Phytogenic products, used as alternatives to antibiotic growth promoters, modify the intestinal microbiota derived from a range of production systems: an in vitro model.

The removal of antibiotics from the feeds used in the livestock industry has resulted in the use of a wide range of alternative antimicrobial products that aim to deliver the productivity and health benefits that have traditionally been associated with antibiotics. Amongst the most popular alternatives are phytogenic product-based extracts from herbs and spices with known antimicrobial properties. Despite embracing such alternatives, the industry is still largely unaware of modes of action, their overall effects on animal health, and interactions with other feed additives such as probiotics. To address some of these issues, three phytogenic products were selected and their interactions with caecal microbiota of layers, grown under six different production systems, were investigated in vitro. Caecal microbiotas were grown with and without phytogenic products, and the changes in microbiota composition were monitored by sequencing of 16S rRNA gene amplicons. Phytogenic products and production system both significantly influenced microbiota composition. The three phytogenic products all altered the relative abundance of species within the Lactobacillus genus, by promoting the growth of some and inhibiting other Lactobacillus species. There were also significant alterations in the Bacillus genus. This was further investigated by comparing the effects of the phytogenic products on the growth of a commercially used Bacillus-based probiotic. The phytogens affected the probiotic mix differently, with some promoting the growth of Bacillus sp. at lower phytogenic concentrations, and fully suppressing growth at higher concentrations, indicating the importance of finding an optimal concentration that can control pathogens while promoting beneficial bacteria. KEY POINTS: • After removal of antibiotics from animal feed, urgent solutions for pathogen control were needed. • Alternative products entered the market without much knowledge on their effects on animal health. • Probiotic products are used in combination with phytogens despite the possible incompatibility.

Further characterisation of cytokines in macropod marsupials: IL-10 and IL-10Δ3.

Interleukin-10 is an immunomodulatory cytokine that has been implicated, along with IFN-γ, in the disease sequelae of mycobacterial infection. In order to investigate the role of IL-10 in marsupial disease models we sequenced and characterised the IL10 gene in the tammar wallaby (Macropus eugenii) and rufous hare-wallaby (Lagorchestes hirsutus). An isoform IL-10Δ3, in which an in-frame deletion of exon 3 occurs, was discovered in both macropod species. Analysis of wallaby and other reported marsupial IL-10 homologs suggests that while marsupial IL-10 is comparable to that of human IL-10, the predicted IL-10Δ3 protein may play a more complicated role in the modulation of IL-10-directed responses. Expression of the canonical gene and splicing variant was confirmed in both wallabies, and the rufous hare-wallaby showed differential expression across lymph node, spleen and liver, with isoform expression detected in the lymph node. This characterisation and expression of IL-10 in de novo tissues provides a basis for further study into the role of IL-10 in disease models in marsupials.

The macropod type 2 interferon gene shares important regulatory and functionally relevant regions with eutherian IFN-γ.

Interferon-γ (IFN-γ) is an important immune regulatory molecule that plays a significant role in internal and external modulation of the mammalian immune response to intracellular pathogens. Herein, we report the 492 nt expressed sequence for the coding domain of IFN-γ from the immune tissues of two Australian macropod marsupial species: the tammar wallaby (Macropus eugenii) and the vulnerable rufous hare-wallaby (Lagorchestes hirsutus). Both 5' and 3' untranslated regions and the coding domain of M. eugenii IFN-γ revealed the presence of motifs responsible for transcriptional regulation, mRNA regulation, post-translational modifications, and receptor binding in other mammals. Since diagnostic kits for mycobacterial disease commonly rely on the assessment of interferon levels, we can now use this information to develop reagents that can be applied in clinical and laboratory settings to further our understanding of marsupial responses to disease.

Molecular characterisation and expression of interleukin-6 and interleukin-6Δ2 in the Tammar wallaby (Macropus eugenii).

The pro-inflammatory cytokine, Interleukin-6 (IL-6), has not yet been fully characterised in the model macropod, Macropus eugenii, due to incomplete sequence information in publically available genome databases. Using a Rapid Amplification of cDNA Ends strategy we have confirmed the expression and complete nucleotide sequence for this molecule in lymph node tissue and activated leukocytes. Structural conservation of the mature wallaby IL-6 molecule was high when compared with human IL-6, although there was only 34% amino acid sequence identity with the human IL-6 peptide, consistent with reports of the evolutionary divergence of this cytokine. We also report the discovery of MeIL-6Δ2, a splice variant missing exon 2, which directly translates to a truncated non-functional peptide, but which may also code for an alternative peptide that is translated downstream of the canonical IL-6 start site. This putative gene product is predicted to maintain some, if not all, of the functions of macropod IL-6 and is the first IL-6 isoform reported outside of eutherian mammals.