Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10(6) to 10(7) cells ml(−1) in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep

Gregg, Keith

Hamdorf, Brenton

Henderson, Kerrin

Kopecny, Jan

Wong, Cheryl

PubMed

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10(6) to 10(7) cells ml-1 in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep

Gregg, K.

Hamdorf, B.

Henderson, K.

Kopecny, J.

Wong, C.M.

Research Repository

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010Sept. 1998, p. 3496–3498 Vol. 64, No. 9Copyright © 1998, American Society for Microbiology. All Rights Reserved.Genetically Modiﬁed Ruminal Bacteria Protect Sheepfrom Fluoroacetate PoisoningKEITH GREGG,* BRENTON HAMDORF,† KERRIN HENDERSON,JAN KOPECNY,‡ AND CHERYL WONGRumen Biotech, Murdoch University, Murdoch,Western Australia 6150, AustraliaReceived 30 March 1998/Accepted 9 June 1998Four strains of Butyrivibrio ﬁbrisolvens, transformed with a gene encoding ﬂuoroacetate dehalogenase,maintained a combined population of 106 to 107 cells ml21 in the rumens of test sheep. Five inoculated sheepshowed markedly reduced toxicological symptoms after ﬂuoroacetate poisoning when behavioral, physiological,and histological effects were compared with those of ﬁve uninoculated control sheep.Monoﬂuoroacetate occurs in some Australian plants at lev-e l so fu pt o5gk g(dry weight)21 (8) and has a 50% lethal dosein ruminants of around 0.3 mg kg of body weight21 (2). Thetoxin appears to be cumulative, and a series of sublethal dosescan result in fatal poisoning (2). It is not surprising, therefore,that loss of livestock from ﬂuoroacetate poisoning is econom-ically signiﬁcant (12). The work described here is part of aproject to develop genetically modiﬁed ruminal bacteria thatwill provide protection against ﬂuoroacetate poisoning.Three Butyrivibrio ﬁbrisolvens strains, OB156, OB291, and10/1, were transformed with plasmid pBHf by electroporation(3, 7) and expressed ﬂuoroacetate dehalogenase activity at 10nmol of ﬂuoride released min21 mg of bacterial protein21 (7).Strain OR85 expressed dehalogenase activity at (20 6 1.7) nmolmin21 mg21 when transformed with a pBHf derivative withfour base pairs removed from between the putative 210 and235 regions of the gene promoter (pBHf24) (unpublisheddata). OB156, OB291, and OR85 were supplied by R. Forsterand R. M. Teather (4) (CFAR, Agriculture and Agrifood Can-ada, Ottawa, Ontario, Canada). B. ﬁbrisolvens 10/1 was isolatedfrom sheep (11) at the Institute of Biotechnology (Universityof New England, Armidale, New South Wales, Australia).Recombinant bacteria were grown in ruminal ﬂuid medium(10) containing yeast extract in place of peptone. Erythromycinwas added (50 mgm l21) except in subcultures prepared forinoculation into the rumen. In previous observations (5, 6), thepopulation of individual bacterial strains within the rumenﬂuctuated within the range ,103 to 107 cells ml21. Therefore,a combination of four modiﬁed strains was used to ensure thateffective numbers of detoxifying bacteria would be presentconsistently. Fifty-milliliter stationary-phase cultures of eachstrain (108 to 109 cells ml21) were pooled, and 60 ml of themixture 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 werehoused in metabolism crates in a PC2-classiﬁed room. Sheepreceived four meals daily (150 g of oat chaff plus 50 g of alfalfachaff) between 10 a.m. and 4 p.m. Water was provided adlibitum.Sheep rumens were surgically cannulated for removal ofsamples. These included particles ,1 mm in diameter. Sam-ples were heated (95°C for 10 min) to inactivate nucleases andcentrifuged (12,000 3 g for 90 s), and the supernatant wasremoved. The pellet was resuspended in water (10 times thevolume of the original sample) and recentrifuged. This proce-dure was repeated once, and the ﬁnal pellet was suspended in10 volumes of water. Dilutions of 1022,1 023,1 024, and 1025were prepared and tested by PCR for the presence of dehalo-genase gene sequences (1).PCR primers used for dehalogenase gene detection wereCC5 (59-TGCGAGGCTATGGCGATTCGGACA-39) andCC6.2i (59-CTGACCGATCATGTGCTCGGGGAA-39),both of which ampliﬁed a 322-bp fragment from the codingregion of the dehalogenase gene (9). PCR conditions were asfollows: 2 cycles of 95°C for 5 min, 55°C for 40 s, and 72°C for60 s followed by 28 cycles of 95°C for 60 s, 55°C for 40 s, and72°C for 60 s. Tests on ruminal samples to which transformedbacteria had been added showed that pBHf was detectable inthe presence of a single target bacterium. Inclusion of 1 mlo fsample in the PCR mixture allowed a threshold of detection of103 cells ml21. Detection of the plasmid at a dilution of 1024(for example) indicated a ruminal population of $107 cells* Corresponding author. Mailing address: Rumen Biotech, Mur-doch University, South Street, Murdoch, WA 6150, Australia. Phone:(61) 8 9360 2122. Fax: (61) 8 9360 6303. E-mail: K.Gregg@murdoch.edu.au.† Present address: Division of Immunology and Cell Biology, JohnCurtin School of Medical Research, Australian National University,Canberra, ACT 0200, Australia.‡ Present address: Institute of Animal Physiology and Genetics,Prague, Czech Republic.FIG. 1. Agarose gel with PCR products from 10-fold serial dilutions of ru-minal particles from sheep T1 (trial I). Lanes and fold dilutions of ruminalsamples are as follows: A, 1021;B ,1 022;C ,1 023;D ,1 024;E ,1 025. Aliquots of1 ml were used in each reaction. Lane F is the positive control product, and laneG contains l HindIII size markers. The diffuse lower band was a commonlyobserved artifact in PCR that used complex biological mixtures as a source oftemplate.3496ml21 in the original sample (Fig. 1). Preinoculation ruminalsamples yielded no dehalogenase gene PCR products and wereroutinely used as negative controls.Fluoroacetate solution was injected into snow peas or ab-sorbed into feed pellets or alfalfa biscuits. These were usuallyeaten voluntarily, even when chaff was refused. The biscuitswere '0.5 g each and were made from alfalfa which had beenblended to a coarse slurry with a 10% sucrose solution, pressedinto discs to remove most of the ﬂuid, and dried overnight at60°C. On the three occasions that control sheep 2 (C2) (trialII) refused to eat them, the biscuits were placed into the ru-mens of both C2 and test sheep 2 (T2) via the cannula. Con-tinuous assessments were made of feeding, drinking, responseto external stimuli, stance, movement, and sleeping behavior.In trial II, heart rates were measured with an electrocardio-graph.In trial I, sheep were challenged with ﬂuoroacetate 10 daysafter inoculation (Table 1), when the modiﬁed strains hadestablished a stable combined population of .107 cells ml21 inT1 and .106 cells ml21 in T2. In trial II, the recombinantbacteria were allowed ﬁve weeks to stabilize their populationto .106 cells ml21 in all three sheep before challenge withﬂuoroacetate. Recombinant bacteria were undetectable incontrol sheep.Trial I. At a cumulative dose of 0.255 mg of ﬂuoroacetatekg21, control sheep exhibited alarm at familiar noises, com-pulsive agitated feeding, periodic muscle spasms, “paddling”movements of the legs, skin ﬂushing, panting, and frequentattempts to escape from their crates by jumping. Inoculatedtest sheep showed occasional head twitching and rapid breath-ing and periodically increased alertness but remained calm,eating and drinking normally.At 0.492 mg of ﬂuoroacetate kg21, control sheep showedintensiﬁcation of all symptoms and died. Behavior of both testsheep remained unchanged. T2 displayed no signs of acutetoxicity but, four hours after the death of the control sheep,died of pulmonary edema, which is common in chronic ﬂuoro-acetate poisoning (3a). Sheep T1 survived the test.Trial II. At 0.255 mg of ﬂuoroacetate kg21, control sheepbecame lethargic and refused food and water. Hyperexcitabil-FIG. 2. Heart rate measurements from sheep in trial II commencing 12 hafter the ﬁrst dose of ﬂuoroacetate in the series (Table 1). Heart rates measuredfor these sheep the day before dosing were in the range of 60 to 80 beats min21.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.TABLE 1. Fluoroacetate dosage schedule for toxicity trials I and IIToxicitytrialTimeperiodDoseevery:Dose increment(s),mg kg21Accumulateddose, mg kg21I 0–10 h 2 h 1 3 0.045, 5 3 0.03 0.19520–22 h 2 h 2 3 0.03 0.25545–57 h 6 h 2 3 0.015, 1 3 0.012 0.297140–152 h 1 h 13 3 0.015 0.492II 0–12 h 2 h 6 3 0.03, 1 3 0.015 0.19522–24 h 2 h 2 3 0.03 0.25538–42 h 2 h 3 3 0.015 0.30046 h NA 1 3 0.03 0.330a NA, not applicable.TABLE 2. Pathological and histological observationsTissue and featureResult for sheep:C1 C2 C3 T1 T2 T3LiverColor Dark Dark Dark Normal Normal NormalCongestion Marked Marked Marked Mild Mild ModerateMononuclear cell inﬁltration None Mild Mild Very mild None NoneLungsCongestion Moderate Severe Moderate Mild Mild SevereTexture Consolidated ﬁrm Consolidated ﬁrm Normal Normal Normal NormalAtelectasia and focal emphysema Mild Mild None None None NonePerivascular mononuclear cell inﬁltrate Mild Mild None None None NoneKidneysColor Dark Dark Dark Normal Normal NormalProtein inclusions: proximal tubules Present None Present None None NoneHeart (multifocal mononuclear cell inﬁltration) Mild/moderate Mild/moderate Moderate/focally severe None Very mild Very mildVOL. 64, 1998 MODIFIED RUMINAL BACTERIA PROTECT SHEEP FROM POISONING 3497ity and compulsive eating did not occur, but muscle weaknesswas evident from their faltering stance. Test sheep showed notoxicity symptoms.At 0.33 mg of ﬂuoroacetate kg21, control sheep continued torefuse food and remained lethargic, with pronounced unsteadi-ness when standing. Electrocardiograms showed heart ratesrising to 140 to 200 beats min21. Test sheep reduced their feedintake approximately 30% but otherwise behaved normally,with maximum heart rates of 80 to 100 beats min21 (T3 only[Fig. 2]). Prior to administration of ﬂuoroacetate, heart rates ofall sheep were in the range of 60 to 80 beats min21. All sheepsurvived trial II.Control sheep displayed reduced appetite to day 6, contin-ued unsteadiness on standing up to day 5, and involuntarymuscle activity (head twitching) through day 4. T1 and T2returned to normal eating habits by day 4, and T3 continued toeat approximately 30% less than usual up to day 6. In all otherrespects, test sheep remained asymptomatic.Trial II sheep were euthanized with pentobarbitone (Letho-barb), and necropsies were performed by Rob Woodgate andBruce Chick of the Veterinary Health Research Laboratory,Armidale, New South Wales, Australia. Tissue samples wereﬁxed in 10% formaldehyde for histological examination (S.Hum, Regional Veterinary Laboratory, Menangle, New SouthWales, Australia). The control sheep displayed measurablygreater tissue damage (Table 2).The intraruminal levels of modiﬁed bacteria achieved hereallowed us to demonstrate their protective effect upon hostanimals, but an increased level of protection would be desir-able for ﬁeld trials with larger numbers of animals. This wouldrequire additional recombinant strains in the rumen and/ormore efﬁcient expression of dehalogenase from each strain.Current work is aimed to achieve these improvements.This work was conducted at the University of New England,Armidale, New South Wales, Australia. Funding was providedby Applied Biotechnology Ltd. (Queensland), The AustralianMeat Research Corporation, and The Faculty of the Sciences,University of New England.We thank B. Entsch for administration of project funds, S.Atkinson for veterinary services, B. Chick and R. Woodgatefor veterinary pathology, and staff of the Animal Sciences De-partment and the University of New England animal house fortheir assistance.REFERENCES1. Allen, G. 1998. Polymerase chain reaction techniques for the study of rumenbacterial ecology and biotechnology. Ph.D. thesis. University of New En-gland, Armidale, New South Wales, Australia.2. Annison, E. F., K. J. Hill, D. B. Lindsay, and R. A. Peters. 1960. Fluoroac-etate poisoning in sheep. J. Comp. Pathol. 70:145–155.3. Beard, C. E., M. A. Hefford, R. J. Forster, S. Sontakke, R. M. Teather, andK. Gregg. 1995. A stable and efﬁcient transformation system for Butyrivibrioﬁbrisolvens OB156. Curr. Microbiol. 30:105–109.3a.Chick, B. Personal communication.4. Forster, R. B., J. Gong, and R. M. Teather. 1997. Group-speciﬁc 16S rRNAhybridization probes for determinative and community structure studies ofButyrivibrio ﬁbrisolvens in the rumen. Appl. Environ. Microbiol. 63:1256–1260.5. Gregg, K. 1995. Engineering gut ﬂora of ruminant livestock to reduce foragetoxicity: progress and problems. Trends Biotechnol. 13:418–421.6. Gregg, K., G. Allen, T. Bauchop, A. Klieve, and M. Lincoln. 1993. Practicalgenetic engineering of rumen bacteria, p. 13–21. In D. J. Farrell (ed.),Recent advances in animal nutrition in australia—1993. University of NewEngland, Armidale, New South Wales, Australia.7. Gregg, K., C. L. Cooper, D. J. Schafer, H. Sharpe, C. E. Beard, G. Allen, andJ. Xu. 1994. Detoxiﬁcation of the plant toxin ﬂuoroacetate by a geneticallymodiﬁed rumen bacterium. Bio/Technology 12:1361–1365.8. Hall, R. J. 1972. The distribution of organic ﬂuorine in some toxic tropicalplants. New Phytol. 71:855–871.9. Kawasaki, H., K. Tsuda, I. Matsushita, and K. Tonomura. 1992. Lack ofhomology between two haloacetate dehalogenase genes encoded on a plas-mid from Moraxella species strain B. J. Gen. Microbiol. 138:1317–1323.10. Klieve, A. V., J. F. Hudman, and T. Bauchop. 1989. Inducible bacteriophagesfrom ruminal bacteria. Appl. Environ. Microbiol. 55:1630–1634.11. Kopecny ￿, J., K. Fliegerova ￿, and K. Gregg. 1998. Transformation of B. ﬁbri-solvens strains with pBHf plasmid. Reprod. Nutr. Dev. (Suppl. 1), p. 72–73.12. McCosker, T. 1989. Ruminal detoxiﬁcation of ﬂuoroacetate. Agric. Sci. NewSer. 2:46–47.3498 GREGG ET AL. APPL.E NVIRON.M ICROBIOL.

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Genetically modified ruminal bacteria protect sheep from fluoroacetate poisoning

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Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning

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