Methanotrophs from natural ecosystems as biocontrol agents for ruminant methane emissions

In ruminant cattle, the anaerobic fermentation of ingested plant biomass results in the production of methane (CH4). This CH4 is subsequently eructated to the environment, where it acts as a potent greenhouse gas and is one of the leading sources of anthropogenic CH4 in Australia. Methane oxidising microorganisms are an important environmental sink for CH4; however the possibility that methanotrophs are native to the rumen has received little attention. This project aimed to characterise methanotrophs from a range of environments, and to subsequently determine the metabolic activity of these microorganisms under in vitro rumen-like conditions. This study is the first to characterise rumen methanotrophs using molecular methodology. Using a combination of denaturing gradient gel electrophoresis and phylogenetic analysis, it was found that simple communities of Proteobacterial methanotrophs can be native residents of the rumen microbial community in grain-fed Bos indicus steers. A putative methanotrophic Gamma-proteobacterial Methylobacter species was also enriched from grain-fed whole rumen contents using novel techniques. However, the activity of these organisms in situ remains to be fully understood. Furthermore, the possibility that a grain-based dietary affect influences the diversity and activity of methanotrophs in situ is intriguing. Future work to address these questions is necessary to evaluate the potential for methanotrophs to act as biocontrol agents for ruminant CH4 emissions.

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

Increasing productivity and reducing methane emissions by supplementing feed with dietary lipids

Methane (CH4) is a by-product in the digestion of plant material by all cattle and sheep. Effectively it is wasted feed material and energy that could otherwise be available for animal production. It is also a major greenhouse gas (14% of Australia’s emissions). Beef cattle contribute 50% of these emissions. Many lipid containing feed materials are known to reduce methane emissions, as well as increasing productivity when used as supplementary feeds. The purpose of this project is to investigate the impact of lipid containing feed additives on the suppression of methane emissions and improvements in the growth rate of steers fed a basal diet of tropical pastures

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

Ammonia-hyperproducing bacteria from New Zealand ruminants

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria,Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genusPeptostreptococcus, that D4 and D5 belonged to the familyBacteroidaceae, where D4 was similar to Fusobacterium necrophorum; and that C2 was most similar to an unidentified species from the genus Eubacterium. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h−1, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h−1, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants

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

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

Aim: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. Methods and Results: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 107 cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 106 CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 108 CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. Conclusion: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7–10 days earlier than in uninoculated cattle. Significance and Impact of the Study: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR

Adsorbents for the sequestration of the Pimelea toxin, simplexin

Pimelea poisoning affects cattle grazing arid rangelands of Australia, has no known remedy and significant outbreaks can cost the industry $50 million per annum. Poisoning is attributable to consumption of native Pimelea plants containing the toxin simplexin. Charcoal, bentonite and other adsorbents are currently used by the livestock industry to mitigate the effects of mycotoxins. The efficacy of such adsorbents to mitigate Pimelea poisoning warrants investigation. Through a series of in vitro experiments, different adsorbents were evaluated for their effectiveness to bind simplexin using a simple single concentration, dispersive adsorbent rapid screening method. Initial experiments were conducted in a rumen fluid based medium, with increasing quantities of each adsorbent: sodium bentonite (Trufeed®, Sibelco Australia), biochar (Nutralick®Australia) and Elitox® (Impextraco, Belgium). Data showed the unbound concentration of simplexin decreased with increasing quantities of each adsorbent tested. Sodium bentonite performed best, removing ~95% simplexin at 12 mg/mL. A second experiment using a single amount of adsorbent included two additional adsorbents: calcium bentonite (Bentonite Resources, Australia) and a synthetic adsorbent (Waters, USA). The concentration of simplexin remaining in the solution after 1 h, the amount able to be desorbed off the adsorbent-toxin matrix with replacement fresh fluid, and the amount remaining bound to the adsorbent were measured. All samples containing an adsorbent were statistically different compared to the blank (p < 0.05), indicating some binding activity. Future work will explore the binding mechanisms and behaviour of the toxin-adsorbent complex in the lower gastrointestinal tract

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

Effects of adsorbents and probiotics in mitigating simplexin poisoning effects in cattle fed Pimelea

Pimelea poisoning of cattle occurs only in arid inland Australia and is caused by the toxin, simplexin found in some native Pimelea plant species. Pimelea-affected cattle show distinctive physical symptoms including subcutaneous oedema under the jaw and brisket, diarrhoea and anaemia. Simplexin was thought to be circulated in the bloodstream to exert its toxic effect, but due to its hydrophobic nature the extent of its blood carriage is unknown. Fletcher et al. (2014) postulated a possible role of rumen microorganisms adapting to detoxify simplexin in cattle fed Pimelea over a prolonged period of time. Anecdotal reports have suggested cattle supplemented with biochar and bentonite showed resistance towards Pimelea poisoning. In this project, a pen trial (QAFFI/QASP/337/20/DAF) was conducted to determine the efficacy of adsorbents (biochar and bentonite) and a bacterial inoculum for reducing the effects of Pimelea poisoning in steers