Leucaena feeding
Leucaena (Leucaena leucocephala) is a highly productive tropical leguminous fodder tree. It is long-lived and drought tolerant and is a high-value feed for ruminant animals due to its palatability and excellent nutritional characteristics i.e. high crude protein content, high digestibility and non-bloating attributes (Jones, 1979). These productive traits consistently deliver excellent live-weight gains superior to other tropical forage systems (Shelton and Dalzell, 2007).
Leucaena does however contain a toxin, the non-protein amino acid mimosine.
Mimosine and its primary rumen derivative 3-hydroxy-4(1H)-pyridone (3,4-DHP) are responsible for inhibiting animal production and the former compound may cause mortality (Peixoto et al., 2008). In 1979, a rumen bacterium, later named Synergistes jonesii, was discovered that could degrade these toxins into harmless by-products (Jones, 1981a, Allison et al., 1992). The bacterium was introduced into Australian ruminants in the 1980s enabling inoculated animals to achieve high dietary intakes of leucaena and display superior performance suggesting that leucaena toxicity was resolved (Jones and Megarrity 1983;
Quirk et al., 1988; Pratchett et al., 1991). However, there have been consistent reports of continuing toxicity in Australian cattle herds even in those previously inoculated and therefore thought protected from DHP toxicity (Dalzell et al., 2012). This producer survey involved extensive herd testing across Queensland, Australia and indicated that approximately 50% of the 44 herds tested displayed sub-clinical toxicity as indicated by high levels of DHP in urine. Toxicity may not always manifest itself as visible symptoms (Jones
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and Winter, 1982) and subclinical toxicity often goes unnoticed while reducing animal performance and profitability. Despite lack of visible symptoms, Quirk et al., (1988) showed that liveweight gain of cattle on leucaena pastures was doubled following inoculation.
The longevity and long-term efficacy of the S. jonesii inoculum is poorly understood and therefore a study was conducted recently to investigate the efficiency and persistence of inoculating S. jonesii into Australian cattle herds (Graham et al., 2013). The experiments conducted in eight commercial herds showed (1) a very slow rate of degradation of DHP isomers; (2) frequent occurrence of high levels of 2,3-DHP in urine indicating partial toxin degradation, both before and after inoculation; (3) a low incidence of detection of S. jonesii in rumen fluid after inoculation based on PCR amplification of 16S rDNA sequence of the type strain 78-1 thus indicating low populations in rumen fluid (4) no evidence of DHP degradation post inoculation on one of two properties tested; and (5) loss of protection from subclinical toxicity on some properties after <4 months on alternative non-leucaena pastures (Graham et al., 2013). It was concluded that while most herds showed some capability to degrade DHP due to some residual capability from previous exposure, they did not achieve the same rapid and complete DHP degradation reported in the 1980s.
Based on this information, our current hypothesis is that S. jonesii was broadly dispersed in Australia prior to the release of the imported strain but was phenotypically different in ability to degrade leucaena toxins which were not present in indigenous Australian plants. This is supported by the observation that detoxification of leucaena in ruminants has been documented on several continents other than Australia. We propose that the S. jonesii species are not geographically constrained but rather have evolved varying phenotypes for metabolism of xenobiotics based on the different habitats they occupy globally.
Fluoroacetate Poisoning
Globally, fluoroacetate is found in over 40 plant species (Harper et al., 2003) and is responsible for intoxication of commercial livestock species (McIlroy, 1982). Due to this problem associated with fluoroacetate-accumulating plants, dehalogenase genes have also been used to develop transgenic rumen bacteria (Gregg et al., 1994) which detoxify fluoroacetate within the gastrointestinal tract of ruminant livestock (Gregg et al., 1998;
Padmanabha et al., 2004). Concerns over the release of these transgenic bacteria, and the lack of information on fluoroacetate-degrading anaerobes prompted us to search for naturally- occurring anaerobic microorganisms that could reduce fluoroacetate toxicity in ruminants.
Despite the interest in the microbial degradation of fluoroacetate, there are few reports of fluoroacetate being degraded naturally under anaerobic conditions (Kim et al., 2000).
We have recently isolated from the bovine rumen a bacterium (strain MFA1) which belongs to the phylum Synergistetes and metabolizes fluoroacetate under anaerobic conditions (Davis et al., 2012). The growth characteristics of strain MFA1 indicate that the bacterium may gain energy via the process of reductive dehalogenation (dehalorespiration).
In the course of our studies, we found four other fluoroacetate-degrading strains with a 99 % similarity of 16S rDNA sequence to the MFA1 bacterium in the gut of emus (Dromaius novaehollandiae), kangaroos (Macropus spp.) and other cattle. The occurrence of strain MFA1 and other related strains (98% similarity) from the gut of animals demonstrates that these members of Synergistetes may be widely distributed in the gastrointestinal tracts of herbivorous mammals. The fact that these sequences could only be detected using nested PCR indicates they are at low numbers, and possibly at the limit of detection for a PCR-based method.
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Growth requirements and genomics of Synergistetes bacteria
Synergistetes species are anaerobic amino-acid fermenters, and this is reflected in their genome content, which has a greater abundance of amino acid transport and metabolism genes compared to other bacteria. These organisms are also known to utilise peptides for growth requirements (Allison et al., 1992; Baena et al., 1999). A better understanding of the nutritional and physiological requirements and genome content of bacteria in this phylum will provide insights into the ecology of these organisms in the rumen which could be managed to protect animals from toxicity. We have therefore examined the diversity of the peptidolytic systems of 15 Synergistetes bacteria at the genome level to identify the predominant genes involved in peptide and amino acid metabolism of rumen Synergistetes.
All of the Synergistetes examined possessed a conserved set of peptidase genes consisting of pepM, ggt, pepF, lap, dpp1, and pepP. Phylogenetically similar species were observed to cluster together based on their peptidolydic systems (Figure 2). Synergistes jonesii, strain MFA1 and closely related species (Cluster IA) were the only bacteria to contain endopeptidase O (pepO) and Proline iminopeptidase (pip) genes. Cluster II members were found to have a higher abundance of peptide transport systems compared to all other groups.
Conclusions
Rumen Synergistetes bacteria appear to have a preference for peptides compared to amino acids for growth. They also have distinctive enzyme systems for the metabolism of peptides of specific amino acid composition. Provision of diets that are optimised to provide nitrogenous nutrients specific for the Synergistetes bacteria may stimulate growth and abundance of these organisms in the rumen and provide greater protection against toxicity from leucaena and fluoroacetate and perhaps other plant secondary compounds.
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Figure 2. Heat map representing distribution of genes associated with peptidolytic systems among 15 Synergistetes species. Alphabet in the parentheses next to the species indicated the genomic sequencing status: D, draft; and F, finished.
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