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Molecular Profiling of Bacterial Species in the Caecum of Geese

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Molecular Profiling of Bacterial Species in the Caecum of Geese Original Paper Czech J. Anim. Sci., 56, 2011 (4): 192–203 Molecular profiling of bacterial species in the caecum of geese B.Y. Liu, Z.Y. Wang, H.R. Wang, P. Hu, D. Xu, Q. Wang College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu Province, P.R. China ABSTRACT: The purpose of this study was to analyse the microbial diversity in the caecum of geese using a 16S ribosomal RNA gene (rRNA) clone library approach. A total of 160 clones and 124 clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a 98% similarity criterion were classified in the contents of goose caecum, as compared to 29 OTUs based on a 97% similarity criterion in the mucosa of goose caecum. The sequences were assigned to 7 and 5 groups in the contents and mucosa of goose caecum, respectively. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum. Keywords: geese; caecum; microbiota; phylogenetic analysis Unlike other avian species, the goose is a kind is considered to be beneficial not only to their nu- of waterfowl with the relatively developed paired trition, but also to the health of animals. Reports caecum, which can take advantage of fibrous on the microbial ecology in the caecum of geese plant materials partly. And birds can digest fibre are scarce. The dominant bacteria in the caecum only through fermentation, mainly in the caecum of geese, detected from Denaturing Gradient Gel (McNab, 1973). The caecum is also known as the Electrophoresis (DGGE) fingerprints, as reported site for fibre digestion, as reported by Yang et by Wang et al. (2009), were related to Pseudomonas al. (2009), the metabolic rates of NDF, ADF and sp. and Bifidobacterium sp. However, the complete hemicellulose were decreased significantly after the description of microbiota in the caecal or mucosal caecum was removed from geese fed basal diet. contents of geese is missing. Moreover, the microbiota in the caecum is known The earlier identification methods rarely allowed to actively ferment carbohydrates that have escaped definitive determinations of bacterial culture, spe- digestion in the upper part of the gastrointestinal cies and they did not often allow the identification (GI) tract. However, the microbiota in the GI tracts even on the genus level. The diversity and com- of non-ruminant species is a diverse population of plexity of the community structure of caecal bac- organisms composed primarily of bacteria (Mackie teria were much higher than it had been reported et al., 1999). Bacterial populations may also be clas- previously by culture-based studies (Gong et al., sified into contents and mucosal populations, and 2007). Since culture-based studies can provide only the mucosal microbiota may further be divided a limited picture of natural microbial communi- into epithelial or cryptal (Ewing and Cole, 1994). ties, it is necessary to rely on alternate methods Although the microbiota in the GI tracts of animals like the sequence analysis of 16S rRNA gene clone Supported by Technology Pillar Program Project of Jiangsu Province, P.R. China (Project No. BE2009351) and by the modern technology system of the waterfowl industry of China. 192 Czech J. Anim. Sci., 56, 2011 (4): 192–203 Original Paper libraries. So far, there have been few reports on the tal conditions (temperature: 26.0 ± 3.0°C, relative molecular diversity of microbiota in the caecum of humidity: 65.5 ± 5.0%) from 5–10 W. Geese had geese. However, the analysis of the PCR-derived free access to diets and water. Geese were fed the 16S rDNA clone libraries has shown that micro- mash diet (Table 1). bial communities are highly diverse and complex At 10 weeks of age, ten geese were selected ran- in ruminants (Whitford et al., 1998; Tajima et al., domly and killed by cutting the carotid arteries. 1999) and in the GI tracts of other animals, includ- The caecum was removed aseptically, clamped with ing pigs (Leser et al., 2002), chickens (Gong et al., forceps, and placed into sterile plastic bags on ice. 2007) and turkeys (Scupham, 2007). After the caecum was opened longitudinally, cae- In order to reveal the fibre digestion and health cal contents were immediately sampled and stored of geese, it is essential to analyse the complex mi- at –70°C (Apajalahti et al., 1998). Mucosa samples crobial communities in the caecum of geese. The were collected after digesta had been removed by purpose of the present study was to provide a de- washing with saline containing 0.1% Tween 80. The scription of the microbial community composition mucous layer attached to the caecal wall was gently in caecal contents and mucosa of geese using the scraped off with a small sterile spatula (Zhu et al., 16S rRNA gene sequence analysis. 2002). All contents or mucosa samples from the ten geese were mixed separately, frozen in liquid nitrogen and stored at –70°C. MATERIAL AND METHODS Animals and sampling DNA extraction All procedures were approved by the Institutional Genomic DNA was isolated from frozen samples Animal Care and Use Committee of Yangzhou using a QIAamp DNA Stool Mini Kit (QIAGEN) University. Yangzhou goose is a medium-sized following the manufacturer’s instructions. DNA goose species in China, with characteristics of extracts were stored at –70°C. stable genetic performance, high reproduction rate, rapid early growth, good meat quality, strong tolerance and adaptability to coarse feed and so PCR procedures on. Ten Yangzhou geese were raised in concrete pens with straw litter (2–3 cm thickness). The birds 16S rRNA genes were amplified by PCR from the were reared in the indoor house with environmen- genomic DNA samples of contents-associated and Table 1. Ingredient and nutrient composition of the experimental diets Ingredient Ingredient content (%) Nutrient composition Nutrient level Corn 61.18 AME (MJ/kg) 11.16 Soybean meal 16.22 crude protein (%) 16.53 Fish meal 4 crude fiber (%) 6.75 Alfalfa meal 5 calcium (%) 0.90 Cellulose 4.5 available phosphorus (%) 0.42 Soybean oil 1.7 Dicalcium phosphate 1.05 Limestone 1.00 Salt 0.35 Vitamin and trace mineral1 5.0 1 Supplied per kilogram of total diet: vitamin A 20 000 IU; vitamin D3 4500 IU; vitamin E 300 IU; vitamin K3 20 mg; vitamin B1 10 mg; vitamin B2 120 mg; vitamin B6 20 mg; vitamin B12 0.2 mg; nicotinic acid 600 mg; pantothenic acid 180 mg; folic acid 10 mg; folate 10 mg; biotin 0.8 mg; choline, 7 g; Fe 1.2 g; Cu 0.2 g; Mn 1.9 g; Zn 1.8 g; I 10 mg, Se 6 mg 193 Original Paper Czech J. Anim. Sci., 56, 2011 (4): 192–203 mucosa-associated bacteria using bacterial prim- RESULTS ers F8 (5'-AGAGTTTGATCCTGGCTCAG-3') and R1492 (5'-GGTTACCTTGTTACGACTT-3') (Eden Bacteria associated with caecum contents et al., 1991). The complete gene of 16S rRNA from bacteria was obtained (about 1500 bp). 160 cloned sequences were distributed in 46 dis- Thermocycling reactions contained 1000nM of tinct OTUs at the 2% difference level by DOTUR each primer, 2 µg of purified template DNA, 5 µl of to define an OUT. The presumptive relationships 10 × Ex Taq reaction buffer (Mg2+ free), 200mM of of these sequences were obtained from a database dNTP, 75µM of MgCl2 and 1.25 U of Ex Taq DNA- comparison. According to assigning to the closest polymerase (TaKaRa, Dalian, China), per 50-µl genus, as shown in Table 2, the highest similarity reaction. Reaction parameters included 4-min ini- of cloned sequences was 100%. However, the lowest tial denaturation at 94°C. Cycling consisted of 50 s was 90%. The BLAST data indicated that among of 94°C denaturation, 50 s of 56°C annealing and the 46 OTUs, 25 OTUs did not correspond to any 2 min of 72°C elongation. Reactions were finished recorded entries in the NCBI database. These se- with 10-min elongation at 72°C. Genes were ampli- quences can be considered as novel sequences with fied from caecal DNAs using the fewest number of an identity of < 97% with the sequences of the da- cycles possible to generate a visible product, gener- tabase. The other 21 sequences had 97% or higher ally 15 cycles. identity with an already characterized sequence. Ten clones had a high identity (99%) with the cul- tured species, Bacteroides coprocola. Four clones Cloning of the PCR amplified products also had a high identity (97%) with Clostridiales and sequence analysis lactatifermentans. Except for T. sanguinis, all the sequences related to ours with a high similarity and PCR products were purified using a PCR prod- had digestive origins from different areas of the uct purification kit (Invitrogen, Beijing, China) gastrointestinal tract of ruminant or monogastric and subcloned [pGEM-T-Easy] (Promega), using animals. In the contents of caecum, Clostridia were a Topo TA cloning kit (Invitrogen). Cloned ampli- the most abundant (94 of 160 clones), representing cons were sequenced using vector-specific primers 58.7% of the clones. Bacteroidetes were the second and an ABI PRISM 377 sequencer (Perkin-Elmer) group (representing 26.9% of the clones) followed in Invitrogen company (Invitrogen, China). by Erysipelotrichi (11.2%). There were 80 cloned Our sequences were analysed by the CHECK- sequences (50%) with less than 97% of relatedness CHIMERA programme to remove chimeric rDNA to database sequences and which may thus repre- clones.
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