The MAZE.ROE research project aims at characterizing the gut microbiota and the diet composition of roe deer (Capreolus capreolus) in the Italian Alps, by applying a metataxonomic approach coupled with high-throughput sequencing (Illumina). A total amount of 384 fecal samples, collected from roe deers in the Trento province (years 2013-2014) and the Valle d'Aosta region (2013), were selected for the study. Preliminary results obtained during a pilot study on a limited number of fecal samples deriving from 3 roe deers individuals confirmed the feasibility of the whole project, which is currently in progress

Ballardini, M.

Cagnacci, F..2.

Crestanello, B.

Girardi, M.

Guidetti, C.

Hauffe, H.

Orusa, R.

Peters, W.

Pindo, M.

Rizzoli, A.

Robetto, S.

English

Cagnacci, Francesca

Ballardini, Marco

Peters, Wibke Erika Brigitta

Crestanello, Barbara

Girardi, Matteo

Rizzoli, Annapaola

Hauffe, Heidi Christine

Pindo, Massimo

Archivio istituzionale della ricerca - Fondazione Edmund Mach

“MAZE.ROE” PROJECT: STUDY OF GUT MICROBIOTA AND DIET COMPOSITION OF ROE DEER (CAPREOLUS CAPREOLUS) IN THE ITALIAN ALPSORUSA R1*, BALLARDINI M1, PETERS W2,3, CRESTANELLO B2, GIRARDI M2, ROBETTO S1, GUIDETTI C1, RIZZOLI A2, HAUFFE H2, PINDO M2, CAGNACCI F21 Centro Nazionale di Referenza per le Malattie degli Animali Selvatici (CERMAS), Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Quart (AO), Italy2 Dipartimento Biodiversità ed Ecologia Molecolare (DBEM), Fondazione Edmund Mach, San Michele all'Adige (TN), Italy3 Ungulate Ecology Research Lab, University of Montana, USACorresponding and speaker on Symposium: * riccardo.orusa@izsto.it (​mailto:riccardo.orusa@izsto.it​) ABSTRACT: The MAZE.ROE research project aims at characterizing the gut microbiota and the diet composition of roe deer (Capreolus capreolus) in the Italian Alps, by applying a metataxonomic approach coupled with high-throughput sequencing (Illumina). A total amount of 384 fecal samples, collected from roe deers in the Trento province (years 2013-2014) and the Valle d'Aosta region (2013), were selected for the study. Preliminary results obtained during a pilot study on a limited number of fecal samples deriving from 3 roe deers individuals confirmed the feasibility of the whole project, which is currently in progress.INTRODUCTION: Roe deer (Capreolus capreolus) populations in Italy have been expanding during the last 30 years (Carnevali et al. 2009). This herbivorous species is involved in the transmission cycles of several infectious and parasitic diseases, including zoonoses (Carpi et al. 2009, Solarczyk et al. 2012); nevertheless, the role of roe deer as a reservoir of intestinals pathogens has not been investigated until now. Concurrently, both the ecological impact on vegetation and the diet composition -considered as a proxy of its health status- of C. capreolus remain largely unknown. The MAZE.ROE project therefore intends to study the gut bacterial community (microbiota) and the diet composition of roe deer in the Italian Alps, by applying a metataxonomic approach coupled with high-throughput sequencing (HTS). Herein, the genereal outline of the project and the results of a pilot study performed on a limited number of samples are reported.MATERIALS AND METHODS: Rectal fecal samples, ear biopsies and hair samples were collected from 19 roe deer individuals captured and radio-collared in the Trento province, NE Italy, in 2013-2014, and preserved at -80°C. After radiotracking of roe deers, a total amount of 341 fresh fecal samples found on the ground were putatively attributed to the marked indiduals according to their location and collected as follows: 2-3 sampling sessions per 3 sampling seasons (early summer, mid summer, late summer) of each year. Ground-collected samples were splitted into 2 aliquotes each and preserved in silica gel and RNAlater, respectively, and then freezed at -20°C. Sixty-six fecal swabs were also collected from hunted roe deers in Valle d'Aosta Region, NW Italy, in 2013, and preserved at -80°C, as an outgroup for the gut microbiota characterization. A total amount of 384 fecal samples (ground samples plus fecal swabs), including some replicates and negative controls, were finally selected for the study.For the metataxonomic phase, the V3-V4 hypervariable regions of the bacterial 16S rRNA gene (Schwartz et al. 2014) and the P6 loop of the trnL(UAA ) intron of cpDNA (Taberlet et al. 2006) were selected as barcodes for bacteria and vascular plants, respectively. As genus- and species-level indentification based on the P6 loop is problematic for some plant families, ITS1 for Asteraceae, Cyperaceae and Poaceae, and ITS2 for Rosaceae were chosen as additional barcodes (De Barba et al. 2014). A reference database of plant species barcode sequences, based on a checklist of local plant species prepared during the field phase of the study, was obtained through a search on GenBank public database. Given their lower cost and the high number of reads provided, Illumina MySeq for microbiota and Illumina HiSeq 2000 for plants were chosen as HTS platforms. To obtain individual roe deer identification for individual life-history analyses and verify whether ground-collected fecal samples belonged to the putative individuals, a panel of 24 nuclear dinucleotidic microsatellite markers and the amelogenin (AMEL X,Y) gene (Gurgul et al. 2010) were tested on genomic DNA extracted from ear biopsies/hair samples and rectal fecal samples. Genotyping data were analysed using GeneMapper software v3.7 (Life Technologies, USA).To evaluate the feasibility of the whole study, a pilot study on a limited number of samples, belonging to 2 roe deer individuals (1 adult male and 1 adult female) from Trento province was performed; an adult female from Valle d'Aosta was also included in a few analyses. Three commercial kits (Thermo Scientific KingFisher Cell and tissue Kit, QIAmp DNA stool Minikit from QIAgen and Mag-Bind Stool DNA kit from Omega bio-tek) and protocol modifications for genomic DNA extraction were tested on both silica gel- and RNA-later preserved fecal samples, to simultaneously recover host, bacterial and plant DNA. Extracted DNA was quantified on a spectrophotometer. Amplifications of roe deer mtDNA D-loop control region and of  bacterial and vascular plant barcode regions were performed on thermocyclers. PCR products were separated and visualised using QIAxcel (QIAgen, Germany). Whereas fragments of the expected size were obtained, amplicons were purified and sequenced on an ABI 3130XL genetic analyzer (Life Technologies, USA). In the case of microbiota and plants, several overlapping sequences were expected. Sequences obtained were maually checked using Sequencher software (Gene Codes Corporation, USA). In the case of any clear sequence fragment, a BLAST search on GenBank was performed for taxonomic identification.RESULTS AND DISCUSSION: For what concerns genotyping for individual identification, 2 STR loci out of 24 did not amplify and were discarded, while 4 more loci revealed to be monomorphic. The remaining 18 loci turned out to be polymorphic (3-10 alleles); data analyses is currently in progress. During the pilot study, all DNA extraction kits and protocols allowed to simultaneously obtain host, bacterial and plant DNA, however the Mag-Bind stool DNA kit (Omega bio-tek, USA) gave more satisfactory results in terms of DNA yeld, costs and the possibility to be coupled with an automated extractor. As RNAlater-preserved samples performed much better than silica gel-preserved ones, especially in the case of the diet component, the former were chosen for the whole study. Amplification of the host control region provided the expected fragment; one of the haplotypes obtained was highly similar to the one of a C. capreolus individual previously found in the Trentino province (Vernesi et al. 2002). Amplification of bacterial and plant barcode sequences was also successful and several overlapping sequences were obtained from most samples. On the microbiota side, the BLAST search on GenBank allowed to identify Pseudomonas sp. in a few samples from Trentino. Interestingly, the sample from Valle d'Aosta was characterised by a unique pattern of overlapping bacterial sequences. For what concerns diet composition, a few sequence fragments deriving from Trentino samples revealed the presence of Fragaria sp., Vaccinium myrtillus, Rubus chamaemorus, Rubus sp.CONCLUSION: The pilot study demonstrated the feasibility of the MAZE.ROE project. Analyses are currently in progress and the HTS phase will be soon performed.REFERENCES Carnevali L., Pedrotti L. et al. Banca Dati Ungulati: Status, distribuzione, consistenza, gestione e prelievo venatorio delle popolazioni di Ungulati in Italia. Rapporto 2001-2005. Biol. Cons. Fauna 2009; 117:1-168.Carpi G., Bertolotti L. et al. Anaplasma phagocytophilum groEL Gene Heterogeneity in Ixodes ricinus Larvae Feeding on Roe Deer in Northeastern Italy. Vector Borne Zoonotic Diseases. 2009; 9(2):179-84. doi: 10.1089/vbz.2008.0068.De Barba M., Miquel C. et al. DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet. Molecular Ecology Resources 2014; 14:306-323Gurgul A., Radko A., Slota E. Characteristics of X- and Y-chromosome specific regions of the amelogenin gene and a PCR-based method for sex identification in red deer (Cervus elaphus). Molecular Biology and  Reproduction 2010; 37:2915-2918.Schwartz J.D., Lachman M. et al. Characterization of the vaginal microbiota of ewes and cows reveals a unique microbiota with low levels of lactobacilli and near-neutral pH. Frontiers in Veterinary Science 2014; 1. doi: 10.3389/fvets.2014.00019.Solarczyk P., Majewska A.C. et al. Multilocus genotyping of Giardia duodenalis isolates from red deer (Cervus elaphus) and roe deer (Capreolus capreolus) from Poland. Folia Parasitologica 2012; 59(3):237-240.Taberlet P., Coissac E. et al. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleid Acid Research 2006; 35(3):e14. doi:10.1093/nar/gkl938.Vernesi C., Pecchioli E. et al. The genetic structure of natural and reintroduced roe deer (Capreolus capreolus) populations in the Alps and central Italy, with reference to the mitochondrial DNA phylogeography of Europe. Molecular Ecology 2002; 11:1285-1297.ACKNOWLEDGEMENTS: The MAZE.ROE project has been funded by the Italian Ministry of Health. We wish to thank all the people who participated in the field work and Dr. Cristiano Vernesi, head of the Conservation Genetics group at DBEM.

“MAZE.ROE” project: study of gut microbiota and diet composition of roe deer (Capreolus capreolus) in the Italian Alps

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“MAZE.ROE” project: study of gut microbiota and diet composition of roe deer (Capreolus capreolus) in the Italian Alps

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