Life inside tigers: exploring the tiger gut microbiome

Methods

Summary

The tiger's microbiota has not previously been studied under controlled conditions, and therefore our project sets out to characterise the tiger's gut microbiome. To achieve this aim we have recruited nine tigers held in an AZA zoo, which will be studied using non-invasive techniques.

After collecting fresh fecal samples from tigers , we will preserve them for analysis whilst we transport them to the laboratory. Once at the laboratory, the microbial DNA will be extracted and microbes will be identified (diversity and composition) using samples of captive tiger feces.). Microbial DNA will be extracted from feces and used to amplify 16S rRNA genes. Gene sequences will be obtained using a high throughput Illumina Miseq platform that will allow us to identify common and rare members of the tiger gut microbiomes. Sequence data will be analyzed using the mothur bioinformatics pipeline followed by a battery of statistical analyses that will enable comparisons among tigers, and potentially reveal impacts of dietary changes.

This project forms part of a much larger investigation which integrates behavior, welfare science, nutritional physiology, and veterinary health. By taking this multi-disciplinary approach we will be exploring ways to improve the captive diet of tigers (and potentially other endangered felids as well). As part of the larger study (funded separately) we will also capture data on tiger behavior, as well as gastrointestinal function and health. Using a cross-over study design, each tiger will be studied for two distinct feeding periods - one where they are fed their basal diet of ground meat, and a second period in which fiber is added to their basal diet.

Therefore, whilst this project's principal aim is to characterise the tiger's microbiota under controlled conditions, we will also be able to evaluate our microbiological findings against the measured health parameters on two different diets. This will assist us in identifying ways in which to optimise tiger health and welfare in captivity. Results will be made available to the public through websites and peer-reviewed publications.

Challenges

Since we’re working with tigers, even though our sampling regime is non-invasive (meaning we don’t have to actually touch the tigers in order to obtain the samples), we still need to ensure the samples can be safely collected from the tiger’s habitat in the zoo. Therefore, we will have to wait until the tigers are secured in another part of their enclosure before we can collect the faecal material. In order to preserve the microbes we will need to collect this faecal material as soon after it was voided as possible, so we’ll be working closely with an expert team of tiger keepers to ensure the sampling protocol is a success. These keepers know the tigers very well, e.g.their personalities and daily routines, including their toileting habits, so we have a good idea of when to expect a fresh sample to become available. The keeping team are also experts in managing the tigers’ activities so they can request the tigers move to certain secured areas of the enclosure during the collection times, keeping everyone safe and comfortable during the study.

Once we’ve got the sample, it’s possible that the differences between individuals will be great (as is seen in other species, including humans). This could mean that defining a “typical” tiger gut microbiome is challenging. However, since currently nothing is known about the tiger’s gut microbiota – any finding that we can determine is a valuable finding in itself. If inter-individual variation is high, then we can start to explore possible reasons for this in future studies. Likewise, since we are planning to collect samples from tigers before and after a dietary change, then we may be able to pin down the influence of this dietary change within an individual. This will also be highly valuable information which can help us determine how diet influences the tiger’s gut microbiota, which in turn will have an influence on the tiger’s overall gut health. On the other hand, we may alternatively see very few impacts of dietary changes on gut microbes. Again, this would be a noteworthy finding given that the majority of literature in other species indicates that diet is a major influencing factor – so our next challenge would be to find out why the tiger’s gut microbiota is different to these other species! Lastly, microbial composition and biodiversity of tigers in a closely managed environment such as a zoo, may differ considerably from that found in free-ranging animals. This study represents an initial step at quantifying some of these differences which we ultimately hope to contrast with samples from free-ranging tigers in nature, in order to better understand how a captive environment may influence the tiger gut microbiome.

Pre Analysis Plan

Sequences from all fecal samples will be processed to identify all operational taxonomic units (OTUs) to the lowest possible level (typically genus). Compositions of individual animals will be summarized at genus, family and phylum/class levels to identify major and minor members of communities in individual tigers. For each sample, a set of alpha diversity measures will be generated to include the Shannon index, the inverse Simpson index, ACE and Chao richness estimates and coverage. Rarefaction plots will also be generated to assess adequacy of the sampling effort. Differences in measures of alpha diversity among individuals within treatment groups and between treatment groups (i.e., fiber supplement) will be assessed using packages in Xlstat or R. These analyses will also include animal age and gender as potential factors that might explain observed variability.

Beta diversity will be measured with dissimilarity indices such as the Jaccard Index and the Yue/Clayton measure of dissimilarity. Dissimilarity indices can be represented in tree form, with parsimony and unifrac tests for significance of differences among treatment groups. We will also use several multivariate approaches including principal coordinates analysis with unifrac distances, principal component and non-metric multidimensional scaling analysis for OTU incidence, and MANTEL and ANOSIM analyses to test for significance of differences among groups, treatments and relevant variables (age, gender, etc.). These analyses will be performed for the datasets as a whole, and for specific phyla, families or genera as dictated by the results.

The core hypothesis that will be tested is that dietary fiber additions to tiger diets have no impact on gut microbiome composition. Given the paucity of data on tigers, it is not possible to predict a priori whether the results will lead to a definitive outcome. However, at a minimum, the results will provide a basis for designing more extensive studies that include larger numbers of tigers, samples and dietary treatments. If distinct responses to fiber addition are observed, it will also be possible to develop further targeted studies to better understand relationships between microbiome composition and function in tigers.