA using CPSs extracted from Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis was 87.7, 89.0 and 83.6%, respectively. The specificity of this ELISA was 93.4, 92.9 and 93.1%, respectively. ELISA using CPSs antigens could therefore serve as a valuable aid in serodiagnosis of Klebsiella pneumonia.