Disease studies: Bighorn sheep research continues

Casper – At Washington State University (WSU) in Pullman, Wash., Subramaniam Srikumaran has continued his focused research on pneumonia in wild sheep, and recent research has shown which bacteria causes the disease, and may yield a vaccine to prevent wild sheep die-offs as a result.
    “Dr. Srikumaran is doing cutting edge research related to the domestic sheep/bighorn sheep question,” explained Wyoming Wild Sheep Foundation Executive Director Kevin Hurley at the 2012 Wyoming Wild Sheep Annual Convention in Casper on June 2, where Srikumaran, known as Dr. Sri, presented his latest findings.
    Srikumaran, is a professor at WSU and holds the Rocky Crate Foundation for North American Wild Sheep Endowed Chair.
Finding the enemy
    Sri noted that there are several bacterial species that have been identified in the lungs of deceased bighorn sheep, but he said it is important to identify the bacteria primarily responsible for causing disease.
    “The bacteria that have been isolated and detected in pneumonia lungs are Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida and Mycoplasma ovipneumoniae, but which of these gives the kiss of death?” asked Sri. “If you look at the pneumonic lungs, we asked which bacterium could cause lesions, breakdown of the alveoli wall and kill the white blood cells.”
    For a microorganism to be accepted as a pathogen, Sri mentioned that a series of qualifications, called Koch’s Postulates, must be met. Organisms must be isolated from the sick animal, be cultured in pure form, and the purified culture should cause disease in healthy animals. The organism should be also be re-isolated from the sickened animals.
    “Isolation does not mean an organism causes disease,” clarified Sri, “and failure to isolate an organism from a sick or dead animal does not mean that it does not cause disease.”
    Based on the virulence factors of the bacteria, Sri said research has indicated the Mannheimia haemolytica consistently kills bighorn sheep.
    “When we put bacteria in the lungs, 100 percent of the animals die in 48 hours,” he explained. “Most importantly, M. haemolytica secretes a toxin that kills the white blood cells of the host, called a leukotoxin.”
Friendly fire
    Leukotoxins cause white blood cells, called PMNs, to self-destroy, in turn causing damage to the bighorn sheep.
    “A cell called PMN is the most important cellular defense agent in a host organism because it will swallow bacteria,” explained Sri. “PMNs have a granule containing toxic material.”
    After engulfing bacteria, PMNs release the toxic material, killing the bacteria and themselves.
    However, leukotoxins bind to PMNs and kill them, causing the release of the toxin-filled granules, which in turn cause damage to the host, in this case, bighorn sheep.
    “You could call it friendly fire,” he adds. “The acute inflammation and lung injury are caused by toxic substances released by white blood cells.”
The culprit
    Despite all the signs pointing toward M. haemolytica, Sri mentioned culture methods failed to isolate the bacteria in pneumonic lungs.
    “From the die-off that occurred in 2009/10, from the samples from Montana, M. haemolytica was isolated from only one out of 100 animals. From Nevada, out of 15 animals, only two had M. haemolytica, and from the state of Washington, out of 29 animals, they were able to isolate it from one,” said Sri. “That led some people to believe that maybe we were missing the agent causing death.”
    But the answer wasn’t that simple. Sri and his team then asked why M. haemolytica was the only organism causing death in experimental conditions, but was isolated less frequently in samples.
    “When we cultured M. haemolytica and B. trehalosi together, B. trehalosi grew uninhibited, whereas the M. haemolytica started to go down in numbers as early as six hours and was undetectable by 24 hours,” he explained.
    Additionally, more sensitive tests from the samples that had previously shown no presence of M. haemolytica saw that 91 percent of the samples had the leukotoxin secreted by M. haemolytica present.
    “Only 2.2 percent of healthy animals had toxin-positive M. haemolytica,” said Sri. “This is the most important organism that causes bighorn sheep pneumonia.”
    Other organisms did not have the full potential to result in the deaths of bighorn sheep, as was shown in lab trials.
    “P. multocida does not possess the virulence factors that causes lesions in the lungs,” he said, explaining that the organism would not be responsible as a result. “M. ovipneumoniae alone cannot cause fatal pneumonia. Two out of two lambs did not die when inoculated with the bacterium. It can increase the spread and severity of M. haemolytica-caused pneumonia, but it is not a necessary predisposing agent.”
    Continued research with M. haemolytica and B. trehalosi showed the leukotoxin secreted by bacteria is the main factor in bighorn sheep death.
    “Most bighorn sheep do not carry M. haemolytica. If they do carry it, they carry leukotoxin negative strains,” he added.
Developing a solution
    “The question is, what can we do?” asked Sri. “Spatial separation of domestic sheep and bighorn sheep works because we can prevent the transmission of M. haemolytica, or we can attempt to vaccinate the bighorn sheep.”
    Researchers are working on a vaccination for bighorn sheep that would be given nasally or in feed and would also be transmitted between the wild sheep, but, thus far, results have been unsuccessful.
    “One approach that is more promising uses a harmless virus that carries a non-lethal copy of the leukotoxin,” explained Sri. “The expectation is that, if we give it to a few bighorn sheep, the virus will be transmitted so they get vaccinated.”
    However, because of the difficultly of vaccinating wild animals, Sri’s team is looking at eliminating the organism in domestic animals or minimizing the shedding of M. haemolytica to reduce transmission.
    “Remember, we have showed that the bacteria B. trehalosi can inhibit growth of M. haemolytica. Can we use this property to our advantage?” he asked.
    Research is underway to see if domestic sheep could be inoculated with B. trehalosi, which would eliminate the toxin-producing bacteria.
    “We have done some experiments, and the results are very encouraging,” Sri commented. “After inoculation, we couldn’t detect any M. haemolytica in the nasal secretion.”
    Additionally, research is being pursued to elucidate the mechanism by which B. trehalosi inhibits M. haemolytica.
    “If we know what is inhibiting the M. haemolytica, we can genetically engineer B. trehalosi to produce more of the killer protein to enhance the inhibitory effect,” he said.
    With the difficult of developing vaccines, testing them and getting the permissions to use the vaccines, Sri noted that it would take time.
    “This type of vaccine takes a long time to develop,” he said. “I won’t have a vaccine in the next five years. We should be able to do it in less than 20, but not in five.”
    Saige Albert is editor of the Wyoming Livestock Roundup and can be reached at saige@wylr.net.

Sri verifies disease transmission
    In continuing his research to solve the problems in bighorn sheep and domestic sheep interactions, Washington State University professor Subramaniam Srikumaran, known as Dr. Sri, needed to document transmission of M. haemolytica from domestic sheep to bighorn sheep.
    “There have been anecdotal reports of die-offs following contact with domestic sheep, which prompted commingling studies,” Sri explained at a June 2 lecture in Casper. “These studies failed to convince everyone that domestic sheep transmitted pathogens, because they did not show conclusively that the pathogens were acquired from domestic animals.”
    To attempt to irrefutably prove transmission, Sri and his research team developed a strategy to tag bacteria from domestic sheep with a marker.
    M. haemolytica from domestic sheep were genetically modified to express a green fluorescent protein (GFP) and resistance to the antibiotic ampicillin. Four domestic sheep were inoculated with the tagged bacteria.
    “We kept the four animals 30 feet away from four bighorn sheep, which were shown to be negative for M. haemolytica,” said Sri. “Nothing happened to the animals. We then allowed fenceline contact through a chain link fence.”
    Following fenceline contact, he noted that two of the bighorn sheep exhibited colonization of the GFP tagged bacteria in their nasal pharynx, coughing and nasal discharge.
    “Then we allowed commingling. Within two days, one died. Three days later, two more died, and the last died after nine days,” he mentioned, noting that the GFP-tagged organisms were isolated from the bighorn sheep. “This is irrefutably proves that domestic sheep can transmit disease.”
    However, Sri clarified, “This study was not performed to point fingers at domestic sheep, but to determine conclusively whether there is transmission of M. haemolytica.”
    This study was published in the Journal of Wildlife Diseases in July 2010.