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The Tibetan swine (TIS) is a non-ruminant herbivore with high disease resistance. Also, it has the ability to digest plants with high fiber content. However, it is not known whether any relationship exist between these characteristics of the TIS and its cecal microbiota. Thus, this study aims to investigate the cecal microbiota of the adult TIS using high-throughput sequencing techniques in order to explore possible relationships between these unique characteristics of the TIS (high disease resistance and ability to digest high fiber plants) and its cecal microbiota. PIC pigs (lean type) were chosen as controls. The results show that 75,069 valid sequences of the 16S rRNA gene at V4-V5 region were obtained in the cecal content of TIS. They were composed of 15 phyla, 70 genera and divided into 660 Operational Taxonomic Units (OTUs). Bacteroidetes and Firmicutes were the predominant phyla in both breeds, but TIS had more Bacteroidetes than Firmicutes. Also, 42.4% of the cecal bacteria were found to be unclassified and uncultured. Many cellulolytic bacteria were also found in the two breeds. TIS (88.10%) had much higher abundance in the core bacterial communities than PIC pigs (81.29%), and the proportion of Bacteroides and Spirochaetes that can effectively degrade cellulose were 6.01 and 6.40% higher than PIC pigs, respectively, while Proteobacteria that are closely related to gastrointestinal diseases were 1.61% lower than PIC pigs. Thus, the disease resistance of the TIS and its ability to digest plants with high fiber content may be related to high abundance of core bacterial communities as well as the large number of unknown and unclassified bacteria. . . . . . Keywords Cecal microbiota 16S rRNA gene High-throughput sequencing Tibetan swine Disease resistance Herbivorous characteristics Highlights • Comparing the cecal microbiota of a Tibetan and PIC (lean- type) pig. � The core bacterial communities were different in these two breeds. � Tibetan swine had a higher proportion of digestion related bacteria phyla. Weiping Yang and Haiyun Xin are joint first authors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1329-z) contains supplementary material, which is available to authorized users. * Binyun Cao Institute of Animal Science, Guangdong Academy of Agricultural email@example.com Sciences, No.1 Da feng Road, Tianhe District, Guangzhou, Guangdong 510640, People’s Republic of China College of Animal Science and Technology, Northwest A&F University, No.22 Xinong Road, Yangling 712100, Shaanxi, Animal Engineering Branch, Yangling Vocational & Technical People’s Republic of China College, No. 10 Xinong Road, Yangling, Shaanxi 712100, People’s Republic of China Life sciences college, Luoyang Normal University, No.6 Jiqing Road, Yibin District, Luoyang 471934, Henan, People’sRepublic of China 186 Ann Microbiol (2018) 68:185–194 Introduction farms. All the five pigs of each breed were reared under the same standard feeding and management conditions. The diet The microbial communities in the gastroenteric tracts of of the TIS consisted of 90% of green fodder and 10% of humans and animals greatly influence their digestion, metabo- soybeans and wheat bran, while the diet of PIC pigs consisted lism, and disease resistance (Turnbaugh et al. 2009). Firmicutes of compound feed. The pigs had no history of gut infectious and Bacteroidetes are the two dominant phyla in the gut bacte- disease, and no antimicrobial administration occurred during rial communities of many ruminant and non-ruminant animals. the feeding process. They were fed an antibiotic-free diet. The Changes in their composition and ratio greatly affect digestion, cecal content samples from the TIS and PIC pigs were collect- metabolism, and substance absorption of animals (Turnbaugh ed from the Tibetan swine Slaughterhouse of Shaanxi Huayi et al. 2009; Lietal. 2012;Looft etal. 2012). Moreover, changes Industrial Co., Ltd. and the Shaanxi Benxiang Pig in the proportion of Lactobacilli and Enterobacteria could be Slaughterhouse, respectively. After the pigs were killed with used as a gut health indicator for animals (Castillo et al. 2007). sodium pentobarbital (50 mg/1 kg BW), the cecum was ligat- The diversity and composition of the gut microbial communi- ed at both ends (~ 100 g/sample) and was immediately re- ties of animals are related to several factors, including animal moved from the peritoneal cavity, placed into aseptic ziplock species (Pajarillo et al. 2014), age (Castillo et al. 2007), envi- baggies, and stored in foamed plastic containers filled with dry ronment (Wu et al. 2012), diet type (Yan et al. 2013;Chen etal. ice. All samples were transported to the laboratory within 2014), dietary fiber content (Castillo et al. 2007;Liu et al. 2012; 30 min for microbial genomic DNA extraction. The DNA Zhang et al. 2016), as well as antibiotics content in the diet was kept frozen at − 80 °C until it was needed. (Looftetal. 2012, 2014). The experimental design and procedures were approved by The Tibetan swine (TIS) is a Qinghai (China) native, the Animal Care and Use Committee of Northwest A&F plateau-dwelling herbivore. Ninety percent of its nutrients University. The cecal samples were collected with the permis- can be obtained from forage grasses. The TIS has a large sion of Hongzhou Wang and Junfang Yan, the director of intestine like other non-ruminant monogastric animals, and Tibetan Pig Slaughterhouse of Shaanxi Huayi Industrial Co., its structure and function are at an intermediate stage between Ltd. and Shaanxi Benxiang Pig Slaughterhouse, respectively. those of carnivorous and herbivorous animals. Most undigest- The study did not involve endangered or protected species. ed feed components and endogenous secretions are fermented by microorganisms in the large intestine to provide the neces- DNA extraction and PCR amplification sary nutrients for the animal (Wenk 2001). 11 12 About 10 –10 microbial cells live in each gram of cecal After mixing the cecal contents of each pig, the genomic DNA content of pigs, comprising of 400 to 500 different types was extracted from 200 mg of samples using the E.Z.N.A.® (Castillo et al. 2007). However, more than 80% of the bacterial Stool DNA Kit (OMEGA, USA) according to the manufac- species are not yet identified (Leser et al. 2002). At present, turer’s protocols and the concentration was measured using a studies on TIS microbiota mainly focus on the isolation and NanoDrop Spectrophotometer ND1000 (Thermo Scientific, identification of bacteria that can degrade cellulose (Meng USA). The V4-V5 region of the bacterial 16S rRNA gene et al. 2014;Yanget al. 2014;Ma et al. 2015) or secrete anti- was amplified by PCR using primers 515F 5′-barcode- bacterial peptide (Xin et al. 2017). Xiao et al. (2017)found GTGCCAGCMGCCGCGG)-3′ and 907R 5′-CCGT that the immunologic characteristics can be transferred by gut CAATTCMTTTRAGTTT-3′ (Sun et al. 2013; Pitta et al. microbiota, suggesting the vital role of microbiota in immune 2014). The PCR procedure was as follows: initial denaturation phenotype programming, and making us suppose the relation- at 95 °C for 2 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, ship of host microbiota with disease resistance and other char- and 72 °C for 30 s, and a final extension at 72 °C for 5 min acteristics. Therefore, we investigated the diversity and com- where the barcode is an eight-base sequence unique to each position of the bacterial community in the cecum of the TIS sample. PCR reactions were performed in triplicate involving using Miseq high-throughput sequencing analysis in order to 20 μL mixtures containing 4 μL of 5× FastPfu buffer, 2 μLof explore possible relationships between the bacterial commu- 2.5 mM dNTPs, 0.8 μLof each primer (5 μM), 0.4 μLof nity of the TIS and its unique characteristics. FastPfupolymerase, and 10ngoftemplateDNA. Illumina MiSeq sequencing Materials and methods Amplicons were extracted from 2% agarose gels and purified Collection of cecal content samples using the AxyPrep DNA Gel Extraction Kit (TaKaRa) accord- ing to the manufacturer’s instructions. They were quantified Five healthy male adult TIS (8 months) and five healthy male using QuantiFluor™ -ST (Promega, USA). The purified adult PIC (5 months) pigs were obtained from two different amplicons were pooled in equimolar concentrations and Ann Microbiol (2018) 68:185–194 187 paired-end sequenced (2 × 250) on an Illumina MiSeq plat- chimeric sequences were removed using UCHIME; 75,069 form according to the standard protocol. and 74,759 sequences were left for further analysis of the TIS and PIC pigs, respectively. Further analysis identified Processing and analysis of sequencing data 660 and 668 OTUs from the TIS and PIC pigs, respectively. The total number of optimized reads, OTUs, statistical species Raw fastq files were demultiplexed and quality-filtered using richness, and diversity estimations for each sample are pre- QIIME (Caporaso et al. 2010) (version 1.17) in accordance sented in Table 1. with the following criteria: (i) 250 bp reads were truncated at OTUs and microbial diversity indices were used to con- any site receiving an average quality score of < 20 over a 10- struct rarefaction and Shannon curves to compare the micro- bp sliding window, and the truncated reads below 50 bp were bial community richness and diversity of the different samples discarded. (ii) The exact barcode matching, two nucleotide at different sequencing depths by means of the Mothur soft- mismatch in primer matching, and reads containing ambigu- ware. The rarefaction curves (Fig. 1) and the community rich- ous characters were removed. (iii) Only sequences with an ness indices (chao1 and ace) (Table 1) indicate that the com- overlap of more than 10 bp were assembled according to their munity richness of the TIS was higher than that of PIC pigs, overlap sequence. Reads that could not be assembled were slightly. However, the Shannon curves (Fig. 2) and indices of discarded. community diversity (Shannon and Simpson) (Table 1)indi- OTUs were clustered at 97% similarity cutoff using cate that the two breeds of pig were similar with respect to UPARSE (Edgar 2013)(version7.1, http://drive5.com/ their microbial community diversities. All the Shannon curves uparse/), and chimeric sequences were identified and tended to reach a plateau shape when the sequencing depth removed using UCHIME (Edgar et al. 2011) (version 4.2. was 10,000 reads for each sample (Fig. 2). Therefore, the 40, http://drive5.com/usearch/manual/uchime_algo.html). sequencing results could completely reflect the microbial The phylogenetic affiliation of each 16S rRNA gene community richness and diversity of the samples and can be sequence was analyzed by RDP Classifier (http://rdp.cme. used for the following analysis. msu.edu/) and compared with the Silva (Release115 http:// www.arb-silva.de) 16S rRNA database using a confidence Taxonomic composition of bacterial communities threshold of 70% (Wang et al. 2007) to determine the bacterial community composition of each sample at the different levels. All the sequences were classified by the RDP classifier. The OTUs at a 97% similarity level were used for alpha diversity cecal bacteria were divided into 15 different phyla in both (Shannon, Simpson), richness (ACE and Chao1), Venn dia- breeds. In the TIS, almost 98.42% of the bacteria accounted gram, rarefaction curve, and Shannon curve analyses using the for more than 1% of the total cecal bacterial sequences Mothur program (http://www.mothur.org). (Bacteroidetes, Firmicutes, Spirochaetae, and Tenericutes), while the corresponding proportion was 98.98% in the PIC Statistical analysis pigs (Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetae, Fusobacteria, Planctomycetes, and unclassi- According to the relative abundance values of the bacterial fied). Bacteroidetes and Firmicutes were the most abundant community, metastats (http://metastats.cbcb. umd.edu/) was bacteria in both breeds, but Bacteroidetes was more abundant used to assess the differences between the two groups at the than Firmicutes. However, the TIS had a higher Bacteroidetes different taxonomic levels. Alpha levels below 0.05 were abundance (59.79%) and a lower Firmicutes abundance considered significant. All data were shown as mean ± SD. (28.90%) compared with the PIC pigs (Fig. 3, S1 Table). Statistical significance test showed that Verrucomicrobia and Fusobacteria were the unique bacterial taxa for the TIS Results and PIC pigs, respectively; the TIS had a significantly higher abundance of Spirochaetae (p< 0.01) and lower Community richness and diversity Fibrobacteres as well as Proteobacteria (p< 0.01) than the PIC pigs. The other bacteria phyla did not show any signifi- The raw reads of Miseq high-throughput sequencing were cant difference between the two breeds (S1 Table). deposited into the NCBI Sequence Read Archive (SRA) da- All the cecal bacteria could be divided into 70 different tabase (accession numbers: SRP058661). genera in the TIS, among which 14 genera accounted for more Up to 169,766 valid sequences at V4-V5 region of 16S than 1% of the total cecal bacterial sequences and a total pro- rRNA gene were obtained from the 10 samples using portion of 92.32% of all the bacteria, which mainly included Illumina Miseq high-throughput sequencing analysis. The av- the uncultured bacteria (27.06%), unclassified bacteria erage length of these valid sequences was 393.8 bp. OTUs (15.30%), S24-7_norank (11.89%), RF16_norank (7.28%), were analyzed at a 97% similarity level using UPARSE; Prevotella (6.48%), Spirochaeta (5.31%), RC9_gut_group 188 Ann Microbiol (2018) 68:185–194 Table 1 Numbers, abundance, Sample name Optimize reads OTUs Chao1 ace Shannon index Simpson index and diversities of OTUs in the cecal microbiota of Tibetan pigs T1 10,425 418 515.68 485.34 4.66 0.02 and PIC pigs. The identity value used for all the analyses in this T2 19,043 543 600.75 590.53 4.89 0.02 study were 97% T3 12,026 440 542.10 503.08 4.44 0.03 T4 9342 462 528.94 535.11 4.68 0.02 T5 24,233 549 614.07 595.5 4.85 0.02 P1 22,923 423 498.00 473.35 4.19 0.04 P2 11,727 525 586.12 585.46 5.11 0.01 P3 11,048 412 473.39 464.8 4.80 0.02 P4 11,048 395 480.00 460.81 4.59 0.02 P5 18,013 583 686.06 659.85 5.16 0.01 Note: T1–T5 are the cecal samples from five Tibetan pigs; P1–P5 are the cecal samples from five PIC pigs, respectively (3.68%), Bacteroides (3.57%), Clostridiales of the incertae Among all the identified genera in the two breeds, 24 gen- sedis (2.26%), Parabacteroides (2.26%), Treponema era showed significant or extremely significant differences (2.25%), Phascolarctobacterium (1.86%), Anaerovibrio between the two breeds (p <0.05 or p <0.01), while all the (1.83%), and RF9_norank (1.29%). Meanwhile, in the PIC other genera displayed similar percentages. A number of cel- pigs, 74 genera were identified, among which up to 88.88% lulolytic bacteria were found in the cecum of both breeds, such of all the bacteria made up more than 1% of total cecal bacte- as Ruminococcus, Bacteroides, Prevotella, Clostridium, rial sequences, including the uncultured bacteria (28.67%), Butyricicoccus, Fibrobacter, Lachnospira, Anaerovibrio, Prevotella (20.78%), S24-7_norank (9.60%), unclassified Parabacteroides,and Pseudobutyrivibrio (S2 Table). bacteria (8.24%), Parabacteroides (3.00%), Clustered heatmap analysis depending on the bacterial Phascolarctobacterium (2.95%), RC9 gut group (2.73%), community profiles at the genus level revealed that the Treponema (1.80%), Clostridiales of the incertae sedis samples obtained from the TIS (T1–T5) were highly sim- (1.77%), Oscillospira (1.58%), Clostridium (1.48%), p- ilar in bacterial community composition and were classi- 1088-a5_gut_group (1.48%), Anaerovibrio (1.38%), fied as a single group, while the samples from the PIC Roseburia (1.29%), Fusobacterium (1.12%), and pigs were classified into two groups (Fig. 5). The heatmap Bacteroides (1.07%) (Fig. 4, S2 Table). rows not only reflect the relative abundance and Fig. 1 Rarefaction curves of different samples. The abscissa represents Fig. 2 Shannon-Wiener curves of the different samples. The abscissa the different sequencing depths and the ordinate represents the numbers represents the different sequencing depths and the ordinate represents of the OTUs the Shannon indices Ann Microbiol (2018) 68:185–194 189 Fig. 3 Compositions of the bacterial communities at the phylum level. Relative abundance of bacterial groups (phylum level) in the cecum of the five Tibetan pigs and the five PIC pigs clustering of OTUs in the different samples but also show Core bacterial communities the similarities and differences of the bacterial community compositions in the different samples. The red regions in The presence of core bacterial communities was assayed the heatmap indicate bacterial communities with high rel- further in both breeds. The result shows that 256 OTUs ative abundance. Bacterial communities with high relative were shared among the different TIS (Fig. 6), and their abundances in the different TIS included the unclassified sequences accounted for 88.1% of all the bacterial se- bacteria, S24-7_norank, RF16_norank, Spirochaeta, quences. The shared reads made up 86.23, 86.12, 90.00, RC9_gut_group, Bacteroides, Incertae_Sedis, 89.87, and 88.29% in T1–T5 samples of the TIS, respec- Treponema, Anaerovibrio,and RF9_norank, while those tively (Fig. 6,Table 2). Moreover, the shared 256 OTUs in the different PIC pigs included the uncultured bacteria, were identified in seven phyla, and Bacteroidetes, Prevotella, Parabacteroides, Phascolarctobacterium,and Firmicutes,and Spirochaetes were the dominant phyla Ruminococcu (Fig. 5). (Table 2). For all PIC pigs, 242 OTUs were shared, and Fig. 4 Compositions of the bacterial communities at the genus level. Relative abundance of bacterial groups (by genus) in the cecum of the five Tibetan pigs and the five PIC pigs 190 Ann Microbiol (2018) 68:185–194 Fig. 5 Bacterial distributions of the gut bacteria communities by heatmap analysis. Bacterial distributions in the ten samples. Columns represent the different samples, while rows represent the OTUs Ann Microbiol (2018) 68:185–194 191 and Sutterella genera. The Planctomycetaceae family belongs to the Planctomycetes phylum. Discussion Diversity of bacterial communities in the cecum of the two breeds Bacterial 16S rRNA genes contain nine Bhypervariable regions^ (V1–V9) that demonstrate considerable sequence di- versities among different bacteria. Species-specific sequences within a given hypervariable region are useful targets of diag- nostic assays and other scientific investigations, but a single region cannot tell all the bacteria (Chakravorty et al. 2007). A large number of scientific reports demonstrate that the combi- nation of V4–V5 is the optimal region combination for diver- Fig. 6 Shared OTUs in the five TIS. Venn diagram shows the unique and shared OTUs in the different Tibetan pigs sity and evenness identifications of microbes (Sun et al. 2013; Pitta et al. 2014). Using the Miseq high-throughput sequenc- ing, 660 and 668 OTUs were founded in the cecal bacteria of the TIS and PIC pigs, respectively. These values are lower their sequences accounted for 81.29% of all the bacterial than the number of OTUs reported in rhinoceros’ rumen sequences. The shared reads were different among the (Jami and Mizrahi 2012) and the gut of pigs (Kim et al. samples obtained from the PIC pigs (P1–P5) (Fig. 7, 2012) but higher than the number in the gut of pandas (Zhu Table 3). The shared 242 OTUs involved six phyla, with et al. 2011). In addition, the percentage of OTUs (59.23%) Bacteroidetes, Firmicutes, Planctomycetes,and presented in some of the TIS (less than 5) were lower com- Proteobacteria as the dominant phyla (Table 3). pared with those of the PIC pigs (62.87%). The higher OTUs Firmicutes and Bacteroidetes were the core bacterial com- number in PIC pigs mainly because most OTUs were found in munities in both breeds, and Bacteroidetes made up a higher individuals, not in all the samples, which was also reported in proportion than Firmicutes (Tables 2 and 3). Firmicutes a study performed on cattle (Jami and Mizrahi 2012). Thus, contained some dominant families, including the TIS had more stable cecal bacterial communities. Ruminococcaceae, Lachnospiraceae,and Erysipelotrichaceae, while Bacteroidetes were mainly dominated by S24-7_norank, Rikenellaceae,and Prevotellaceae families. In other core bacte- Core bacterial communities in the cecum of the two rial communities, Spirochaetae was dominated by the pig breeds Treponema and Spirochaeta genera in the TIS, while Proteobacteria in the PIC pig samples was dominated by In animals, the core cecal microbiota have a great effect on the Campylobacter, Helicobacter, Succinivibrio, GR-WP33-58, normal gut functions (Turnbaugh et al. 2009). Our study found Table 2 Core bacteria in the cecum of five Tibetan swines Phylum Shared OTUs Reads of shares OTU Reads of shared OTU/total reads (%) T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 Tenericutes 10 67 234 65 39 160 0.64 1.23 0.54 0.42 0.66 Spirochaetae 14 733 1436 749 737 1746 7.03 7.54 6.23 7.89 7.21 Proteobacteria 2 18 63 9 5 60 0.170.33 0.070.050.25 Planctomycetes 1 131 5 7 30 70 1.260.03 0.060.320.29 Firmicutes 138 2697 4047 3002 2244 6070 25.87 21.25 24.96 24.02 25.05 Cyanobacteria 1 1 3 7 2 13 0.01 0.02 0.06 0.02 0.05 Bacteroidetes 90 5342 10,612 6985 5339 13,276 51.24 55.73 58.08 57.15 54.78 Total shared sequences 256 8989 16,400 10,824 8396 21,395 86.23 86.12 90.00 89.87 88.29 192 Ann Microbiol (2018) 68:185–194 2012), in the intestine is related to the improvement of diet utilization efficiency. Proteobacteria are the most diverse in bacterial phyla. They are well-known for their clinical importance in human gastroin- testinal disease diagnosis; they play a role in luminal dysbiosis and in the imbalance between pathogenic bacteria and function- ally defensive commensal bacteria (Walujkar et al. 2014). In the PIC pig, Proteobacteria were dominant in the cecum and reached 2.96% of all the bacteria, significantly higher than the value in the TIS (p< 0.01). Also, lots of Burkholderiales, Campylobacterales, Desulfuromonadales,and Aeromonadales belonging to Proteobacteria were found to be the core microbi- ota in the PIC pig. The proportion of Escherichia bacteria was also very high, close to 0.75% in the PIC pig. Previous studies indicate that adding antibiotics (Looft et al. 2012; Looft et al. 2014) and soybean fiber (Chen et al. 2014) to pig diet induced an increase in Proteobacteria in the intestine, especially an in- Fig. 7 Shared OTUs in the five PIC pigs. Venn diagram shows the unique crease in Escherichia coli. and shared OTUs in the different individual of the PIC pigs Spirochaete, a phylum of bacteria capable of degrading polymers (xylan, pectin, arabinogalactan) and hemicellulose effectively, was found to be dominant among the core micro- that the relative abundance of core bacterial communities was biota in the cecum of the TIS. Treponema, a genus of higher in the cecum of the TIS (88.10%) than in the PIC pigs Spirochaete phylum, not only participated in cellulose degra- (81.29%). Both Bacteroidetes and Firmicutes were the most dation (Shinkai et al. 2010) but also degraded pectin in the abundant bacteria in both breeds, but the former was more abun- plant cell wall to produce acetic acid, propionic acid, or other dant than the latter. Moreover, the distribution of these two dom- short-chain fatty acids to provide energy for the animals (Liu inant bacteria is contrary to other reports involving rhino (Bian et al. 2014; Niu et al. 2015). Additionally, Treponema is a vital et al. 2013), pig (Kim et al. 2012), and herbivorous rodents and beneficial genus in cattle rumen because of its capability (Kohl et al. 2014) but consistent with the studies on the rumen to inhibit Salmonella and Escherichia coli (Edrington et al. of dairy animal (Jami and Mizrahi 2012; Lietal. 2012) and pigs 2012). This may be the reason why the TIS has a high disease (Looftetal. 2012). In contrast to the situation in TIS and PIC resistance. pigs, Firmicutes was more abundant than Bacteroidetes in the gut of obese mice and obese people. Firmicutes showed greater ability to obtain energy from the diet and get volatile fatty acids Bacterial community compositions in the cecum (SCFAs) during fermentation, thereby promoting the deposition and grazing characteristics of the TIS of fat, while increased Bacteroidetes was significantly associated with weight-loss in humans (Turnbaugh et al. 2009). Looft et al. The bacterial community compositions in the cecum of the also reported that changes in Firmicutes/Bacteroidetes propor- TIS and PIC pigs were highly similar, but their distribution tion, which may be affected by antibiotics additives (Looft et al. and quantities differed significantly. Cellulolytic bacteria were Table 3 Core bacteria in the cecum of five PIC pigs Phylum Shared OTUs Reads of shares OTUs Reads of shared OTUs/total reads (%) P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Spirochaetae 5 64 105 157 42 168 0.28 0.90 1.42 0.38 0.93 Proteobacteria 7 909 59 151 231 262 3.97 0.50 1.37 2.09 1.45 Planctomycetes 1 378 255 219 141 12 1.65 2.17 1.98 1.28 0.07 Firmicutes 147 5201 3363 3203 2904 5818 22.69 28.68 28.99 26.29 32.30 Cyanobacteria 1 1 5 5 6 2 0.00 0.04 0.05 0.05 0.01 Bacteroidetes 81 13,357 4478 5091 6762 7779 58.27 38.19 46.08 61.21 43.19 Total shared sequences 242 19,910 8265 8826 10,086 14,041 86.86 70.48 79.89 91.29 77.95 Ann Microbiol (2018) 68:185–194 193 detected in the cecum of the two breeds, including References Ruminococcus, Bacteroides, Prevotella, Clostridium, Butyricicoccus, Fibrobacter, Lachnospira, Anaerovibrio, Bian G, Ma L, Su Y, Zhu W (2013) The microbial community in the feces of the white rhinoceros (Ceratotherium simum) as determined by Parabacteroides, and Pseudobutyrivibrio (Zhu et al. 2011; barcoded pyrosequencing analysis. PLoS One 8:e70103. https:// Jami and Mizrahi 2012;Wuetal. 2012;Bianetal. 2013). doi.org/10.1371/journal.pone.0070103 The number of Bacteroidales was significantly higher in the Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, TISthaninthePICpigs(p< 0.01), but Prevotella, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, Clostridium,and Fibrobacter were significantly lower in the McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, TIS than in the PIC pigs (p< 0.05); the other bacterial genera Tumbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld capable of degrading cellulose displayed no significant J, Knight R (2010) QIIME allows analysis of high-throughput com- difference between the two breeds. At the genus level, the munity sequencing data. Nat Methods 7:335–336. https://doi.org/ 10.1038/Nmeth.F.303 uncultured and unclassified bacteria accounted for 42.4% of Castillo M, Martin-Orue SM, Anguita M, Perez JF, Gasa J (2007) all the bacteria in the TIS and 36.9% of the bacteria in the PIC Adaptation of gut microbiota to corn physical structure and different pigs. Studies have shown that high fiber diets can promote gut types of dietary fibre. Livest Sci 109:149–152. https://doi.org/10. development in pigs, including increasing the integrities of the 1016/j.livsci.2007.01.129 small intestinal mucosa, the height of the intestinal villi, the Chakravorty S, Helb D, Burday M, Connell N, Alland D (2007) A de- tailed analysis of 16S ribosomal RNA gene segments for the diag- number of beneficial gut microorganisms, and, finally, nosis of pathogenic bacteria. J Microbiol Methods 69:330–339. changing the intestinal mucosal digestive physiology of the https://doi.org/10.1016/j.mimet.2007.02.005 pig (Wenk 2001;Chenet al. 2014). Thus, the advantageous Chen H, Mao XB, Che LQ, Yu B, He J, Yu J, Han GQ, Huang ZQ, Zheng characteristics of TIS are related not only to the breed proper- P, Chen DW (2014) Impact of fiber types on gut microbiota, gut environment and gut function in fattening pigs. Anim Feed Sci ties but also to the unique intestinal microflora formation Technol 195:101–111. https://doi.org/10.1016/j.anifeedsci.2014.06. resulting from long-term dietary fiber intake (Edrington et al. 2012). Edgar RC (2013) UPARSE: highly accurate OTU sequences from micro- bial amplicon reads. Nat Methods 10:996. https://doi.org/10.1038/ nmeth.2604 Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics Conclusions 27:2194–2200. https://doi.org/10.1093/bioinformatics/btr381 Edrington TS, Dowd SE, Farrow RF, Hagevoort GR, Callaway TR, Our study analyzed the diversity and composition of cecal Anderson RC, Nisbet DJ (2012) Development of colonic microflora microbiota in the TIS and presents initial observations for as assessed by pyrosequencing in dairy calves fed waste milk. J Dairy Sci 95:4519–4525. https://doi.org/10.3168/jds.2011-5119 further understanding of the microbial ecology of this intesti- Jami E, Mizrahi I (2012) Composition and similarity of bovine rumen nal habitat. Our results indicate that the high disease resistance microbiota across individual animals. PLoS One 7:e33306. https:// of the TIS and its ability to digest high fiber plants may be doi.org/10.1371/journal.pone.0033306 related to its gut microbiota. Future studies should focus on Kim HB, Borewicz K, White BA, Singer RS, Sreevatsan S, Tu ZJ, the characterization of the community composition variations Isaacson RE (2012) Microbial shifts in the swine distal gut in re- sponse to the treatment with antimicrobial growth promoter, tylosin. with the changes in temporal and spatial factors as well as Proc Natl Acad Sci U S A 109:15485–15490. https://doi.org/10. dietary changes. 1073/pnas.1205147109 Kohl KD, Miller AW, Marvin JE, Mackie R, Dearing MD (2014) Acknowledgments We thank Hongzhou Wang (Tibetan swine Herbivorous rodents (Neotoma spp.) harbour abundant and active Slaughterhouse of Shaanxi Huayi Industrial Co., Ltd.) and Junfang Yan foregut microbiota. Environ Microbiol 9:2869–2878. https://doi. (Shaanxi Benxiang Pig Slaughterhouse) for their help in sampling. We are org/10.1111/1462-2920.12376 also grateful to Juan Qin (Shanghai Majorbio Bio-Pharm Technology Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Moller K Co., Ltd.) for technical help. (2002) Culture-independent analysis of gut bacteria: the pig gastro- intestinal tract microbiota revisited. Appl Environ Microbiol 68: Funding This study was sponsored by grants from the Key Agricultural 673–690. https://doi.org/10.1128/aem.68.2.673-690.2002 Science and Technology Promotion Project in Shaanxi province, China Li RW, Wu ST, Baldwin RL, Li WZ, Li CJ (2012) Perturbation dynamics (ZDKJ-2014-27) and the Annual National Undergraduate Innovative of the rumen microbiota in response to exogenous butyrate. PLoS Entrepreneurial Training Program of China (201410712008), and One 7. https://doi.org/10.1371/journal.pone.0029392 Science and Technology Plan Project in Henan Province Liu H, Ivarsson E, Dicksved J, Lundh T, Lindberg JE (2012) Inclusion of (162102310475). chicory (Cichorium intybus L.) in pigs’ diets affects the intestinal microenvironment and the gut microbiota. Appl Environ Microbiol 78:4102–4109. https://doi.org/10.1128/AEM.07702-11 Compliance with ethical standards Liu J, Wang JK, Zhu W, Pu YY, Guan LL, Liu JX (2014) Monitoring the rumen pectinolytic bacteria Treponema saccharophilum using real- Conflict of interest None declare time PCR. FEMS Microbiol Ecol 87:576–585. https://doi.org/10. 1111/1574-6941.12246 194 Ann Microbiol (2018) 68:185–194 Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul Walujkar SA, Dhotre DP, Marathe NP, Lawate PS, Bharadwaj RS, Shouche YS (2014) Characterization of bacterial community shift WJ, Stedtfeld TM, Chai B, Cole JR, Hashsham SA, Tiedje JM, Stanton TB (2012) In-feed antibiotic effects on the swine intestinal in human ulcerative colitis patients revealed by Illumina based 16S microbiome. Proc Natl Acad Sci U S A 109:1691–1696. https://doi. rRNA gene amplicon sequencing. Gut Pathogens 6. https://doi.org/ org/10.1073/pnas.1120238109 10.1186/1757-4749-6-22 Looft T, Allen HK, Cantarel BL, Levine UY, Bayles DO, Alt DP, Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classi- Henrissat B, Stanton TB (2014) Bacteria, phages and pigs: the ef- fier for rapid assignment of rRNA sequences into the new bacterial fects of in-feed antibiotics on the microbiome at different gut loca- taxonomy. Appl Environ Microbiol 73:5261–5267. https://doi.org/ tions. ISME J 8:1566–1576. https://doi.org/10.1038/ismej.2014.12 10.1128/aem.00062-07 Ma L, Yang WP, Meng FX, Ji SY, Xin HY, Cao BY (2015) Wenk C (2001) The role of dietary fibre in the digestive physiology of the Characterization of an acidic cellulase produced by Bacillus subtilis pig. Anim Feed Sci Technol 90:21–33. https://doi.org/10.1016/ BY-4 isolated from gastrointestinal tract of Tibetan pig. J Taiwan s0377-8401(01)00194-8 Inst Chem Eng 56:67–72. https://doi.org/10.1016/j.jtice.2015.04. Wu SG, Wang GT, Angert ER, Wang WW, Li WX, Zou H (2012) Composition, diversity, and origin of the bacterial community in Meng FX, Ma L, Ji SY, Yang WP, Cao BY (2014) Isolation and charac- grass carp intestine. PLoS One 7. https://doi.org/10.1371/journal. terization of Bacillus subtilis strain BY-3, a thermophilic and effi- pone.0030440 cient cellulase-producing bacterium on untreated plant biomass. Lett Xiao Y, Yan HL, Diao H, Yu B, He J, Yu J, Zheng P, Mao XB, Luo YH, Appl Microbiol 59:306–312. https://doi.org/10.1111/lam.12276 Chen DW (2017) Early gut microbiota intervention suppresses Niu Q, Li PH, Hao SS, Zhang YQ, Kim SW, Li HZ, Ma X, Gao S, He LC, DSS-induced inflammatory responses by deactivating TLR/NLR Wu WJ, Huang XG, Hua JD, Zhou B, Huang RH (2015) Dynamic signalling in pigs. Sci Rep 7:3224. https://doi.org/10.1038/s41598- distribution of the gut microbiota and the relationship with apparent 017-03161-6 crude fiber digestibility and growth stages in pigs. Sci Rep 5. https:// Xin H, Ji SY, Peng JY, Han P, An XP, Wang S, Cao BY (2017) Isolation doi.org/10.1038/Srep09938 and characterisation of a novel antibacterial peptide from a native Pajarillo EAB, Chae JP, Balolong MP, Kim HB, Seo KS, Kang DK swine intestinal tract-derived bacterium. Int J Antimicrob Agents 49: (2014) Pyrosequencing-based analysis of fecal microbial communi- 427–436. https://doi.org/10.1016/j.ijantimicag.2016.12.012 ties in three purebred pig lines. J Microbiol 52:646–651. https://doi. Yan H, Potu R, Lu H, de Almeida VV, Stewart T, Ragland D, Armstrong org/10.1007/s12275-014-4270-2 A, Adeola O, Nakatsu CH, Ajuwon KM (2013) Dietary fat content Pitta DW, Kumar S, Veiccharelli B, Parmar N, Reddy B, Joshi CG (2014) and fiber type modulate hind gut microbial community and meta- Bacterial diversity associated with feeding dry forage at different bolic markers in the pig. PLoS One 8. https://doi.org/10.1371/ dietary concentrations in the rumen contents of Mehshana buffalo journal.pone.0059581 (Bubalus bubalis) using 16S pyrotags. Anaerobe 25:31–41. https:// Yang WP, Meng FX, Peng JY, Han P, Fang F, Ma L, Cao BY (2014) doi.org/10.1016/j.anaerobe.2013.11.008 Isolation and identification of a cellulolytic bacterium from the Shinkai T, Ueki T, Kobayashi Y (2010) Detection and identification of Tibetan pig’s intestine and investigation of its cellulase production. rumen bacteria constituting a fibrolytic consortium dominated by Electron J Biotechnol 17:262–267. https://doi.org/10.1016/j.ejbt. Fibrobacter succinogenes. Anim Sci J 81:72–79. https://doi.org/ 2014.08.002 10.1111/j.1740-0929.2009.00698 Zhang LL, Mu CL, He XY, Su Y, Mao SY, Zhang J, Smidt H, Zhu WY Sun DL, Jiang X, Wu QLL, Zhou NY (2013) Intragenomic heterogeneity (2016) Effects of dietary fibre source on microbiota composition in of 16S rRNA genes causes overestimation of prokaryotic diversity. the large intestine of suckling piglets. FEMS Microbiol Lett 363. Appl Environ Microbiol 79:5962–5969. https://doi.org/10.1128/ https://doi.org/10.1093/femsle/fnw138 aem.01282-13 Zhu L, Wu Q, Dai J, Zhang S, Wei F (2011) Evidence of cellulose Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley metabolism by the giant panda gut microbiome. Proc Natl Acad RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M, Sci U S A 108:17714–17719. https://doi.org/10.1073/pnas. Henrissat B, Heath AC, Knight R, Gordon JI (2009) A core gut microbiome in obese and lean twins. Nature 457:480–484. https:// doi.org/10.1038/nature07540
Annals of Microbiology – Springer Journals
Published: Mar 9, 2018
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