Leucaena Toxicity: A New Perspective on the Most Widely Used Forage Tree Legume

The tree legume Leucaena leucocephala (leucaena) is a high quality ruminant feed, vitally important for livestock production in the tropics despite the presence of mimosine in the leaves. This toxic non-protein amino acid has the potential to limit productivity and adversely affect the health of animals. The discovery and subsequent distribution in Australia of the ruminal bacterium Synergistes jonesii as an oral inoculum was shown in the 1980s to overcome these toxic effects. However, recent surveys of the status of toxicity worldwide; improved understanding of the chemistry and mode of action of the toxins; new techniques for molecular sequencing; and concerns about the efficacy of the in vitro inoculum; have cast doubt on some past understanding of leucaena toxicity and provides new insights into the geographical spread of S. jonesii. There is also confusion and ignorance regarding the occurrence and significance of toxicity in many countries worldwide. Ongoing research into the taxonomy and ecology of the Synergistes phylum, improved methods of inoculation, improved management solutions, along with awareness-raising extension activities, are vital for the future success of leucaena feeding systems

Rheumatoid arthritis synovium contains plasmacytoid dendritic cells

We have previously described enrichment of antigen-presenting HLA-DR(+ )nuclear RelB(+ )dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123(+)HLA-DR(+ )plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c(+ )myeloid DCs and CD123(+ )pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB(-)CD123(+ )DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB(+)CD123(- )DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-γ, IL-10, and tumor necrosis factor α were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB(+ )synovial DCs. pDCs may contribute significantly to the local inflammatory environment

Functional gene analysis suggests different acetogen populations in the Bovine Rumen and Tammar Wallaby Forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems

Is There Genetic Diversity in the ‘Leucaena Bug’ \u3cem\u3eSynergistes jonesii\u3c/em\u3e Which May Reflect Ability to Degrade Leucaena Toxins?

Leucaena leucocephala, a nutritionally rich forage tree legume, contains a non-protein amino acid, mimosine, which is degraded by ruminal bacteria to toxic metabolites 3,4-DHP and 2,3-DHP resulting in goitre-like symptoms in animals, severely restricting weight gain. Raymond Jones, in the early 1980s, discovered the ‘leucaena bug’ in the rumen of goats in Hawaii that degraded these toxic DHP metabolites into non-toxic compounds (Jones and Lowry 1984) which was named Synergistes jonesii (Allison et al. 1992) Subsequently, a rumen inoculum containing S. jonesii was used as an ‘oral drench’ for cattle, kept in continuous culture (Klieve et al. 2002) and supplied to farmers to dose cattle foraging on leucaena. Studies on Queensland herds that received this oral drench showed that up to 50% of 44 herds grazing on leucaena had apparent subclinical toxicity based on high 3,4- and 2,3-DHP excretion in urine (Dalzell et al., 2012). In another study by Graham et al. (2013), a 16S rDNA nested PCR showed that rumen digesta from 6 out of 8 properties tested had a variant DNA profile from S. jonesii ATCC 78.1 strain, which suggested a different strain of the bacterium. It was postulated that either the continually cultured oral inoculum may have undergone genetic modification and/or that animals could harbor other DHP degrading bacteria or S. jonesii strains with differential DHP degrading potential (McSweeney et al. unpublished). The present study looks at changes in the 16S rDNA gene at the molecular level that may suggest divergence from the type strain S. jonesii 78.1 (ATCC) in Queensland cattle as well as in cattle and other ruminants, internationally. These changes can appear as discrete mutations or ‘single nucleotide polymorphisms’ (SNPs) and may be correlated to their ability to degrade DHP, relative to the type strain

Methanogen Diversity in Indigenous and Introduced Ruminant Species on the Tibetan Plateau

Host factors are regarded as important in shaping the archaeal community in the rumen but few controlled studies have been performed to demonstrate this across host species under the same environmental conditions. A study was designed to investigate the structure of the methanogen community in the rumen of two indigenous (yak and Tibetan sheep) and two introduced domestic ruminant (cattle and crossbred sheep) species raised and fed under similar conditions on the high altitude Tibetan Plateau. The methylotrophic Methanomassiliicoccaceae was the predominant archaeal group in all animals even though Methanobrevibacter are usually present in greater abundance in ruminants globally. Furthermore, within the Methanomassiliicoccaceae family members from Mmc. group 10 and Mmc. group 4 were dominant in Tibetan Plateau ruminants compared to Mmc. group 12 found to be highest in other ruminants studied. Small ruminants presented the highest number of sequences that belonged to Methanomassiliicoccaceae compared to the larger ruminants. Although the methanogen community structure was different among the ruminant species, there were striking similarities between the animals in this environment. This indicates that factors such as the extreme environmental conditions and diet on the Tibetan Plateau might have a greater impact on rumen methanogen community compared to host differences

Prevalence of DHP Toxicity and Detection of \u3cem\u3eS. jonesii\u3c/em\u3e in Ruminants Consuming \u3cem\u3eLeucaena leucocephala\u3c/em\u3e in Eastern Indonesia

Leucaena leucocephala (leucaena) is a productive forage tree legume widely used in eastern Indonesia. While highly nutritious, it possesses the toxin mimosine which adversely affects animal production. In ruminants, mimosine is readily converted to the two isomers of dihydroxypyridine (3,4-DHP and 2,3-DHP) known to cause goitre, suppress appetite, and cause severe mineral deficiencies. These adverse symptoms may be partially responsible for the reluctance of some farmers to feed leucaena. A bacterium capable of complete degradation of DHP, Synergistes jonesii, originally discovered in Hawaii in goats consuming leucaena (Jones and Megarrity 1986), was later found in Indonesia which led to the assumption that all Indonesian ruminants were protected from leucaena toxicity even on 100% leucaena diets. The objective of this study, conducted during October-November 2011, was to confirm this hypothesis via an extensive survey of the toxicity status of ruminants consuming leucaena in eastern Indonesia

A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division

Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms

Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle

Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrate

Seasonal and Nutrient Supplement Responses in Rumen Microbiota Structure and Metabolites of Tropical Rangeland Cattle

This study aimed to characterize the rumen microbiota structure of cattle grazing in tropical rangelands throughout seasons and their responses in rumen ecology and productivity to a N-based supplement during the dry season. Twenty pregnant heifers grazing during the dry season of northern Australia were allocated to either N-supplemented or un-supplemented diets and monitored through the seasons. Rumen fluid, blood, and feces were analyzed before supplementation (mid-dry season), after two months supplementation (late-dry season), and post supplementation (wet season). Supplementation increased average daily weight gain (ADWG), rumen NH3&ndash;N, branched fatty acids, butyrate and acetic:propionic ratio, and decreased plasma &delta;15N. The supplement promoted bacterial populations involved in hemicellulose and pectin degradation and ammonia assimilation: Bacteroidales BS11, Cyanobacteria, and Prevotella spp. During the dry season, fibrolytic populations were promoted: the bacteria Fibrobacter, Cyanobacteria and Kiritimatiellaeota groups; the fungi Cyllamyces; and the protozoa Ostracodinium. The wet season increased the abundances of rumen protozoa and fungi populations, with increases of bacterial families Lachnospiraceae, Ruminococcaceae, and Muribaculaceae; the protozoa Entodinium and Eudiplodinium; the fungi Pecoramyces; and the archaea Methanosphera. In conclusion, the rumen microbiota of cattle grazing in a tropical grassland is distinctive from published studies that mainly describe ruminants consuming better quality diets

Identification and isolation of synovial dendritic cells

In rheumatoid arthritis patients, three compartments need to be considered: peripheral blood, synovial fluid, and synovial tissue. Dendritic cells characterized from each compartment have different properties. The methods given are based on cell sorting for isolation of cells, and flow cytometry and immunohistochemical staining for analysis of cells in these compartments