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Monofluoroacetate occurs in some Australian plants at levels of up to 5 g kg (dry weight)¹ (8) and has a 50% lethal dose in ruminants of around 0.3 mg kg of body weight¹ (2). The toxin appears to be cumulative, and a series of sublethal doses can result in fatal poisoning (2). It is not surprising, therefore, that loss of livestock from fluoroacetate poisoning is economically significant (12). The work described here is part of a project to develop genetically modified ruminal bacteria that will provide protection against fluoroacetate poisoning.

Three Butyrivibrio fibrisolvens strains, OB156, OB291, and 10/1, were transformed with plasmid pBHf by electroporation (3, 7) and expressed fluoroacetate dehalogenase activity at 10 nmol of fluoride released min¹ mg of bacterial protein¹ (7). Strain OR85 expressed dehalogenase activity at (20 ± 1.7) nmol min¹ mg¹ when transformed with a pBHf derivative with four base pairs removed from between the putative 10 and 35 regions of the gene promoter (pBHf⁴) (unpublished data). OB156, OB291, and OR85 were supplied by R. Forster and R. M. Teather (4) (CFAR, Agriculture and Agrifood Canada, Ottawa, Ontario, Canada). B. fibrisolvens 10/1 was isolated from sheep (11) at the Institute of Biotechnology (University of New England, Armidale, New South Wales, Australia).

Recombinant bacteria were grown in ruminal fluid medium (10) containing yeast extract in place of peptone. Erythromycin was added (50 µg ml¹) except in subcultures prepared for inoculation into the rumen. In previous observations (5, 6), the population of individual bacterial strains within the rumen fluctuated within the range <10³ to 10⁷ cells ml¹. Therefore, a combination of four modified strains was used to ensure that effective numbers of detoxifying bacteria would be present consistently. Fifty-milliliter stationary-phase cultures of each strain (10⁸ to 10⁹ cells ml¹) were pooled, and 60 ml of the mixture was inoculated into the rumen of each test animal.

Trial I used four Merino/Border-Leicester crossbred sheep, and trial II used six pure-bred Merino sheep; all sheep were housed in metabolism crates in a PC2-classified room. Sheep received four meals daily (150 g of oat chaff plus 50 g of alfalfa chaff) between 10 a.m. and 4 p.m. Water was provided ad libitum.

Sheep rumens were surgically cannulated for removal of samples. These included particles <1 mm in diameter. Samples were heated (95°C for 10 min) to inactivate nucleases and centrifuged (12,000 × g for 90 s), and the supernatant was removed. The pellet was resuspended in water (10 times the volume of the original sample) and recentrifuged. This procedure was repeated once, and the final pellet was suspended in 10 volumes of water. Dilutions of 10², 10³, 10⁴, and 10⁵ were prepared and tested by PCR for the presence of dehalogenase gene sequences (1).

PCR primers used for dehalogenase gene detection were CC5 (5'-TGCGAGGCTATGGCGATTCGGACA-3') and CC6.2i (5'-CTGACCGATCATGTGCTCGGGGAA-3'), both of which amplified a 322-bp fragment from the coding region of the dehalogenase gene (9). PCR conditions were as follows: 2 cycles of 95°C for 5 min, 55°C for 40 s, and 72°C for 60 s followed by 28 cycles of 95°C for 60 s, 55°C for 40 s, and 72°C for 60 s. Tests on ruminal samples to which transformed bacteria had been added showed that pBHf was detectable in the presence of a single target bacterium. Inclusion of 1 µl of sample in the PCR mixture allowed a threshold of detection of 10³ cells ml¹. Detection of the plasmid at a dilution of 10⁴ (for example) indicated a ruminal population of 10⁷ cells ml¹ in the original sample (Fig. 1). Preinoculation ruminal samples yielded no dehalogenase gene PCR products and were routinely used as negative controls.

View larger version (74K): [in this window] [in a new window]   FIG. 1.   Agarose gel with PCR products from 10-fold serial dilutions of ruminal particles from sheep T1 (trial I). Lanes and fold dilutions of ruminal samples are as follows: A, 101; B, 102; C, 103; D, 104; E, 105. Aliquots of 1 µl were used in each reaction. Lane F is the positive control product, and lane G contains  HindIII size markers. The diffuse lower band was a commonly observed artifact in PCR that used complex biological mixtures as a source of template.

Fluoroacetate solution was injected into snow peas or absorbed into feed pellets or alfalfa biscuits. These were usually eaten voluntarily, even when chaff was refused. The biscuits were 0.5 g each and were made from alfalfa which had been blended to a coarse slurry with a 10% sucrose solution, pressed into discs to remove most of the fluid, and dried overnight at 60°C. On the three occasions that control sheep 2 (C2) (trial II) refused to eat them, the biscuits were placed into the rumens of both C2 and test sheep 2 (T2) via the cannula. Continuous assessments were made of feeding, drinking, response to external stimuli, stance, movement, and sleeping behavior. In trial II, heart rates were measured with an electrocardiograph.

In trial I, sheep were challenged with fluoroacetate 10 days after inoculation (Table 1), when the modified strains had established a stable combined population of >10⁷ cells ml¹ in T1 and >10⁶ cells ml¹ in T2. In trial II, the recombinant bacteria were allowed five weeks to stabilize their population to >10⁶ cells ml¹ in all three sheep before challenge with fluoroacetate. Recombinant bacteria were undetectable in control sheep.

Trial I. At a cumulative dose of 0.255 mg of fluoroacetate kg¹, control sheep exhibited alarm at familiar noises, compulsive agitated feeding, periodic muscle spasms, "paddling" movements of the legs, skin flushing, panting, and frequent attempts to escape from their crates by jumping. Inoculated test sheep showed occasional head twitching and rapid breathing and periodically increased alertness but remained calm, eating and drinking normally.

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Trial II. At 0.255 mg of fluoroacetate kg¹, control sheep became lethargic and refused food and water. Hyperexcitability and compulsive eating did not occur, but muscle weakness was evident from their faltering stance. Test sheep showed no toxicity symptoms.

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View larger version (51K): [in this window] [in a new window]   FIG. 2.   Heart rate measurements from sheep in trial II commencing 12 h after the first dose of fluoroacetate in the series (Table 1). Heart rates measured for these sheep the day before dosing were in the range of 60 to 80 beats min1. The three lines (from the front to the back of the three-dimensional diagram) represent measurements from sheep T1 to T3 and C1 to C3, respectively.