Introduction, impact and retention of Synergistes jonesii in cattle herds grazing Leucaena leucocephala

Mr Samuel Graham (2010). Introduction, impact and retention of Synergistes jonesii in cattle herds grazing Leucaena leucocephala MPhil Thesis, School of Agriculture and Food Sciences, The University of Queensland.

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Author Mr Samuel Graham
Thesis Title Introduction, impact and retention of Synergistes jonesii in cattle herds grazing Leucaena leucocephala
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2010-07
Thesis type MPhil Thesis
Supervisor Associate Professor Max Shelton
Dr. Scott Dalzell
Dr. Rafat Al Jassim
Total pages 158
Total colour pages 19
Total black and white pages 139
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Leucaena leucocephala (leucaena) is a tropical leguminous forage tree; when planted with companion grasses it forms a highly valuable and productive grazing system for animal production. Leucaena however contains the toxic, non-protein, amino acid mimosine. Post-ingestion, mimosine is rapidly degraded by plant enzymes and rumen microbes into 3-hydroxy-4(1H)-pyridone (3,4-DHP), and sometimes the isomer 2,3-dihydroxypyridine (2,3-DHP), which are detrimental to animal health. In the 1980’s, the rumen bacterium Synergistes jonesii was found to degrade 3,4-DHP into harmless by-products. Subsequent inoculations with S. jonesii were found to increase animal performance and indicated that the problem of leucaena toxicity was resolved. Leucaena toxicity has re-emerged in cattle grazing leucaena pastures across Queensland. In a 2003/2004 study, approximately 50% of cattle herds tested were found to have very high urinary DHP concentrations and were therefore suffering subclinical toxicity which was largely unnoticed by graziers. There is concern within the beef industry about the protection status of herds and the effectiveness of the currently recommended in vitro inoculum, especially when cattle are removed from leucaena pastures for several months. Possible factors contributing to the re-occurrence of toxicity may relate to reduced effectiveness and persistence of the current in vitro source of S. jonesii inoculum. This study investigated the efficacy and persistence of the in vitro S. jonesii inoculum to prevent leucaena toxicity on commercial cattle properties in southern Queensland. Leucaena availability, animal performance and inoculation history on each of 8 properties were evaluated. The first experiment investigated the efficacy of the in vitro inoculum. Treatments were minus/plus inoculation, arranged sequentially over time. Prior to inoculation, the presence of toxicity in 14-15 animals from each property grazing leucaena pastures was monitored for 10-17 weeks to ensure high DHP levels in urine. Paired blood (DHP concentrations), urine (DHP concentrations), faecal (% leucaena in diet) and rumen fluid samples [for polymerase chain reaction (PCR) analysis of S. jonesii DNA] were collected from each animal. Herds excreting DHP in urine at concentrations >200 µg/mL were categorized as not ‘protected’. Following inoculation, the same 14-15 animals continued grazing leucaena and were monitored to determine if toxicity had been controlled. Faecal delta carbon analysis revealed animals selected diets of 30-50% leucaena ensuring moderate-high levels of mimosine intake throughout the trial as feed intake was adequate with animals continuing to gain weight (0.15-1.1 kg/head/day). Blood DHP concentrations were very low [at the limits of detection by high performance liquid chromatography (HPLC)] and not useful in determining toxicity status of the animals. Urine DHP concentrations were much higher and useful indicators of toxicity. The efficacy of inoculation could be assessed by urine DHP analysis on two properties only, with inoculation eliminating toxicity on one property while apparently failing on the other. Remaining properties acquired DHP degrading capabilities from unknown bacteria which were slow to colonise the rumen. Real-time PCR failed to detect type strain S. jonesii DNA in any of the rumen fluid samples collected indicating populations (if present) were <105 cells/mL. Nested PCR detected slightly dissimilar DNA profiles of S. jonesii compared to the type strain in only 6.1% of rumen fluid samples. This indicated that a S. jonesii variant was present on 6 of the 8 properties studied. The delayed (10-17 weeks) degradation activity of this variant and the absence of detectable levels of the in vitro produced S. jonesii type strain post-inoculation confirmed that the S. jonesii bacteria involved, both the variant and the in vitro inoculum, lacked ability to rapidly colonise the rumen to detectable population levels. However, the endemic DHP-degrading bacteria were able to degrade DHP to low levels and prevent toxicity in cattle grazing leucaena for sufficient lengths of time. In a second experiment, the persistence of DHP-degrading bacteria post-inoculation with the in vitro S. jonesii inoculum was studied in cattle grazing leucaena-free pastures on 4 properties (5 herds). Eight animals were removed from leucaena pastures to graze alternative diets for 3-4 months. Populations of S. jonesii were enumerated by real-time PCR analysis of rumen fluid samples collected regularly after the animals left the leucaena pasture. The capacity of the bacteria to degrade DHP isomers was determined by urine DHP and faecal delta carbon analysis 3 weeks after the animals re-commenced grazing leucaena pastures. Failure of the real-time PCR to detect S. jonesii DNA in any of the rumen fluid samples prevented the observation of changes in bacterium population levels (cells/mL) over time. Urine DHP analysis indicated that only 3 herds (on 2 of 4 properties) retained capacity to degrade DHP and demonstrated that herds could lose ‘protection’ after 80-111 days off leucaena. Persistence of S. jonesii may be affected more by the type of diet than by the time off leucaena. Herds which lost protection grazed lush oats pastures; although one herd which grazed oats with additional roughage retained protection. It was hypothesised that the physical and chemical properties of the alternative diets, which dictate the rumen environment and microbial growing conditions, are likely to affect the persistence of S. jonesii. Urine DHP analysis was a better indicator of toxicity than blood analysis and current PCR techniques for detecting ruminal populations of S. jonesii lacked the required sensitivity to adequately monitor the low (<105 cells/mL) S. jonesii populations observed in rumen fluid. The effectiveness and practicality of alternative methods for analysis of DHP in urine and percentage of leucaena in diet were also investigated. Urine samples (368 samples) were analysed using both HPLC and a colorimetric technique (acidified ferric chloride) to assess the robustness of the colorimetric test and its capacity to quantify urine toxin concentrations. Faecal samples (100 samples) were analysed using both the delta carbon and faecal near infrared reflectance spectroscopy (faecal NIRS) methods to assess ability of faecal NIRS to reliably predict the percentage of leucaena in diet. The results indicated that the alternative methods for analysis of urinary DHP concentrations and prediction of the percentage of leucaena in diet lacked accuracy. Both tests demonstrated too much variability for research application. It was concluded that the colorimetric urine test can be applied as a qualitative analysis method for indicating presence or absence of toxicity provided sufficient samples were taken (>10 per herd). The current faecal NIRS test gives a quantitative measure of dietary composition useful for graziers managing leucaena pastures provided sufficient samples (>20 samples per herd) are collected to minimise variability. It is recommended that the faecal NIRS method be further improved by increasing the number of paired data in the underlying calibration equations. It is anticipated that many herds grazing leucaena pastures, post-inoculation with the in vitro source of S. jonesii, may have lost ‘protection’ during periods ‘off’ leucaena and may be suffering undiagnosed subclinical toxicity that may be limiting animal productivity and profitability. It is recommended that graziers: (a) periodically test their herds’ toxicity status; and (b) and minimise time ‘off’ leucaena to reduce the risk of losing viable populations of S. jonesii. Further research is required to investigate the possible reasons for the low vigour and persistence of the in vitro produced inoculum.
Keyword Leucaena
Synergistes jonesii
leucaena toxicity
Additional Notes 27, 54, 56, 59, 60, 66, 67, 69, 82, 83, 95, 98, 115, 116, 117, 118, 119, 120 and 123 (page number/158). 53, 65, 68, 71, 72, 74, 76, 99, 100 (page number/158).

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Created: Thu, 30 Dec 2010, 09:06:49 EST by Mr Samuel Graham on behalf of Library - Information Access Service