New DNA-Based Technique To Check For Deadly Pneumonia

To help wildlife biologists prevent transmission of disease during future relocation operations, Drs. Al Ward and Glen Weiser are once again evaluating tonsil swab samples from Northwest bighorn sheep this winter.

Of the 348 bighorn sheep they sampled last year from Idaho, Oregon, Nevada, Alaska and Canada, Ward and Weiser found that about 20 percent carried bacteria that potentially threaten the health of the wild sheep. These infected animals bore Pasteurella haemolytica and Pasteurella trehalosi bacteria armed with the "lktA" gene-a gene that enables the bacteria to release a toxin that attacks bighorns' white blood cells. As many as 75 percent of bighorns infected with lktA-positive bacteria can die of pneumonia.

Last year's samples were first cultured in the laboratory-the slower, conventional technique-then frozen and retested using the new, rapid polymerase chain reaction, or PCR, method. In the frozen samples, the PCR test came to the same conclusions as the older method 92 percent of the time.

Ward and Weiser are confident that this year's study-conducted entirely with fresh samples-will detect the lktA gene with even greater accuracy and frequency. Laboratory cultures, which use selective antibiotics to help isolate the Pasteurella bacteria, depend on follow-up steps to identify the bacteria and evaluate their ability to produce leukotoxin. By contrast, PCR is a direct test for the presence of the lktA gene. "Theoretically, one of the beauties of this type of procedure is that an intact gene can be detected and an infected animal can be identified even if the bacteria themselves have died," says Ward.

Mark Drew, wildlife veterinarian for the Idaho Department of Fish and Game, calls Pasteurella pneumonia "probably one of the major factors" in death and illness among bighorn sheep. "Conceivably, if we could get results in a reasonable amount of time and with reasonable accuracy, we could potentially pick and choose the sheep that we wanted to move or transplant," he says.

All PCR tests depend on primers-short pieces of DNA that recognize only the specific sequence of the gene scientists are seeking. To locate any targeted gene, scientists need primers for both the beginning and end of its sequence. UI scientists first determined which primers were needed to find the lktA gene by using the National Institute of Health's electronic genebank for Pasteurella. Then, building on previous work by Ward, they developed a liquid enrichment medium that allows the disease organisms to multiply rapidly during overnight incubation. The formula works so well that as few as 50 live Pasteurella bacteria produce "profuse" overnight growth, Ward says, while multiplication of most other bacteria is prevented or retarded.

Ward believes the test will prove useful in two significant ways: It should be able to protect "clean" bighorn populations from infection by disease-carrying newcomers, and it should prevent chronically infected animals from being moved and dying unnecessarily of stress-induced flare-ups. But Ward and Weiser won't know for sure until this year's research is completed. "This stage of the testing is critical," says Ward. "We need to make sure that our technique works, that it works well and that it works quickly."