Faecal Microbiota of Forage-Fed Horses in New Zealand and the Population Dynamics of Microbial Communities following Dietary Change Karlette A. Fernandes 1 *, Sandra Kittelmann 2 , Christopher W. Rogers 1 , Erica K. Gee 1 , Charlotte F. Bolwell 1 , Emma N. Bermingham 2 , David G. Thomas 1 1 Institute of Veterinary, Animal, and Biomedical Sciences, College of Sciences, Massey University, Palmerston North 4442, New Zealand, 2 AgResearch Ltd, Grasslands Research Centre, Palmerston North 4442, New Zealand Abstract The effects of abrupt dietary transition on the faecal microbiota of forage-fed horses over a 3-week period were investigated. Yearling Thoroughbred fillies reared as a cohort were exclusively fed on either an ensiled conserved forage- grain diet (‘‘Group A’’; n = 6) or pasture (‘‘Group B’’; n = 6) for three weeks prior to the study. After the Day 0 faecal samples were collected, horses of Group A were abruptly transitioned to pasture. Both groups continued to graze similar pasture for three weeks, with faecal samples collected at 4-day intervals. DNA was isolated from the faeces and microbial 16S and 18S rRNA gene amplicons were generated and analysed by pyrosequencing. The faecal bacterial communities of both groups of horses were highly diverse (Simpson’s index of diversity .0.8), with differences between the two groups on Day 0 (P,0.017 adjusted for multiple comparisons). There were differences between Groups A and B in the relative abundances of four genera, BF311 (family Bacteroidaceae; P = 0.003), CF231 (family Paraprevotellaceae; P = 0.004), and currently unclassified members within the order Clostridiales (P = 0.003) and within the family Lachnospiraceae (P = 0.006). The bacterial community of Group A horses became similar to Group B within four days of feeding on pasture, whereas the structure of the archaeal community remained constant pre- and post-dietary change. The community structure of the faecal microbiota (bacteria, archaea and ciliate protozoa) of pasture-fed horses was also identified. The initial differences observed appeared to be linked to recent dietary history, with the bacterial community of the forage-fed horses responding rapidly to abrupt dietary change. Citation: Fernandes KA, Kittelmann S, Rogers CW, Gee EK, Bolwell CF, et al. (2014) Faecal Microbiota of Forage-Fed Horses in New Zealand and the Population Dynamics of Microbial Communities following Dietary Change. PLoS ONE 9(11): e112846. doi:10.1371/journal.pone.0112846 Editor: Robert J. Forster, Agriculture and Agri-Food Canada, Canada Received January 29, 2014; Accepted October 17, 2014; Published November 10, 2014 Copyright: ß 2014 Fernandes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Massey University Internal Research Funding (DGT) and AgResearch CORE (A17976) funds (ENB). An educational fellowship from the Ministry of Business Innovation and Employment: Science and Innovation Group (www.msi.govt.nz) and a Massey University Doctoral Scholarship (www.massey.ac.nz) provided financial assistance for the duration of the doctoral study (KAF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: k.a.fernandes@massey.ac.nz Introduction The horse is a cursorial grazer with the ability to efficiently utilise high-fibre grass and other forages [1,2]. The majority of ingested plant fibre is comprised of structural carbohydrates such as cellulose, hemicellulose, and lignin, which cannot be digested by host enzymes in the foregut. As a result, the undigested plant material reaches the hindgut where breakdown of cellulose and hemicellulose occurs through the process of microbial fermenta- tion, generating energy-yielding products such as volatile fatty acids (VFAs) [3,4]. It is estimated that forage-fed horses may obtain 50–70% of their energy requirements from VFAs [5]. Several species of microbes including bacteria, archaea and eukarya (protozoa and fungi) inhabit the equine gastrointestinal tract [6,7]. However, the bacterial community, which represents the major proportion of the hindgut microbiota, has been the focus of much of the published literature and has predominantly been investigated using culture-based techniques [8,9,10]. Other molecular techniques independent of culture, such as polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), fluorescent in-situ hybridization (FISH) and terminal restriction fragment length polymorphism (TRFLP), have also been used [8,11,12]. More recently, a few investigations have used metagenomics involving high-throughput next generation se- quencing (pyrosequencing of 16S and 18S rRNA gene amplicons) to explore the equine hindgut microbiome (eHGM) in vivo [13,14,15]. However, there are still gaps in the current knowledge on the composition and the function of the microbial communities that inhabit the equine hindgut. This may be due to the difficulty in culturing anaerobic microorganisms and comparing results from studies that have used different experimental designs, sequencing methodologies and phylogenetic and statistical data analyses [8]. Because of the need to fistulate the horse to obtain hindgut samples, the majority of the work on the eHGM has focused on the use of material obtained from faecal samples [8]. From the limited comparative work conducted on the bacterial community of the equine hindgut and faeces, there appears to be good agreement between the microbiomes of the colon and faeces [15,16]. PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e112846