Development and application of DNA probes and PCR tests for Haemophilus paragallinarum

Screening a genomic DNA library of H. paragallinarum strain Modesto identified 4 clones that reacted specifically with all 56 isolates of H. paragallinarum tested and failed to react with 24 closely related bacteria from the genera Pasteurella and Actinobacillus . All 4 clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae . The probes based on these 4 clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The 4 probes detected between 7.8 and 31.25 ng of purified DNA from homologous strains with no obvious correlation between probe size and sensitivity. The smallest probe, termed P601, was partially sequenced, and primers for 2 polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR rests were able to detect 1 pg DNA and between 102 and 103 cells. A method for using the HPG-2 PCR test directly on sinus swabs was developed. Using this method, there was 100% agreement between culture and the 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 infectious coryza. The tests can be used as confirmation following the isolation of a haemophilic organism. The HPG-2 PCR test also appears to be a potential alternative to culture