Diversity of Intestinal Bacterial Microbiota of Indigenous and Commercial Strains of Chickens Using 16S rDNA-Based Analysis

Simple Summary Little is known about how the bacterial community differs among different genetic breeds of chickens, especially those with various growth rates, such as in local native chickens. Our data, generated by molecular detection revealed the heterogeneity of bacterial populations existing in different intestinal segments for the two strain of chickens. Abstract The objective of this study was to assess the relative abundance of bacteria microflora in different segments of the gastrointestinal tract (duodenum, jejunum, ilium, and cecum) of indigenous (local Omani) and commercial (Cobb 500) chicken strains. Birds were raised under an intensive management system fed a nonmedicated corn-soybean meal diet from Day 0–35 days of age. Using 16S rDNA-based analysis the study showed that in both breeds of birds Bacilli were the most abundant class of bacteria in the duodenum, jejunum, and ileum. Local Omani chickens had significantly higher numbers of Clostridia at most time periods. Actinobacteria were found in higher numbers and reached 54.9% of the bacteria in the jejunum at Day 35 in Cobb 500 versus only 5.42% in the Omani chickens. The bacterial microbiota relative abundance differed significantly (p < 0.05) across different intestinal segments of the two strains, suggesting that each region developed its own bacterial community and the relative abundances of these were quite different.


Introduction
The study of gut microbiota in birds is finally beginning to blossom within the present scientific literature. A number of studies have revealed not only their importance in nutritional utilization, immunological priming of the intestinal tract but also their role as a driving force in growth and development.
The composition of the intestinal microflora in chickens continues to change over time under the influence of different factors, such as bird age and dietary factors [1,2]. There is also significant diversity in bacterial populations among different parts of the digestive tract of the birds [3]. Each region of the GI tract develops its own unique microbial profile, and the composition of the microflora becomes more complex and changes in relation to the age of the chickens, different feed ingredients [4][5][6], breed and geographic location [7][8][9].
With the development of animal husbandry in Oman, local "indigenous" chicken production is becoming more widespread. Local chicken production is among the farming activities in the rural communities of Oman that provides opportunities for food security and income for many

DNA Extraction
Total DNA was extracted from contents of each luminal content samples (duodenum, jejunum, ileum, and cecum) using a QIAamp DNA Stool Mini Kit (QIAGEN, CA, Hamburg, Germany) according to the manufacturer's instructions. The DNA concentration was evaluated by measuring optical density using Nano-Drop 2000 (Thermo Electron Corporation, Waltham, MA, USA) at a wavelength of 260 and 280 nm. The integrity of the DNA extracts was assessed visually using 1.0% agarose gel (containing ethidium bromide) electerophoresis.

Polymerase Chain Reaction Amplicon Production and High-Throughput Sequencing
The variable regions V3-V4 of the 16S rDNA gene were amplified and sequenced. The PCRs were performed in triplicate in a total volume of 20 µL containing 5 µM of each primer, 10 ng of DNA template, 4 µL 1× FastPfu buffer, 2.5 mM dNTPs, and 0.4 µL of FastPfu polymerase (TransGen Biotech, Beijing, China). PCR conditions were as follows: Initial denaturation at 95 • C for 2 min; followed by 25 cycles of denaturation 94 • C for 30 s, annealing at 55 • C for 30 s, and extension at 72 • C for 30 s and then, a final extension at 72 • C for 5 min. PCR products were separated on 2% agarose gels, and purified using the DNA gel extraction kit (Axygen Scientific Inc., Union City, CA, USA). Amplicons produced form different intestinal luminal content samples were sent to a commercial company (BGI Genomic

Sequencing Analysis
All the raw sequences obtained from Illumina Miseq were firstly filtered for quality control to get operational sequences. The quality control and analysis of the sequences were performed using the software Quantitative Insights into Microbial Ecology (QIIME, v1.8.0) [18]. The paired-end reads from the DNA fragments were merged using FLASH [19]. Sequences data was treated by read trimming and identification of V3-V4 sequences and set of sequences with ≥97% identity were defined as an operational taxonomic unit (OTU). The UCLUST [20] clustering method was used to cluster operational taxonomic units. The defined OTUs were assigned to different taxonomic levels (phylum, class, genus and families) at a cutoff of 97%. The clustered OTUs were also used to construct the rarefaction curves and calculate the Shannon and Simpson diversity indices, abundance-based coverage estimators (ACE), Chao 1 richness, and coverage percentage by Good's method.

Bioinformatics and Statistical Analyses
Bioinformatics and statistical analyses were performed using the QIIME and R package (v3.1.1). The alpha-diversity indices (Chaol, ACE, Shannon diversity index, and Simpson index) were calculated to establish the relative abundance and diversity of the sequences. Beta diversity was determined using unweighted Unifrac distance metrics to evaluate the structure and distribution of the microbial genetic communities among the samples. Differences in the Unifrac distances for pairwise comparisons among groups were calculated using Student's t-test and the Monte Carlo permutation test with 1000 permutations. Metastats and R package (v3.1.1) [21] were used to compare and determine which taxonomic groups were significantly different between groups of samples based on intestinal segments and age period. The differences were considered to be significant at p < 0.05. The obtained p-value was adjusted by a Benjamini-Hochberg false discovery rate correction (Function 'p.adjust' in the stats package of R (v3.1.1)).

Results
Our data obtained for both breeds of chicken showed that each region of intestinal segments developed its own bacterial community and the diversity of the bacterial community changed from one age period to the next. In addition, Bacilli were the dominant 16S rDNA sequences in the duodenum, jejunum, and ileum libraries, whereas Clostridia were the dominant 16S rDNA sequences in the cecum libraries.
The most intriguing observation in this study is that at the early stage of their lives (five days of age), local Omani chickens had a significantly a smaller percentage of Bacilli in their duodenum (68.5%) when compared to Cobb 500 broiler chickens (98%), whereas Clostridia at the same age was more dominant in the Omani local chickens (25.84%) compared to Cobb 500 broiler chickens (0.88%). The microbial composition of each intestinal segment at different ages for each strain of chicken was summarized in their respective section below.

Microbial Composition of the Duodenum of Cobb 500 Broiler Chickens
Bacteria classified according to their respective Phylum and Class, found in the duodenum of broiler chickens at different ages, are presented in Table 1

Microbial Composition of the Ileum of Cobb 500 Broiler Chickens
Bacteria classified according to their respective Phylum and Class, found in the ileum of broiler chickens at different ages are presented in Table 3. Thirty bacterial microbiota at the Class level were found in the ileum. Of the 200,624 reads, Bacilli were the most abundant, at 75.74% of the total sequences. Actinobacteria and Proteobacteria sequences represented 5.89% and 6.38%, respectively of the total sequences. Clostridia accounted for 11.73% of the total sequences. Across different age periods Bacilli were the dominant group, representing 98.33% at Day 5,88

Microbial Composition of the Cecum of Cobb 500 Broiler Chickens
Bacteria classified according to their respective Phylum and Class, found in the cecum of broiler chickens at different ages are presented in Table 4. Thirty bacterial microbiota at the Class level were in the cecum. Of the 208,941 reads, Clostridia were the most abundant, at 89.42% of the total sequences. Only a few of Coriobacteria (1.09%) and Erysipelotrichia (2.52%) related to the total sequences were detected. Actinobacteria and Proteobacteria represented a very small percentage of 0.20% and 0.020%, respectively, of the total sequences. Bacilli accounted for 6.59% of the total sequences. Across different age periods Clostridia were the dominant group, representing 80.61% at Day 5,92.19%

Differences of Microbial Communities among Samples from Different Intestinal Segments of Cobb 500 Broiler Chickens
The p-value distribution of 16S rDNA gene sequence libraries comparing the quantitative differences of microbial communities among samples from different intestinal segments of broiler chickens are presented in Table 5. Statistical comparisons of the libraries revealed that the composition of the Duodenum-Jejunum, Duodenum-Ileum, Cecum-Duodenum, Cecum-Ileum Cecum-Jejunum bacterial microbiota differed significantly (p < 0.05), suggesting that each region developed its own bacterial community. The number of Actinobacteria, Coriobacteria, Bacilli, Clostrdia, Erysipelotrichia, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria differed significantly across different intestinal segments (p < 0.05). Bacilli were the dominant 16S rDNA sequences in the duodenum, jejunum, and ileum libraries, whereas Clostridia were the dominant 16S rDNA sequences in the cecum libraries. Table 5. P-value distribution of 16S rDNA gene sequence libraries compared the abundance differences of microbial communities among samples from different segments for Cobb 500 Broiler chickens. The p-value distribution of 16S rDNA gene sequence libraries comparing the quantitative differences of microbial communities among samples from broiler chickens at different age groups are presented in Table 6. Statistical comparisons of the libraries revealed that there were no significant differences (p > 0.05) between the microbial composition at different age groups: Day 5-15, Day 5-25, Day 5-35, Day 15-25, Day 15-35, and Day 25-35. The results of the statistical evaluation at certain age groups revealed that the percentage of bacterial microbiota of Acidobacteriia, Betaproteobacteria, and Cytophagia varied significantly (p < 0.05). The average percentage of Acidobacteriia detected at Day 25 (0.01%) of age was significantly higher (p < 0.05) than at Day 35 (0.001%) of age. The average percentage of Betaproteobacteria was detected at significantly higher levels (p < 0.05) at Day 35 (0.67%) of age than at Day 5 (0.05%) of age. The average percentage of Cytophagia at Day 15 (0.02%) was significantly different (p < 0.05) from those of the other age groups. From Figure 1, it can be seen that the diversity of the bacterial community in the intestinal segments of broiler chickens changed from one age period to the next. Species that exhibited an abundance less than 0.5% in all samples were classified into "others". The intestinal segments of duodenum, jejunum, and ileum had a higher relative abundance of Bacilli, and as the birds aged, the percentage of Bacilli decreased, whereas the cecum had a higher relative abundance of Clostridia and as the birds aged, the percentage of Clostridia increased.

Broiler Chickens
From Figure 1, it can be seen that the diversity of the bacterial community in the intestinal segments of broiler chickens changed from one age period to the next. Species that exhibited an abundance less than 0.5% in all samples were classified into "others". The intestinal segments of duodenum, jejunum, and ileum had a higher relative abundance of Bacilli, and as the birds aged, the percentage of Bacilli decreased, whereas the cecum had a higher relative abundance of Clostridia and as the birds aged, the percentage of Clostridia increased.

Microbial Composition of the Duodenum of Local Omani Chickens
Bacteria classified according to their respective Phylum and Class, found in the duodenum of local Omani chickens at different ages are presented in Table 7. Thirty-five bacterial microbiota at the Class level were found in duodenum. Of the 391,488 reads, Bacilli were the most abundant, at 76.8% of the total sequences, while sequences related to Clostridia accounted for 15.4% of the total sequences. Proteobacteria accounted for 5.93% of the total sequences. Actinobacteria, Coriobacteria, Rubrobacteria, Thermoleophilia, Flavobacteria and Deinococci and Tissierellia represented a very small percentage of 0.97%, 0.01%, 0.004%, 0.003%, 0.06%, 0.14%, 0.04%, and 0.03%, respectively of the total sequences. Across different age periods Bacilli were the dominant group, representing 68.52% at Day 5,86

Microbial Composition of the Duodenum of Local Omani Chickens
Bacteria classified according to their respective Phylum and Class, found in the duodenum of local Omani chickens at different ages are presented in Table 7. Thirty-five bacterial microbiota at the Class level were found in duodenum. Of the 391,488 reads, Bacilli were the most abundant, at 76.8% of the total sequences, while sequences related to Clostridia accounted for 15.4% of the total sequences. Proteobacteria accounted for 5.93% of the total sequences. Actinobacteria, Coriobacteria, Rubrobacteria, Thermoleophilia, Flavobacteria and Deinococci and Tissierellia represented a very small percentage of 0.97%, 0.01%, 0.004%, 0.003%, 0.06%, 0.14%, 0.04%, and 0.03%, respectively of the total sequences. Across different age periods Bacilli were the dominant group, representing 68.52% at Day 5,86

Microbial Composition of the Jejunum of Local Omani Chickens
Bacteria classified according to their respective Phylum and Class, found in the jejunum of local Omani chickens at different ages are presented in Table 8. Thirty-five bacterial microbiota at the Class level were found in the jejunum. Of the 307,792 reads, Bacilli were the most abundant, at 89.91% of the total sequences. Clostridia and Actinobacteria represented 6% and 3.09% of the total sequences. Proteobacteria accounted for 0.65% of the total 16S rDNA sequences. Coriobacteria, Rubrobacteria, Thermoleophilia, Chitinophagia, Flavobacteria, Deinococci, and Tissierellia represented a very small percentage at 0.02%, 0.01%, 0.01%, 0.001%, 0.001%, 0.01%, 0.004%, respectively of the total sequences.

Microbial Composition of the Ileum of Local Omani Chickens
Bacteria classified according to their respective Phylum and Class, found in the ileum of local Omani chickens at different ages are presented in Table 9. Thirty-five bacterial microbiota at the Class level were found in the ileum. Of the 268,837 reads, Bacilli were the most abundant, at 82.10% of the total sequences. Actinobacteria and Proteobacteria sequences were less abundant in the ileum and detected at 1.45% and 1.32%, respectively of the total sequences. Clostridia accounted for 14   Chlamydiia

Microbial Composition of the Cecum of Local Omani Chickens
Bacteria classified according to their respective Phylum and Class, found in the duodenum of local Omani chickens at different ages are presented in Table 10. Thirty-five bacterial microbiota at the Class level were found in the cecum. Of the 266,937 reads, Clostridia were the most abundant, at 81.13% of the total sequences. Erysipelotrichia and Proteobacteria represented a small percentage of 2.09% and 2.94%, respectively of the total sequences. Only a few Actinobacteria (0.73%) related sequences were detected. Bacilli accounted for 4.87% of the total sequences. Across different age periods Clostridia were the dominant group, representing 79.08% at Day 5,93

Differences Of Microbial Communities among Samples from Different Intestinal Segments of Local Omani Chicken
The p-value distribution of 16S rDNA gene sequence libraries comparing the relative abundance differences of microbial communities among samples from different intestinal segments of local Omani chickens are presented in Table 11. Statistical comparisons of the libraries revealed that the composition of the Duodenum-Jejunum, Duodenum-Ileum, Cecum-Duodenum, Cecum-Ileum Cecum-Jejunum bacterial microbiota differed significantly (p < 0.05), suggesting that each region developed it is own bacterial community.

Differences of Microbial Communities among Samples from Local Omani Chickens of Different Age Groups
The p-value distribution of 16S rDNA gene sequence libraries comparing the quantitative differences of microbial communities among samples from local Omani chickens of different age groups are presented in Table 12. Statistical comparisons of the libraries revealed that there were no significant differences (p > 0.05) between the microbial composition at different age groups: Day 5-15, Day 5-25, Day 5-35, Day 15Day 15-25, Day 15Day 15-35, and Day 25-35. The results of the statistical evaluation at certain age groups revealed that the percentage of the bacterial microbiota of Acidimicrobiia and Fusobacteriia varied significantly (p < 0.05). At Day 5 of age the percentage of Acidimicrobiia (0.002%) was significantly higher (p < 0.05) than at Day 15Day 15 (0.0%) and Day 35 (0.0%) of age. Fusobacteriia was detected at significantly higher levels (p < 0.05) at Day 35 of age (0.011%) than at Day 5 (0.0%) of age.  Figure 2, it can be seen that the diversity of the bacterial community of intestinal segments of local Omani chickens changed from one age period to the next. Species of that exhibited an abundance less than 0.5% in all samples were classified into "others". The intestinal segment of duodenum, jejunum, and ileum had a higher abundance of Bacilli, and as the birds aged, the percentage of Bacilli decreased, whereas the cecum had a higher abundance of Clostridia, and as the birds aged, the percentage of Clostridia increased.

Discussion
Our data generated by molecular detection and bioinformatics analysis revealed the heterogeneity of bacterial populations existing in different intestinal segments. The 16S rDNA analysis generated a huge database that is beyond the scope of this manuscript. Hence, the findings of the present study have been limited to those classes of bacteria of the most quantitative significance.
In the present study, the statistical comparisons of the libraries of microbial communities within each respective breed among samples from different intestinal segments at different age groups revealed that there were no significant differences (p > 0.05). On the contrary, bacterial microbiota differed significantly (p < 0.05) within each respective breed across different intestinal segments, suggesting that each region developed its own bacterial community and the relative abundances of these were quite different. Similar findings were represented by different studies that showed that the bacterial community is relatively transient and is replaced by a stable bacterial community once the rate of the intestinal development decreased [9,6,12].
In the duodenum of both the Cobb 500, as well as in the Omani chickens, Bacilli were the predominate class of bacteria at all age periods. However, an important difference between the two birds was the fact that local Omani chickens had significantly higher numbers of Clostridia. Indeed, at Day 5 and Day 25 Clostridia comprised 25.8% and 26.75% of the bacteria in the duodenum of local Omani chickens, respectively, versus only 0.88% and 8.388% in the Cobb 500.
It can be clearly hypothesized that the superior performance of the Cobb 500 is associated with the higher proportion of Bacilli in the duodenum or conversely that Clostridia might limit optimal duodenum development in Omani chickens.
A similar microbial picture was mirrored in the duodenum with Bacilli being the predominate class of bacteria in both birds but again with Clostridia being more abundant at Day 15Day 15, 25, and 35 in the Omani chickens. At Day 15Day 15 and 25 the Omani chickens had a 6-and 9-fold greater number of Clostridia, respectively.
In the ileum it was again observed against a backdrop of an overall higher number of Bacilli of both birds Clostridia at Day 5 made up 9.23% of the bacteria versus 0.805% in Cobb 500. This trend

Discussion
Our data generated by molecular detection and bioinformatics analysis revealed the heterogeneity of bacterial populations existing in different intestinal segments. The 16S rDNA analysis generated a huge database that is beyond the scope of this manuscript. Hence, the findings of the present study have been limited to those classes of bacteria of the most quantitative significance.
In the present study, the statistical comparisons of the libraries of microbial communities within each respective breed among samples from different intestinal segments at different age groups revealed that there were no significant differences (p > 0.05). On the contrary, bacterial microbiota differed significantly (p < 0.05) within each respective breed across different intestinal segments, suggesting that each region developed its own bacterial community and the relative abundances of these were quite different. Similar findings were represented by different studies that showed that the bacterial community is relatively transient and is replaced by a stable bacterial community once the rate of the intestinal development decreased [6,9,11].
In the duodenum of both the Cobb 500, as well as in the Omani chickens, Bacilli were the predominate class of bacteria at all age periods. However, an important difference between the two birds was the fact that local Omani chickens had significantly higher numbers of Clostridia. Indeed, at Day 5 and Day 25 Clostridia comprised 25.8% and 26.75% of the bacteria in the duodenum of local Omani chickens, respectively, versus only 0.88% and 8.388% in the Cobb 500.
It can be clearly hypothesized that the superior performance of the Cobb 500 is associated with the higher proportion of Bacilli in the duodenum or conversely that Clostridia might limit optimal duodenum development in Omani chickens.
A similar microbial picture was mirrored in the duodenum with Bacilli being the predominate class of bacteria in both birds but again with Clostridia being more abundant at Day 15Day 15, 25, and 35 in the Omani chickens. At Day 15Day 15 and 25 the Omani chickens had a 6-and 9-fold greater number of Clostridia, respectively.
In the ileum it was again observed against a backdrop of an overall higher number of Bacilli of both birds Clostridia at Day 5 made up 9.23% of the bacteria versus 0.805% in Cobb 500. This trend continued at Day 15Day 15 with Clostridia comprising 13% of the bacteria of the Omani chickens versus 5.9% in the Cobb 500.
An interesting observation in both the duodenum and jejunum was the high number of Actinobacteria in the Cobb 500 and in the jejunum at Day 25 and 35. Indeed, at Day 25 the Actinobacteria made up 54.89% of the bacteria. This very high number of Actinobacteria is compelling and in harmony with reports of Actinobacteria being important in the upper digestive tract of other high producing broilers, such Ross 308 [22]. Again, this observation would suggest that smaller numbers of Actinobacteria in Omani chickens is not in keeping with optimal development of these intestinal segments.
Since the function of each intestinal segment differ and provide different environmental conditions, it is expected that distinct microbiota develop in different intestinal segments [23,24]. In the present study the most profound differences in the microbial population in the intestinal segments of the two breeds was observed between Day 5 to 25. One possible explanation is that at early age periods, immediately after hatching, is the most critical period in the life of the chick. During this period, there is the transition from yolk to oral nutrition associated with major physical and functional development of the digestive tract and organs [25] resulting in unstable environmental conditions of the digestive tract's microecosystem. However, by three weeks of age, the rate of digestive tract development lessens, and the intestinal bacterial community becomes more stable and establishes optimal environmental conditions, allowing for the clear separation of bacterial communities [4].
The observations of the present study permit the suggestion that Bacilli contribute more favorably to intestinal villi development and hence better nutrient absorption during the critical early period in the life of chickens. Some studies have already shown that the ileum was the most reactive site in which villus area changes significantly with nutritional changes or with an increase in nutrient content in the intestinal lumen [26,27].
Furthermore, Clostridia are primarily involved in fermentation [28]. The significantly larger number of Clostridia in the duodenum of local Omani chickens during early intestinal development likely have no or limited function in nutrient turn over and absorption and subsequently the duodenum functions at an impaired level when compared to that of Cobb 500 chickens.
It is noteworthy that the chicken cecum and its mucosal tissue are dominated by Clostridia related species [29][30][31]. It can be hypothesized that the higher abundance of Clostridia that are associated with fermentation act in an inhibitory fashion for nutrient absorption in the intestine of the local Omani chickens and the villus development is slowed down. The morphological analysis in a study by Al-Marzooqi et al. [10] revealed that Cobb 500 broilers had a greater villi height compared to the Omani breed (p < 0.01). It is assumed that an increased villus height is paralleled by an increased digestive and absorptive function of the intestine due to increased absorptive surface area, expression of brush border enzymes, and nutrient transport systems [32]. Enterocyte enzymatic activity and structure are two of the most important features of the intestinal mucosal physiology [33]. Al-Marzooqi et al. [10] concluded that villus development has a profound effect on the growth performance of the chickens. A study by Lumkins et al. [11] showed that differences in bacterial communities between the modern and ACR broiler lines may be due to variances in villi height, which could lead to an increase in the distance of the crypt away from the lumen with greater villus height, providing a niche for certain bacteria [34,35]. Thus, villi height can be considered another factor contributing to the bacterial community's niche.
Therefore, optimal growth and development of the intestinal tract of chicken is likely governed by early development of the intestinal tract. From this data, it would be assumed that optimal development of small intestinal microflora is associated with a preponderance of Bacilli (98%) in the duodenum at Day 5 of Cobb 500 chickens and that the inferior growth rate of the local Omani chickens was assumed to be related to the smaller number of Bacilli (68.5%) in their duodenum at Day 5. Similarly, a small number of Clostridia (0.88%) in the duodenum of the Cobb 500 and the relatively Fast growth rates and improved weight gains and feed efficiency as a result of genetic selection has helped the broiler industry by providing birds that grow to a heavier weight in a shorter period of time, which have also resulted in changes to the micro-ecosystem in the gastrointestinal tract that provides stable environmental conditions to indigenous intestinal bacteria [36].
Our knowledge of gut microbiota remains incomplete. However, it should be noted that a variety of molecular techniques; such as metagenomic, next generation sequencing (NGS) or marker-gene and metagenomic sequencing (MGS), are available to investigate chicken gut microbial community composition and functions, each with different strengths and weaknesses [37]. Direct comparison among studies is difficult and may not be accurate because of variation in platforms used, breed of chickens, sampling method, differences in approaches, and concepts of studies. The high cost of sequencing analysis and the bias of some of the protocols towards detecting some taxa over others are drawbacks of these approaches. However, multiple PCR combined with q-PCR might be the tool to overcome these constrains [38].

Conclusions
The present study is the first to compare the microbial diversity in the small intestine and cecum of indigenous and commercial strain of chickens. Our study indicate that each region of different intestinal segments developed its own bacterial community and the relative abundances of these were quite diverse. The local breed of chicken had a 6-to 9-fold greater number of Clostridia in comparison to the commercial one and therefore, it was concluded that Clostridia were not conducive to optimal development of the intestinal villi and consequently nutrient turnover and absorption, resulting in inferior performance of the local breed. However, understanding the dynamics of the gut microbial community or microbial balance is far from complete. Future work should be directed towards identifying gut bacteria that can be associated with improved/poor chicken performance. In addition, future studies need to be directed towards development of diets such as the utilization of probiotics or manipulation of fiber intake to increase the development of Bacilli during early intestinal development and subsequent better utilization of nutrients in the local chickens.