Sheep lung microbiota

Until recently it was assumed that the healthy mammalian lung did not harbour a microbiota, unlike other body sites. However, through the use of sequencing based technologies this has been shown to not be the case. Low biomass communities of microbes can be identified in the healthy lung and the lung microbiota in various diseases states has been shown to differ form these 'healthy' communities. The sheep respiratory microbiota is of interest from both an animal health perspective and due to the potential use of the sheep as a large animal model for studying the lung microbiota. In this thesis I seek to characterise the composition and variability of the sheep lung microbiota; the differences between the sheep upper and lower respiratory tract bacterial communities and to assess whether exhaled breath condensate collection can be used as a non-invasive lung microbiota sampling method. To study the bacterial communities present in samples I have used 16S rRNA gene sequencing and analysis. In Chapter 3 I examine the inter-individual and spatial variability present within the sheep lung microbiota. Protected specimen brushings were collected from three lung segments in six animals at three time-points. In a separate sheep a greater number of brushings was taken (n=16) in order to examine the amount of variability over a smaller spatial scale. I find that there can be large differences between the bacterial communities isolated from different locations within the lung, even over short distances. Samples also cluster by the sheep from which they were taken, indicating a host specific influence on the lung microbiota. In Chapter 4 I compare whole lung washes and oropharyngeal swabs from 40 lambs in order to examine the differences between the upper and lower respiratory tract microbiotas. I find that oropharyngeal swabs separate into rumen-like or upper respiratory tract-like bacterial communities. Despite the fact that in humans the upper and lower respiratory microbiotas have been shown to have similar compositions, the sheep lung microbiota samples in this study do not resemble either oropharyngeal samples or reagent only controls. In my first two results chapters, lung sampling methods were used which involved either anaesthesia combined with a bronchoscopic procedure (Chapter 3) or samples being taken from dead animals (Chapter 4). In Chapter 5 I assess whether there is a less invasive way of taking lung microbiota samples from a living individual, both to minimise the procedural stress on animals used as models and to increase the pool of potential volunteers for human lung microbiota studies. I compared samples taken via protected specimen brushings to samples taken via exhaled breath condensate collection, a less invasive sampling technique. I find that condensate samples contain less bacterial DNA and different bacteria than brushing samples, indicating that it is unlikely they could be used as a replacement for invasive sampling methods. In my final results chapter I compare the results across Chapters 3, 4 and 5 to identify bacteria which occur consistently in the sheep lung and could therefore potentially be described as core lung microbiota members. In conclusion, while I have found that there are large differences between the sheep lung microbiota and that which has previously been described in humans, the sheep can still be of use as a model in studies where these differences would not have a significant impact, such as in Chapter 5 of this thesis. I have identified several bacterial members of the core sheep lung microbiota which in future it would be interesting to better characterise and to assess whether they play a role in sheep health

Microbiota of healthy dogs demonstrate a significant decrease in richness and changes in specific bacterial groups in response to supplementation with resistant starch, but not psyllium or methylcellulose, in a randomized cross-over trial.

Even though dietary fibres are often used as prebiotic supplements in dogs, the effect of individual types of fibres on canine microbiota composition is unknown. The objective of this study was to assess changes in faecal microbiota richness, diversity and taxonomic abundance with three different fibre supplements in dogs. These were psyllium husk, resistant starch from banana flour and methylcellulose. They were administered to 17 healthy dogs in a cross-over trial after transition to the same complete feed. Faecal scores and clinical activity indices were recorded, and faecal samples were collected before and at the end of supplementation, as well as 2 weeks after each supplement (washout). Illumina NovaSeq paired-end 16S rRNA gene sequencing was performed on all samples. After quality control and chimera removal, alpha diversity indices were calculated with QIIME. Differences in specific taxa between groups were identified using Metastats. Methylcellulose significantly increased faecal scores but had no effect on microbiota. Psyllium resulted in minor changes in the abundance of specific taxa, but with questionable biological significance. Resistant starch reduced microbiota richness and resulted in the most abundant changes in taxa, mostly a reduction in short-chain fatty acid-producing genera of the phylum Bacillota, with an increase in genera within the Bacteroidota, Pseudomonadota, Actinomycetota and Saccharibacteria. In conclusion, while psyllium and methylcellulose led to few changes in the microbiota composition, the taxonomic changes seen with resistant starch may indicate a less favourable composition. Based on this, the type of resistant starch used here cannot be recommended as a prebiotic in dogs. </p

Investigating the impact of database choice on the accuracy of metagenomic read classification for the rumen microbiome

The Roslin Institute forms part of the Royal (Dick) School of Veterinary Studies, University of Edinburgh. This project was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/S006680/1, BB/R015023/1), including institute strategic program grant BBS/E/D/30002276. R.H.S. is supported by an EASTBIO studentship funded by BBSRC (BB/M010996/1). A.W.W. and the Rowett Institute receive core financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (SG-RESAS). We would like to thank all of those who were involved in creating and publicly sharing both the Hungate Collection data and the RUG data.Peer reviewedPostprin

Effect of cecal microbiota transplantation between different broiler breeds on the chick flora in the first week of life

The cecal microbiota plays numerous roles in chicken health and nutrition. Where such microbiota differs between lines exhibiting distinct phenotypes, microbiota transplantation offers scope to dissect the role of gut microbial communities in those traits. However, the composition and stability of transplants over time is relatively ill-defined and varying levels of success have been reported. In this study, we transplanted cecal contents from adult Roslin broilers into chicks from a different broiler line. Within 0.1% abundant (average) in the donor sample, 137 were detected in the treated group (75 were >0.1% abundant (average)) while only 88 were detected in the control group (29 were >0.1% abundant (average)). Our data therefore suggests that stable transplantation of the cecal microbiota between lines is achievable using the methods described in this paper

Age-related differences in the respiratory microbiota of chickens

In this era of next generation sequencing technologies it is now possible to characterise the chicken respiratory microbiota without the biases inherent to traditional culturing techniques. However, little research has been performed in this area. In this study we characterise and compare buccal, nasal and lung microbiota samples from chickens in three different age groups using 16S rRNA gene analysis. Buccal and nasal swabs were taken from birds aged 2 days (n = 5), 3 weeks (n = 5) and 30 months (n = 6). Bronchoalveolar lavage (BAL) samples were also collected alongside reagent only controls. DNA was extracted from these samples and the V2-V3 region of the 16S rRNA gene was amplified and sequenced. Quality control and OTU clustering were performed in mothur. Bacterial DNA was quantified using qPCR, amplifying the V3 region of the 16S rRNA gene. We found significant differences between the quantity and types of bacteria sampled at the three different respiratory sites. We also found significant differences in the composition, richness and diversity of the bacterial communities in buccal, nasal and BAL fluid samples between age groups. We identified several bacteria which had previously been isolated from the chicken respiratory tract in culture based studies, including lactobacilli and staphylococci. However, we also identified bacteria which have not previously been cultured from the respiratory tract of the healthy chicken. We conclude that our study can be used as a baseline that future chicken respiratory microbiota studies can build upon

Assembly of hundreds of novel bacterial genomes from the chicken caecum

BACKGROUND: Chickens are a highly important source of protein for a large proportion of the human population. The caecal microbiota plays a crucial role in chicken nutrition through the production of short-chain fatty acids, nitrogen recycling, and amino acid production. In this study, we sequence DNA from caecal content samples taken from 24 chickens belonging to either a fast or a slower growing breed consuming either a vegetable-only diet or a diet containing fish meal. RESULTS: We utilise 1.6 T of Illumina data to construct 469 draft metagenome-assembled bacterial genomes, including 460 novel strains, 283 novel species, and 42 novel genera. We compare our genomes to data from 9 European Union countries and show that these genomes are abundant within European chicken flocks. We also compare the abundance of our genomes, and the carbohydrate active enzymes they produce, between our chicken groups and demonstrate that there are both breed- and diet-specific microbiomes, as well as an overlapping core microbiome. CONCLUSIONS: This data will form the basis for future studies examining the composition and function of the chicken caecal microbiota