DEVELOPMENT AND APPLICATION OF DNA PROBES AND PCR TESTS FOR HAEMOPHILUS-PARAGALLINARUM

A genomic DNA library of Haemophilus paragallinarum strain Modesto was created. Screening of this library identified four clones that reacte d specifically with all 56 isolates of H. paragallinarum rested and fa iled to react with 24 closely related bacteria from the genera Pasteur ella and Actinobacillus. All four clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae. The probes based on these four clones were appro ximately 1.8, 2.3, 3.5, and 5.5 kb in size. The four probes were able to detect between 7.8 and 31.25 ng of purified DNA from homologous str ains with no obvious correlation between probe size and sensitivity. T he smallest probe, termed P601, was partially sequenced, and primers f or two polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were shown to b e specific, all 41 isolates of H. paragallinarum rested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR te sts were able to detect 1 pg DNA and between 10(2) and 10(3) cells. A method for using the HPG-2 PCR test directly on sinus swabs was develo ped. Using this method, there was 100% agreement between culture and t he direct HPG-2 PCR for the 36 swabs processed. The DNA probes and PCR tests appear to be useful diagnostic tools for the detection of infec tious coryza. The tests can be used as confirmatory tests following th e isolation of a hemophilic organism. As well, the HPG-2 PCR test appe ars to be a potential alternative to culture.