Strategies for improving ruminant utilisation of high grain diets: Pangenome of Streptococcus bovis

The field of microbiology is being transformed by the increased ease and reduced costs of DNA sequencing. An initiative of the United States Department of Energy (USDOE), Joint Genome Institute facilitated the establishment of a Community Sequencing Project, the Hungate 1000, an international collaborative venture which aimed to sequence the genomes of bacteria isolated from the rumen and intestinal tract of herbivores. The DAF Rumen Ecology Unit (REU) contributed 36 rumen bacteria and seven bacteriophages to this genome sequencing effort. Thirteen of these bacteria belonged to the Streptococcus bovis/Streptococcus equinus complex (SBSEC). These are virulent, fast-growing commensal bacteria which over-proliferate in the rumen of cattle when they transition too quickly from forage to high grain feedlot rations, causing rumen lactic acidosis. Costly management strategies based on lengthy feed transition times (up to 20 days), and the use of feed additives including rumen modifiers such as monensin and antibiotics, are currently used to control rumen acidosis. This project used the SBEC genome sequences generated by the Hungate 1000, as well as publically available SBEC genome sequences, to create a combined genome dataset or pangenome. This pangenome of 42 genome sequences was then used to identify: 1. factors which enable these organisms to rapidly proliferate on starch-rich diets, for example, carbohydrate degrading enzymes; and 2. new approaches to control these organisms, for example inhibitory peptides (bacteriocins) and enzymes, carried by naturally-occurring viruses predatory to bacteria (known as bacteriophage or phage), which can specifically target and puncture bacterial cell walls. During the course of the project, technical capacity of the REU was also developed to facilitate the analysis and secure storage of large sequence datasets, with the expansion of high performance computing resources and installation of specialist software. Several of the phage proteins identified by the pangenome analysis were shown to be intact and functional, with the formation and release of phage particles from bacterial host cells verified by transmission electron microscopy and proteomics analysis. These phage proteins represent potential candidates for further investigation as antimicrobials to control SBEC, and will be used as background proof of concept for the development of project proposals for submission to funding agencies

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

Editorial: Advances in the Understanding of the Commensal Eukaryota and Viruses of the Herbivore Gut

Herbivores play an important role in the survival of humanity, contributing food and textiles, as well as social and economic value. For decades, optimizing the productivity, health, welfare, and environmental footprint of herbivorous animals, particularly ruminant livestock, has been the subject of an extensive, global research effort. Much of this research effort has focused on the herbivore gut. The specialized nature of the herbivore digestive tract and its resident microbes enables the breakdown of highly fibrous plant materials, which are unable to be utilized by omnivores and carnivores. In recent years, the bacteria and methanogenic archaea have been the major focus of research efforts, with the other gut microbes being understudied in comparison

Bioresponsive polymer-protection conjugates as a unimolecular drug delivery system

PEGylation has become very popular for the generation of nanomedicines with improved protein delivery properties, despite its lack of biodegradability. Researchers usually try to maximise retained protein activity during PEGylation. However, this proof of principle study aimed to create an inactive peptide or enzyme product, using a biodegradable polymer, that would elicit minimal activity/non-specific toxicity on administration. Following triggered site-specific degradation of the polymer, the hypothesis was that protein activity could be slowly regenerated in the general circulation or localised to a specific target site. Model conjugates were synthesised by coupling dextrin degraded by amylase to trypsin and melanocyte stimulating hormone MSH, to test this concept and targeted delivery for both an enzyme and a receptor-binding ligand. Hyaluronic acid HA degraded by hyaluronidase conjugates of trypsin and ribonuclease A were also synthesised. The latter was intended to develop the possibility of designing novel anti cancer conjugates. A higher molecular weight dextrin 47,200 g/mol, 26 mol succinoylation was shown to best mask 34 trypsin activity and reinstate 58 of the activity by addition of amylase. When a HA fraction molecular weight 130,000 g/mol was prepared by acid hydrolysis and conjugated to trypsin 4 w/w, trypsin activity was masked to 6 and immediately re-instated to 24 on addition of hyaluronidase. Similarly, the dextrin-MSH conjugate reduced melanin production to 11 of the control and only restored to 33 on addition of amylase. RNase A alone was not cytotoxic up to 1 mg/mL, whereas, the HA-RNase A conjugate 0.1 mg/mL RNase A equivalent was cytotoxic in B16F10 and CV-1 cells 72 h. This work provides proof of principle for the concept of using biodegradable polymers to mask and reinstate conjugated protein activity in the presence of the appropriate enzyme 'unmasking' trigger

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

Positives and negatives: reclaiming the female body and self-deprecation in stand-up comedy

Drawing on existing research into feminist humour, this article argues that many of the functions of self-deprecation within comic performance that have been identified and explored in relation to the American context of the late 90s and early 2000s are still evident on the current UK circuit. Self-deprecation in stand-up comedy by women continues to be understood as both positive (as part of the rise of popular feminisms) and negative (as reinforcing patriarchal norms). These contradictory understandings of self-deprecation in stand-up comedy are always inextricably linked to the identities of the audiences for such humour. I consider how emergent female stand-up performers may rationalise and understand the role self-deprecation plays within their own work in the current British context. I then discuss the work of stand-up comedian Luisa Omielan as an example of the rejection of self-deprecatory address. I make the argument that self-deprecation cannot function simply as positive or negative in the current UK context, but must always be considered (for both audiences and performers) as challenging and reinforcing restrictive patriarchal attitudes towards women simultaneously

Changes in mixed microbial inoculums to prevent the toxic side-effects in cattle grazing new varieties of Leucaena.

Leucaena leucocephala is a legume fodder crop that grows in tropical and subtropical environments. Leucaena provides a high quality feed for cattle boosting liveweight gain both per animal and per hectare, improving profitability for steer turnover by 121% compared with the base scenario of grazing buffel grass (Bowen and Chudleigh, 2018). A number of commercial leucaena cultivars, Cunningham, Peru, Taramba, El Salvadore, and Wondergraze, are used in Queensland. Leucaena contains a toxic amino acid, mimosine, which many rumen bacteria can degrade to a toxic metabolite 3-hydroxy-4-(1H)-pyridone (DHP). Productivity from leucaena-pasture can be reduced by DHP-induced depressions in intake. A DHP degrading bacterium, Synergistes jonesii was isolated from a mixed bacterial population isolated from a goat from Hawaii (Allison et al., 1992). For over twenty years DAF has provided a mixed bacterial rumen inoculum, containing S. jonesii, for cattle grazing Leucaenapastures, produced in an in vitro fermentation system with Cunningham cultivar as the feed source. All of the commercial cultivars are susceptible to attack by psyllid insects, limiting their use in higher rainfall areas - to address this limitation, a commercial psyllid-resistant cultivar, Redlands, was released in 2019. To assess if the leucaena inoculum is impacted by Redland’s anti-psyllid, chemical characteristics a series of 30 day in vitro anaerobic fermentations were undertaken feeding either Redlands, Cunningham or Wondergraze cultivars. The fermentations were conducted following the method of Klieve et al. (2002). Fermentations were started with either cryopreserved leuceana inoculum from a single day of a production fermentation; or day 30 of a Wondergraze or Redlands fermentation. Daily samples were taken and assays set up on days 10, 15, 20, 25 and 30 to monitor the fermentation’s ability to break down mimosine, 3,4 DHP and 2,3 DHP. Genomic DNA extracted from daily samples was used in a S. jonesii quantitative PCR and for barcoded amplicon sequencing of the 16S rRNA gene V3 –V4 region using the Illumina MiSeq platform

Cinema as mnemotechnics

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Enriching for rumen bacteria to degrade the Pimelea plant toxin simplexin, in an anaerobic in vitro fermenter

Three species of Australian native plants, Pimelea trichostachya, P. simplex and P. elongata, are endemic to the arid rangelands of Queensland, New South Wales and South Australia and are responsible for Pimelea poisoning, also known as St George or Marree disease. Pimelea poisoning occurs in cattle ingesting Pimelea plants, with the orthoester simplexin identified as the responsible toxin. There is no effective treatment and economic losses have been estimated at over $50 million during significant Pimelea poisoning events. In a previous feeding trial, animals were fed increasing amounts of Pimelea, and after initially showing signs of poisoning, the animals appeared to adapt to ingesting Pimelea, possibly through rumen microbial degradation of the toxin (Fletcher et al., 2014). Kangaroos, forestomach fermenters, often graze pastures containing Pimelea with no apparent ill effects. To investigate the degradation effect further, a series of 30 day in vitro, anaerobic fermentations were undertaken

Role of Staphylococcus agnetis and Staphylococcus hyicus in the Pathogenesis of Buffalo Fly Skin Lesions in Cattle

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions