Summary
Four genetically-modified strains of the rumen bacterium Butyrivibrio fibrisolvens were inoculated in to the rumens of two sheep (T 1 & T2). All four bacterial strains had been shown to produce the detoxifying enzyme: fluoroacetate dehalogenase with activity between 10 - 20 nmol fluoroacetate/minlmg bacterial protein. For direct comparison of poison sensitivity, two uninoculated sheep were used as "control" animals (CI & C2). For the week preceding the toxicity tests, samples of rumen contents were taken daily from all four sheep and analysed to measure the number of bacteria containing the detoxification gene.
Immediately prior to the test, inoculated sheep showed levels around 107 (Tl) and 106 (T2) modified bacteria per millilitre of rumen fluid, whereas C 1 and C2 contained no recombinant bacteria. Sheep were weighed the day before the poisoning experiment and doses were adjusted for each animal, to ensure that each received a specific amount of poison, per kilogram of body weight. The four sheep were fed a series of small doses of fluoroacetate over a period of three days, to a total amount calculated as an average lethal dose. They were allowed to recover for 3 days, and then fed repeat doses over the following day.
Fluoroacetate was fed in a series of small doses to allow observation of any significant difference between the test-sheep and the control-sheep During day 2, the control-sheep both showed symptoms typical of acute fluoroacetate toxicity. Both were extremely nervous, reacting to even familiar noises with a panic response. For several hours on day 2, both control-sheep had to be restrained from breaking loose and possibly damaging themselves. Both test-sheep remained calm throughout this period, but showed some physiological signs of chronic toxicity (raised heart-rate and respiration rate) for I - 2 hours.
On day 3, the control-sheep remained very nervous, again requiring physical restraint on several occasions. However, the test-sheep showed no symptoms of fluoroacetate poisoning on day 3. During the second major poisoning period (day 7) the control-sheep again showed extreme nervousness, and periods of panic. Test-sheep remained calm, with just a minor detectable increase in their wariness of sudden noises. At around 5 a.m. on day 8, the control-sheep succumbed to acute poisoning, underwent a panic response, broke free of the metabolism crates in which they were housed and died. At 8.53 a.m., test-sheep T2 died without muscular or nervous spasms. Autopsy showed that death was from pulmonary oedema (fluid on the lungs) typical of chronic fluoroacetate poisoning. At 9:00 a.m. test-sheep TI was visibly affected by the poison, with a slightly elevated heart-rate (130 beats/min) and respiration rate (30 resp/min). These were approximately 50% above normal resting levels. Within 4 hours, both were back to normal and Tl appeared unstressed.
The conclusion was that test-sheep displayed significantly greater resistance to fluoroacetate poisoning than the control-sheep. Between the two test-sheep, TI showed higher levels of detoxifying bacteria than T2 at the beginning of the experiment. TI also showed greater. toxin resistance than T2. These results are highly encouraging, but need to be tested in a repeat experiment before defmite conclusions can be made about the protective effect of the bacteria.
