Within—country variation in the ability of ruminantsto degrade DHP following the ingestion of Leucaenaleucocephala—a Thailand experience

Brian Palmer & Raymond J. Jones &

Somsak Poathong & Jeerasak Chobtang

Accepted: 9 June 2009 /Published online: 20 June 2009# Springer Science + Business Media B.V. 2009

Abstract Goats fed Leucaena leucocephala (leu-caena) at an experimental site in Thailand wereshown to be excreting DHP in their urine. This wasunexpected as earlier results from another site hadshown that goats and cattle fed leucaena did notexcrete DHP and so possessed DHP—degradingbacteria. Goats sampled near the earlier sample siteexcreted no DHP in their urine. Rumen fluid takenfrom these goats was successfully used to transferDHP—degrading ability to the goats at the Experi-mental site some 350 km away that did not show thepresence of DHP-degrading bacteria. Degradation ofmimosine in-vitro and excretion of DHP in the urineceased 72 hr after addition of rumen fluid and

infusion with rumen fluid from protected goats,respectively. The situation in Thailand may not beunique. Countries where leucaena is fed should checkthat animals are protected. Fortunately, the ferricchloride urine test is simple to use and effective indetecting the problem and also the recovery aftertransfer of rumen fluid from protected animals.

Keywords Leucaena toxicity . 3,4 DHP. Goats .

DHP—degrading bacteria . Rumen fluid transfer

Introduction

Ruminants in virtually all tropical countries are ableto convert the toxic amino acid mimosine, found inthe pan-tropical leguminous shrub Leucaena leuco-cephala, (leucaena) into 3- hydroxy-4(1H)-pyridone(3, 4 DHP) and excrete it in their urine in the freeand conjugated form. In some countries 3, 4 DHP isable to be metabolized by other bacteria of whichSynergistes jonesii, an anaerobic gram negative rodshaped bacterium is one (Allison et al. 1992). Thecompound 3, 4 DHP is a potent goitrogen and isexcreted in the urine of unprotected animals fedleucaena. Prolonged exposure to DHP results inreduced serum thyroxine levels, low appetite andpoor productivity (Jones 1985; Hammond 1995).Thyroid glands become enlarged and in very severecases death can occur.

Trop Anim Health Prod (2010) 42:161–164DOI 10.1007/s11250-009-9398-4

B. Palmer (*)2 /30, Camp Street,Mundingburra, Townsville, Qld 4812, Australiae-mail: bpcnrt@bigpond.com

R. J. Jones15, Norwich Court,Kirwan, Townsville, Qld 4817, Australia

S. PoathongAnimal Research and Development Center,Cha Am, Petchaburi, Thailand 76120

J. ChobtangDepartment of Livestock Development,Animal Nutrition Division,Ratchathewi, Bangkok, Thailand 10400

The problem of leucaena toxicity can be overcomeby infusing the rumens of affected animals withsuitable bacteria or rumen fluid from animals knownto be protected from the toxicity (Jones and Megarrity1986; Hammond et al. 1989).

By analyzing urine samples from ruminants fed onleucaena in various tropical countries it was possible tolist those countries that appeared to have rumen bacteriacapable of degrading DHP, so preventing ruminants inthose countries from leucaena toxicosis, and those thatdid not (Jones 1994). Thailand was listed as a countrythat appeared to have such protection from toxicity.However, as we report here, this is not the whole story.

The study

Goats fed predominantly on shoots of leucaena on theResearch Station at Cha Am, Petchaburi (12.42N;99.12E) 184 km SSW of Bangkok were the basis of thestudy conducted in November 2008. Urine was collectedin plastic bottles from 12 goats and a 0.5 ml sub sampleadded to 5 ml of acidified ferric chloride reagent (0.6 gFeCl3.6H2O+4 ml conc. HCl in 1 L water) (Jones and

Megarrity 1986). All urine samples gave a pronouncedpurple colour in the presence of the reagent indicatingthe presence of free mimosine/DHP and the absence ofDHP-degrading bacteria. This result was unexpected inthe light of earlier samples received from Pak Chong(14.43N; 101.21E) about 170 km NNE of Bangkok(Chanchai Manidool, personal communication, 1983)and tested in Australia (Jones 1994).

Another goat producer in the Nakorn RatchasimaProvince was contacted to assess the status of thiswell-established goat herd. The goats in this herdwere fed a diet solely of leucaena branches, theanimals ate the leaf and stripped and consumed theassociated bark. Two goats were slaughtered andurine from the bladder of each goat tested for thepresence of mimosine/DHP as described earlier. Theurine from both goats gave no colour reactionindicating the presence of DHP-degrading bacteria.Thyroid glands of these goats were also normal inappearance and size.

Transfer of bacteria

Rumen fluid from each of two sacrificed goats at SiKhiew (14.54N; 101.42E) was immediately strainedthrough four layers of muslin, cooled and stored inthermos flasks after flushing with CO2. The rumenfluid was transported by car to Cha Am in an ice chestfor the transfer study. Forty hours after collection,15 ml from both rumen fluid samples were combinedand drenched into each of twelve goats, maintained inmetabolism crates and fed leucaena.

Transfer of DHP-degrading ability after drenchingwas assessed by urine analysis of all 12 goats and bysampling rumen fluid obtained by stomach tube from2 of the goats 8 days after initial drenching.

Table 1 Colour development in urine samples from twelvegoats. (5 = strong purple colour, 0 = absence of purple colour)

Sampling Time (hrs) Animal number

1 2 3 4 5 6 7 8 9 10 11 12

Estimated colour development

0 5 5 5 5 5 5 5 5 5 5 5 5

12 5 5 4 4 4 4 4 4 5 4 5 4

48 3 4 0 0 4 0 0 0 0 4 4 4

72 0 0 0 0 0 0 0 0 0 0 0 0

96 0 0 0 0 0 0 0 0 0 0 0 0

Table 2 Estimated colour development in incubation study. (5 = strong purple colour, 0 = absence of purple colour)

Time of incubation (hr) Rumen Fluid from Animal 1 Rumen Fluid from Animal 2 Boiled Rumen Fluid Control

Rep. 1 Rep. 2 Rep.1 Rep. 2 Animal 1 Animal 2

0 h 5 5 5 5 5 5

24 h 5 5 5 4 5 5

48 h 0 1 1 2 5 5

72 h 0 0 0 0 5 5

96 h 0 0 0 0 5 5

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Urine from all 12 goats was collected 0, 12, 48, 72and 96 hr after drenching and sub samples of 0.5 mladded to 5 ml of acidified FeCl3 solution in 10 mltubes. The purple colour reaction was recorded on ascale of 0–5, where 5 was the initial colour densityand 0 was the absence of colour development.

Two 50 ml samples of the strained rumen fluid fromeach of the 2 goats was incubated anaerobically at 37°Cwith 1.0 g of macerated leucaena shoot tips in 100 mlsealed conical flasks; 0.5 ml of fluid was withdrawndaily by syringe from each flask and added to 5ml of theFeCl3 solution as described above. A control treatmentconsisted of two flasks where the 50 ml rumen fluidfrom each animal was boiled for 1 minute to kill anytoxin-degrading and other bacteria. The purple colourreaction was recorded on a scale of 0 to 5, with 5 beingthe purple colour of the boiled control tubes and 0 theabsence of colour development. The flasks wereshaken periodically during digestion.

Reduction and subsequent disappearance of DHPfrom the urine (Table 1) and the cultures (Table 2)after 72 hr clearly showed that successful transfer ofbacteria capable of degrading DHP had beenachieved.

Discussion

The finding that goat herds within a distance of about350 km could be so different was unexpected. Thaisamples analysed in Australia in 1983 came fromcattle and goats in the Pak Chong area only about30 km from the present sample site so this area hasclearly had an effective population of DHP-degradingorganisms (presumably bacteria) for some time. Justwhy only some areas have such bacteria is not known.

The results are a warning that because some herdsor flocks are shown to be protected from leucaenatoxicosis this does not mean that all in any countrywill be so protected.

Other developing countries should not becomecomplacent about their status with regard to protec-tion and should survey the situation in their respectivecountries. In neighbouring Laos, urine samplescollected from cattle fed leucaena in the VilaboulyDistrict of Savannahket; all showed the presence of3,4 DHP/mimosine (B. Palmer, unpublished data,2007). Most cases of leucaena toxicosis are subclinical and this is the problem. Animals may look

healthy and do not give the farmer cause for concern,but the circulating DHP reduces appetite anddepresses serum thyroxine levels so reducing poten-tial productivity (Quirk et al. 1988).

Lack of spread of the DHP-degrading organismsin Thailand may well be associated with themanagement of the herds. If there is little animaltrade from one farm to another throughout thecountry then the likelihood of natural spread wouldbe low. In Thailand most goats are reared andeither eaten locally or sent directly to slaughter.Further work is necessary to delineate areas whereruminants are protected and where they are not.Once this is established, the solution to the problemwill be a simple matter of transferring rumen fluidfrom protected to unprotected animals as describedabove making sure that the recipient animals have beenon a leucaena diet before drenching. Care should betaken to keep the rumen fluid as free from air aspossible during both transit and administration and tominimize the time between collection of the rumenfluid and its administration to donor animals.

Fortunately, the technology is extremely simpleand well tested (Jones 1994). The simple ferricchloride test will be able to detect problem andprotected herds and also serve to assess the success ofany transfer from protected to unprotected animals.

Acknowledgements The cooperation of Saowanee Phongsuwan,Amphor Si Khiew, Nakorn Ratchasima Province 30140 Thailand forallowing us to take samples from her goats. Appreciation is given toSomchit Indramanee, Director of Animal Nutrition Division,Department of Livestock Development for approving use of thefacilities.

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