Leucaena toxicity solution

In the late 1950s and 1960s the Division of Tropical Pastures was actively introducing and evaluating tropical legumes for use in the beef industry of tropical Australia. Among the most promising was a leguminous shrub from central and South America – Leucaena leucocephala (Leucaena). Its valuable features included:

• high production
• high palatability to stock
• high persistence under grazing
• particularly high forage quality.

Unfortunately it contained a toxic amino acid, mimosine, which caused severe problems in Australian cattle. Plant breeding attempts to reduce the mimosine concentration failed and attempts to treat the toxicity in animals by iodine supplementation and thyroxine therapy also failed.

While attending a conference in Hawaii in 1976, CSIRO’s Dr Raymond Jones noted that penned goats were fed a very full diet of Leucaena without ill effects. He wondered whether their rumen microflora allowed them to detoxify the mimosine and its degradation products. After many frustrations Dr Jones proved that this was so, changing the approach to solving the Leucaena toxicity problem worldwide.

The organism Synergistes jonesii, named after Dr Jones, was new to science and was the first example of rumen bacteria being used to overcome plant toxicity. It was a simple solution that transformed the grazing industry in northern Australia and has been effective in other tropical countries in Asia, Africa and South America.

The problem

Leucaena is one of the most potentially valuable fodder crops for the world’s tropical and sub-tropical regions. However, virtually all parts of the Leucaena plant contain the toxic amino acid mimosine.

Pigs and chickens are more affected by mimosine than ruminants because their acid stomachs enable rapid uptake of the toxin. The ability to degrade mimosine in the rumen led to the belief that ruminants would not suffer from Leucaena toxicity as the breakdown product 3,4-DHP was considered to be harmless.

However, Dr Merv Hegarty and co-workers at CSIRO’s Cunningham Laboratory in Brisbane showed that DHP interferes with the way the thyroid gland uses iodine, inducing goitre and reducing production of the growth-regulating hormone thyroxine. This was a major discovery. Acute toxicity leads to hair loss, profuse salivation, listlessness, appetite loss, poor growth and death.

Plant breeders had shown variation for mimosine concentration in different species of Leucaena with L. pulverulenta containing about half the concentration in L. leucocephala. Furthermore, these species could interbreed. Unfortunately, L. pulverulenta was low yielding and the crosses with L. leucocephala yielded progeny which, although performing reasonably in cutting trials, did poorly when grazed by cattle.

Iodine supplements and thyroxine usually have an effect on enlarged thyroids, but not in this case. Clearly the circulating DHP was having adverse effects other than its effect on thyroxine production. Solving the problem would entail removing the effect of the circulating DHP, but how?

A chance observation

In August 1976 Dr Raymond Jones was invited to present a paper at a workshop in Hawaii. During a break participants were taken on a field trip around the island. On this excursion Dr Jones saw penned goats being fed on what appeared to be an exclusive diet of Leucaena. He was particularly interested in the young goats as they appeared healthy with no signs of goitre.

Although Leucaena research was only a small part of Dr Jones’ scientific interest at that stage, the picture of the healthy kids in Hawaii stuck with him. In 1978 he received approval from CSIRO to explore the Leucaena toxicity problem. The plan was to do the study at the University of Hawaii’s Animal Husbandry Department in Honolulu. The proposal was greeted favourably and a date in 1979 was fixed for the study to begin.

The first hurdle

Unfortunately the University of Hawaii reconsidered Dr Jones’ proposal and wrote to him advising that, regretfully, they could not support it. After all the effort expended in setting up the proposal, Dr Jones was determined somehow or other to carry it through.

This resulted in a rather dubious plan in which he would visit incognito, work at a remote horticultural station on the island of Kauai and return to Honolulu at weekends. Here, at the horticultural laboratory of Dr Jim Brewbaker, he would undertake the mimosine and DHP analyses on plant and urine samples. Of these weekend visits to Honolulu Dr Jones recalls:

Often I had no time to shower. Seats are not specifically allocated on these flights and I rarely had anyone sit by me. Goat urine is an acquired odour. Fortunately for me there was a garbage collectors’ strike in Honolulu during this time. Getting into the taxi from the airport to the University on one occasion the driver said, “Boy will I be glad when this strike is over – the place is beginning to smell!”

The simple experiment Dr Jones undertook compared goats on three diets: Leucaena only, half Leucaena and half lucerne, and lucerne only. To his delight all goats grew well. The thyroids were very similar between treatments, so were the serum thyroxine levels. There was no ulceration of the oesophagus or any internal organs, and even the thyroids of the unborn kids were normal.

The next challenge was to show that these results were due to the goats’ rumen microflora. Dr Jones added strained rumen fluid from the recently killed goats and macerated young Leucaena shoots (≈ 10% mimosine) to a sealed sterilising tank and mixed thoroughly. An initial subsample gave the typical purple colour with ferric chloride indicating the presence of mimosine. This colour faded with subsequent subsamples and eventually disappeared after 24 hours.

Proving the hypothesis

Dr Jones returned to Townsville jubilant and immediately wrote up the experiment for publication. The paper was rejected by the Divisional Editorial Panel. The claim that different microorganisms in the rumens of Hawaiian goats accounted for the difference between the responses to Leucaena feeding in the two countries was not seen to be proven. The goats were different, the Leucaena was different, the soils were different and besides, the accepted wisdom was that rumen bacteria were ubiquitous. He did, however, publish a short note in the Australian Veterinary Journal posing the question, ‘Does ruminal metabolism of mimosine explain the absence of Leucaena toxicity in Hawaii?’ Dr Jones would have to get more proof before he could substantiate his claim.

The second hurdle

Dr Jones had a couple of options to prove his hypothesis. He could:

• bring a goat from Hawaii to Australia, feed it home-grown Leucaena and monitor the degradation of mimosine in comparison with Australian goats
• take Australian goats to Hawaii, feed them with Australian-grown Leucaena and measure any differences
• introduce rumen fluid from a Hawaiian goat into Australia and test in the laboratory the degradation of mimosine using Australian grown Leucaena.

Diseases recorded in goats in the different countries precluded the first two options being conducted on quarantine grounds, so Dr Jones obtained the permission to conduct the third experiment.

Unfortunately CSIRO was unable to fund this work so, convinced his hypothesis was correct, Dr Jones paid his own expenses to Hawaii. There he bought the goat, collected its smelly rumen fluid in a thermos and flew back to Australia. His flight was diverted to Fiji due to a re-fueller’s strike in Sydney. When he eventually arrived in Sydney a pilot’s strike further delayed his arrival in Townsville. Finally in the lab, days later the rumen fluid failed to degrade mimosine beyond 3,4-DHP. The key ‘bugs’ had died in transit.

More disappointment

Dr Jones persisted. At the time, Dr Brian Lowry from the CSIRO Division of Animal Production was working on a project at Bogor, Indonesia. He helped Dr Jones put another project together that would entail flying four goats with Leucaena toxicity to Bogor in specially built crates with sufficient dried Leucaena leaves for the experiment. There, with modern animal nutrition facilities two of the goats would receive rumen fluid from the Indonesian goat and two would act as control. Urine would be measured for mimosine and its degradation products by sophisticated automated liquid chromatography equipment in an air conditioned laboratory. The project was approved.

After some initial hiccups in Indonesia the experiments went ahead. A few days after infusion with rumen fluid, the appearance and appetite of the infused goats improved and their urinary excretion of DHP stopped. They were now eating twice as much Leucaena (and mimosine) as the controls yet were excreting little DHP. It was working! One of these infused goats was used to treat one of the Australian controls and the same result was seen in a few days. Not only had they transferred rumen fluid from an Indonesian goat to an Australian goat, but also from one Australian goat to another.

Publication problems

The results were spectacular and they decided to write up the work for quick publication in Nature, however the journal advised that the work was too specific for them to publish, and suggested Dr Jones try the American Journal of Science.

The first manuscript sent to the American Journal of Science was lost. Reviewer’s reports on the replacement manuscript were not favourable. They questioned the study of mimosine degradation, instead believing that the focus should have been on why mimosine was in Leucaena. There were no critical comments of the data presented. It seemed that it was not easy to accept that a paradigm had been broken. Eventually the work was published in Experientia.

Isolating the bacteria

Dr Jones determined that if he could isolate the organisms responsible for the mimosine degradation then perhaps he could get permission to import them and use them in Australia. With quarantine reasons making it impossible to introduce rumen fluid from Indonesia into Australia, Dr Jones again looked to Hawaii.

Before he could undertake this project, Dr Jones went to the USDA Animal Diseases Centre at Ames, Iowa to work with Dr Milton Allison and other rumen microbiologists. There he learned the tedious and exacting procedures involved in isolating single bacterial colonies for study.

Although most rumen microbiologists were skeptical of Dr Jones’ results, many had now accepted the hypothesis that rumen bacteria were not ubiquitous as once claimed. During this period, Dr Jones met the famous Professor Robert Hungate who had produced the rumen microbiology ‘bible’. According to Dr Jones’ recollection:

He looked at me through his rather thick spectacles, waved his finger and said, “Do you realise young man (I was then in my 40s) that if you are right I will have to write a whole new chapter in my book?” He also went on to say that it would be virtually impossible to isolate the organisms involved in the degradation of DHP.

Dr Jones’ colleague Rob Megarrity set up the analytical side of the project at the University of Hawaii in Honolulu. He would analyse the samples that Dr Jones isolated and sent to him.

Degradation of the DHP in the culture tubes was readily achieved on both fresh and frozen and subsequently thawed rumen fluid. This confirmed that bacteria and not protozoa (which are killed by freezing) were involved in the degradation. Eventually they ended up with 10 enriched cultures capable of rapidly degrading DHP which were brought back to Australia.

Success on Australian soil

The cultures were infused into the rumens of goats and steers in the Queensland Department of Primary Industries’ high security facility at Oonoonba, Townsville. As expected there was a reduction in the animals’ urinary DHP excretion. When the animals were declared free of any introduced diseases, restricted clearance was given for further testing. The cultures were studied further at the CSIRO Davies Laboratory.

A sick steer at the CSIRO Lansdown research station was also treated with the cultures. It responded rapidly to dosing with the bacteria. When cleared from partial quarantine and put into a Leucaena pasture it gained over 1.4 kg/day in the wet season and 275 kg over a whole year. Furthermore, the bacteria spread passively to four other steers grazing with him. These were the best gains on any tropical pasture that had been measured.

A new organism described

In the meanwhile work continued in laboratories in Townsville and Ames to isolate and describe both the bacteria involved and the mimosine degradation pathways. They were able to show that some strains of bacteria could completely degrade mimosine, 3,4-DHP and 2,3-DHP, whereas others could only degrade 3,4-DHP or 2,3-DHP. They also showed that 3,4-DHP was converted by these bacteria to 2,3-DHP.

In Iowa, several DHP degrading strains were isolated. Detailed work by Dr Allison and his co-workers determined that the bacteria were obligately anaerobic, non-motile, gram-negative rods 1.2 X 0.6 µ. These strains differed from other rumen bacteria such that they fitted no other taxon. They were given the name Synergistes jonesii.

Commercial release

For commercial use, two strains were combined – one that degraded mimosine and the other that degraded 2,3-DHP. This combination was used initially across state boundaries for quarantine reasons. Subsequently, fistulated cattle previously infused with the bacteria and fed Leucaena were used to produce the rumen fluid. This was strained through cheese cloth to remove large particles into a modified Coca Cola tank then administered to the donor animals via a rumen injector gun. Rumen fluid was less sensitive than the pure cultures to exposure to oxygen and so much more robust for use on cattle properties.

A key test was whether the extreme toxicity encountered on the Ord Irrigation Area in the north-west of Western Australia could be overcome with the ‘bug’. It was, and a gain of 1 490 kg live weight gain/ha/yr was achieved despite a cold ‘winter’. Ten years earlier Dr Jones had predicted that if they solved the Leucaena toxicity problem they would be able to achieve 1 500 kg LWG/ha/yr. Since then this record has been surpassed.

Over the next few years many herds of cattle in Queensland and the Ord were treated with a 100% success rate. Since then the ‘bug’ has been successfully taken to:

• Africa (Ethiopia, Zimbabwe and South Africa)
• China
• Paraguay.

Nature itself has solved the problem of administering the mimosine-degrading organisms. Untreated animals simply acquire the microbes from associated treated animals or from dust from dung in cattle yards. This research has been worth millions of dollars to northern Australia’s beef industry.

In 2009, Raymond Jones published what he described as probably his last paper on the Leucaena bug story. The paper entitled ‘Survival of the rumen bacterium Synergistes jonesii in a herd of Droughtmaster cattle in north Queensland’ shows that the bug is still active 25 years after its first introduction to the herd with no special management.

Source

• Jones RJ, 2008, Personal communication.