| Summary: | 碩士 === 國立中興大學 === 獸醫病理生物學研究所 === 98 === Actinobacillus pleuropneumoniae (APP) exotoxin (Apx) is one of the strong virulent factors of APP. Apx possesses hemolytic and cytotoxic activities which cause serious lung damage in APP infected pigs. The aim of this study was to evaluate the sensitivity of different cell types or cell lines originated from different species toward Apx. APP serotype 10-derived exotoxin ApxI was used to intratracheally inoculate piglets; lung sections were subjected to H&E, immunohistochemical, TUNEL, and Hoechst stains in order to assess the effects of ApxI on porcine lung tissue. Lung sections of ApxI treated group had mild neutrophil infiltration and interstitial hyperplaisa. TUNEL staining revealed higher percentage of apoptotic cells in ApxI treated group of which 54% were phagocytic cells. Piglets inoculated with heat-inactivated ApxI showed mild inflammation in lungs while no obvious histopathological changes observed in saline treated piglets. Both groups had lower percentage of apoptotic cells in lung sections as compared to ApxI treated group. To further understand the sensitivities of cell lines from different species toward Apx of different serotypes, porcine alveolar macrophage (PAM), BL-3, BL-3.1, RAW 264.7, PK-15, and Vero cells were used for comparison. Results showed that BL-3, BL-3.1, and PAM had the highest sensitivity toward exotoxins derived from APP serotype 1 (APP1) and 2 (APP2), and no significant difference was found between these cells. RAW 264.7 cells were mildly sensitive toward APP1 exotoxin, and PK-15 and Vero cells were not sensitive toward exotoxins of APP.
Further, an APP serotype 10 apxIA mutant was successfully constructed through homologous recombination of APP genomic DNA with a suicide vector carrying apxIA gene inserted with a kanamycin-resistant determinant. The mutant with desired mutation was selected and confirmed by antibiotic resistance, PCR, and DNA sequence analysis. Growth curves, Biolog metabolic profiles, hemolytic assay, cytotoxic assay, TUNEL assay, and Western blot analysis were used to verify the phenotypes of mutant strain. The apxIA mutant had similar growth rate and exhibited similar metabolic profiles compared to the parental strain. In addition, apxIA mutant strain lost its ability to produce ApxI, therefore no hemolytic, cytotoxic, or apoptosis inducing activity was observed in the bacterial culture supernatant.
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