Molecular characterization of the faecal fungal flora in healthy horses

Abstract: There is a lack of knowledge on the composition and dynamics of the intestinal micro-eukaryotes of horse and to date their functional role for the health and physiology of the host has been described merely to some extent. Particularly, no culture-independent surveys of the dynamics of the intestinal fungal flora in horse are available. Two culture-independent approaches, terminal restriction fragment length polymorphism (T-RFLP) and cloning & sequencing, were combined to study the composition and dynamics of the intestinal fungal flora in healthy horses. The fungal internal transcribed regions (ITS) and the eukaryotic 18S rRNA genes, recovered from faecal samples of 16 Swedish Standardbred geldings, were used as phylogenetic marker genes in the analyses. Faecal samples were analyzed by TRFLP and two clone libraries were constructed, one library for the 18S gene and the other for the ITS gene from which later 44 random clones were sequenced. T-RFLP profiles and sequencing data together, indicate that the fungal community is temporally stable, has a low diversity and it is dominated by three genera of the Neocallimastigaceae family including Orpinomyces sp., Anaeromyces sp. and Piromyces species. These fungal species have been isolated and characterized previously in different types of herbivores including the Equidae family. These anaerobic fungi have been suggested to exist prevalently in the GI tract of large herbivores which in this aspect, there is no disparity between the data presented in this experiment and those from previous publications. T-RFLP, as a high-throughput culture-independent method which so far has been implemented mainly on the studies targeting the prokaryotic members of the microbiota, is suitable for the survey of the faecal fungal flora of horses, however requires further adaptations.