Archaeaphage therapy to control rumen methanogens

Phage therapy is becoming increasingly important as a means of eradicating or controlling microbial populations and has been raised as a potential strategy to reduce methane emissions from ruminants. To date, very little is currently known about phages which may infect the methane-producing archaeal strains (methanogens) dominant within the rumen of Australian cattle, such as the Methanobrevibacter ruminantium. This project aimed to assemble a collection of phages to be employed in phage therapy. A range of animal-derived and environmental source samples were tested using culture-based methodology, however no lytic phages of methanogens were isolated. Given the dearth of knowledge regarding phages of rumen methanogens, this project established that these naturally-occurring phages may be present in very low concentrations within the rumen and this will need to be considered in future methanogen-phage isolation investigations. The project has begun the process of developing and adapting new methodologies for detecting and examining these phages

Persistence of orally administered Megasphaera elsdenii and Ruminococcus bromii in the rumen of beef cattle fed a high grain (barley) diet

When cattle are fed grain, acidotic ruminal conditions and decreased efficiency in starch utilisation can result from the rapid production and accumulation of lactic acid in the rumen. The efficacy of drenching cattle with Megasphaera elsdenii and Ruminococcus bromii to improve animal performance was investigated. A feedlot trial was undertaken with 80 Bos indicus crossbred steers (initial liveweight 347.1 (s.d. 31.7) kg) in 10 pens in a randomised complete block design. An empty-pen-buffer was maintained between treated (inoculated) and untreated (control) groups to avoid transfer of inoculant bacteria to the control steers. Inoculated steers were orally drenched with M. elsdenii YE34 and R. bromii YE282, and populations increased rapidly over 3-14 days. The steers were fed for a total of 70 days with commercial, barley-based, feedlot rations. High growth rates (1.91 kg per day) were achieved throughout the experiment in both the inoculated and control steers. Intakes averaged 21.3 g dry matter (DM) per kg liveweight per day. There was probably no acidosis achieved in this trial following challenge (i.e. no change in pH occurred). There were no differences in any production or carcass measurements between the control and inoculated steers overall. However, the control group acquired dense ruminal populations of M. elsdenii by Day 14, while R. bromii populations established at high densities within the first 2 weeks but then declined and were undetectable by Day 50. R. bromii appears to be only transiently dominant, and once its dominance waned, it appeared that Ruminobacter spp. established in the rumen. Ruminobacter spp. became dominant between 14 and 28 days in all the steers examined and persisted through to the end of the study. These Ruminobacter spp. may be of future interest in the development of probiotics for grain-fed cattle

Degradation of the Indospicine Toxin from Indigofera spicata by a Mixed Population of Rumen Bacteria

The leguminous plant species, Indigofera linnaei and Indigofera spicata are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with Indigofera spicata plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC–MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the Indigofera plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of Indigofera plant material by grazing ruminant livestock

Development of a mixed microbial drench for detoxification of three Leucaena cultivars

The adoption of Leucaena leucocephala in Queensland, as a high protein, leguminous fodder shrub, has been hindered by insect infestation, with psyllids thriving on Leucaena planted in high humidity regions. A psyllid-resistant cultivar of Leucaena has therefore been developed (Redlands). Nonetheless, all Leucaena cultivars contain the non-protein amino acid, mimosine, which in the rumen of cattle can be degraded by many different bacteria to the toxic metabolite 3hydroxy-4-(1H)-pyridone (3,4-DHP). For over 20 years, a mixed microbial drench containing Synergistes jonesii has been produced by DAF to degrade mimosine, 3, 4-DHP and its degradation product 2,3-dihydroxypyridine (2,3-DHP), to reduce any toxic side-effects of feeding Leucaena to cattle (Klieve et al. 2002). This drench is produced in an in-vitro fermentation system supplied with leaf material from the Leucaena cultivar, Cunningham. Previous research found replacing the Cunningham leaf with either psyllid-resistant Redlands or psyllid-tolerant Wondergraze leaf, negatively impacted the mixed bacterial populations’ ability to degrade 3,4-DHP (Ouwerkerk et al. 2019). This study aimed to test how supplying leaf material from a combination of three cultivars, Cunningham, Redlands and Wondergraze of Leucaena (TriMix) to the fermentation system, would affect S. jonesii populations, the ability of the mixed microbial populations to degrade mimosine, 3,4-DHP and 2,3 DHP and if these microbial populations would grow and retain activity, in fermentations supplied leaf from each single Leucaena cultivar

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry

Do Queensland cattle possess rumen bacteria capable of degrading Leucaena toxins?

Leucaena leucocephala is a leguminous fodder tree used by northern Australian producers to provide protein and boost the weight gains of extensively grazing cattle. There is a range of commercial Leucaena cultivars available which all contain a toxic non-protein amino acid, mimosine. Many rumen bacteria can degrade mimosine to 3,4-dihydroxypyridine (3,4-DHP), which is also toxic to cattle. To enable cattle to safely gain the full benefits of Leucaena, a bacterium, Synergistes jonesii, was isolated that could degrade the toxic metabolites 3,4 DHP and 2,3-hydroxypyridine (2,3-DHP) (Allison et al. 1992). A fermenter-grown mixed bacterial inoculum, containing S. jonesii, has been produced by DAF for over 20 years as an oral drench for cattle to prevent Leucaena toxicity and maximise weight gains (Klieve et al. 2002)

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Ac-induced disruption of the doubleDs structure in tomato

The maize doubleDs element is stably maintained in the tomato genome. Upon the subsequent introduction of Ac into a plant containing doubleDs, disruption of the doubleDs structure and DNA rearrangements at the site of the doubleDs element were observed. No indications were obtained for excision of the complete doubleDs structure. The consequences of transactivation of doubleDs in these experiments are different from those described for transactivation of single Ds elements in tomato. The mechanisms by which such rearrangements could have occurred in tomato are discussed in relation to complex insertions containing doubleDs in maize