The Establishment Of Detection Methods For Riemerella Anatipestifer

Riemerella anatipestifer infection is a contagious disease of domestic ducks, geese, turkeys and various other domestic and wild birds. It occurs as an acute or chronic septicemian characterized by fibrinous, pericarditis, perihepatitis, and airsacculitis. It is a major disease confronting the duck industry throughout the world. RA infection accounts for major economic losses to the duck industry due to high mortality, weight lose. A definite diagnosis of R. anatipestifer infection is usually made upon isolation and identification of this bacterium , although a presumptive diagnosis might be made from clinical and necropsy findings. Immunofluorescence procedures and enzyme-linked immunosorbent assay (ELISA) have also been applied for the detection of R. anatipestifer in tissue or exudates, and to detect serum antibodies in ducks, respectively.According to the published OmpA gene conservative region a pair of primers was designed by DNAstar. Then optimized the condition, a fast, economic and sensitive PCR method for direct identification of Riemerella anatipestifer are been developed.PCR analysis of R.anatipestifer genomic DNA using the primers, detected the presence of a 1.2 kb DNA band in each of the 23 R. anatipestifer strains tested, including the type strain, serotype reference strains and field strains examined. The 670 bp DNA, corresponding to the size expected for the DNA designed, was detected in R. anatipestifer strains belonging to serotypes 1,2, 10, 11 and 16. When detected E.coli, Pasteurella multocida and other common bacteria of duck the results were negative .Sensitivity test results showed that the PCR detection lower limit is 80 Riemerella atipestifer clon。By comparison, the results demonstrated that the colony PCR and conventional PCR got consistent results, a 670 bp band specific band amplified by both of them.The study further proved the reliability of using PCR to detect bacteria. For detecting the R. anatipestifer-specific antibodies in sera , an rompA-based ELISA was developed.The outer membrane protein A gene of Riemerella anatipestifer isolate was amplified by PCR with a size of 1200bp. The amplified DNA fragment was inserted in pET-28a and transformed into Escherichia coli BL21-DL3, the fragment was expressed after induced, the purity of the recombinant protein was verified by SDS-PAGE with Coomassie blue staining and further confirmed by immunoblotting using protein specific polyclonal duck sera.The 42kD rompA-GST fusion protein was expressed mainly as an soluble protein. Using purified soluble nucleoprotein, an indirect ELISA for detection of anti-RA antibodies was developed and its optimal reaction conditions were determined: antigen(12.5μg/mL) coated at 4℃overnight ,serum sample(1:80) incubated at 37℃for 40 min and enzyme-labbeled goat IgG against duck,(1:600)incubated at 37℃for 40 min, the substrate for ELISA incubated at 37℃for 15min. The ELISA assay was confirmed to have a good reiteratively, specificity and sensitivity by reiteratively test, cross test and competitive-inhibition test.The use of this ELISA in combination with PCR methods would therefore constitute an improved diagnostic assay for the detection of R. anatipestifer infection.