Found Of Putative Virulent Gene Fragments Of Riemerella Anatipestifer Type 2 Strains And Isolation And Identification Of Non-Pathogenic Strain Of The Bacterium

The infection of Riemerella anatipestifer (RA) is one of the most important diseases in duck. The disease occurs as an acute or chronic septicemia, characterized by fibrinous pericarditis, perihepatitis, air sacculitis, meningitis, caseous salpingitis, arthritis, and conjunctivitis. Currently, 21 serotypes have been identified in the world, and little cross reaction in different serotypes. At home and abroad, serotype 2 has been frequently isolated. Some research has been done in RA, but little on virulence, pathogenicity and protective antigen.The strain of gram-negative bacteria, LY-58, was isolated from pharyngeal mucous membranes of healthy Pekin ducklings on a duck farm in Hexian of Anhui province. The culture character, morphology observations, physiological and biochemical test, serology and PCR identification showed that LY-58 strain was identified to be type 2 Riemerella anatipestifer. Virulence test showed LD₅₀ for Pekin ducklings of type 2 Riemerella anatipestifer reference strain, RAF2, was 5.57×10⁷ CFU, and LY-58 was more than 1.0× 10¹⁰ CFU. Therefore, LY-58 was regarded as a non-pathogenic strain of Riemerella anatipestifer.In order to find putative virulent genes of RA, a subtractive library including 600 clones was constructed by suppression subtractive hybridization(SSH) between virulent strain RAF2 and avirulent strain LY-58. Ninety-six clones selected at random from the subtractive library were amplified by PCR, the result of colony PCR showed that these clones contained 100-1000bp inserted fragments, then these fragments were sequenced and blasted with the published sequences in Genbank, the result of blast showed 15 gene...