6. Bacterial community composition in migratory birds

A comprehensive analysis of the bacterial community structure in migratory birds using culture-independent methods is introduced below.

## 6.1. Confirmation of avian host

In this decade, a comprehensive analysis of gene sequences using next-generation sequencing (NGS) has spread rapidly [26, 27]. The NGS is a powerful technology capable of concurrently determining nucleotide sequences for tens of millions to hundreds of millions of fragmented DNA strands. Originally, NGS was used for high-throughput sequencing of a single biological genome, but now it is possible to perform high-speed processing, allowing multiple samples to be sequenced simultaneously. Therefore, a wide variety of applications have been proposed for NGS. The price of NGS contract analysis service also has declined greatly in the past few years, making it easier to use so that it is now more useful for research on genetic diseases, clinical diagnoses, relationships between human intestinal flora and diseases, analyses of environmental bacterial community composition and succession in both time and space, and searches for useful microorganisms in various environments. Metagenomic methods provided by NGS technology have facilitated a remarkable expansion of our knowledge regarding uncultured bacteria [28]. A more recent detection method, quantitative real-time PCR, is known for its excellent accuracy and sensitivity when detecting known zoonotic pathogens [29]. On the other hand, it is difficult to identify target pathogens that are not previously known with this method, and often too many samples must be handled simultaneously for it to be convenient. A comprehensive analysis by NGS enables us to comprehend a whole picture of the bacterial community contained in a sample, so it is possible to carry out further analysis with specific pathogenic

The 16S rRNA gene sequence was first used in 1985 for phylogenetic analysis [30]. Because it contains both highly conserved regions for primer design and hypervariable regions to identify phylogenetic characteristics of microorganisms, the 16S rRNA gene sequence became the most widely used marker gene for profiling bacterial communities [31]. Full-length 16S rRNA gene sequences consist of nine hypervariable regions that are separated by nine highly conserved regions [32]. Limited by sequencing technology, the 16S rRNA gene sequences used in most studies are partial sequences. Therefore, the selection of proper primers is critical for

Recent studies utilizing high-throughput technology also have demonstrated that the use of suboptimal primer pairs results in the uneven amplification of certain species, causing either an under- or overestimation of some species in a microbial community [32, 33]. Integrated bioinformatics tools were used to evaluate the phylogenetic sensitivity of the hypervariable regions compared with the corresponding full-length sequences. Results showed that using a combination of V4–V6 regions represented the optimal subregions for bacterial phylogenetic

Bird migration is the regular seasonal journey undertaken by many species of birds. Bird movements occur as a response to changes in food availability, habitat, or weather. Approximately 1800

bacteria based on the taxonomic information obtained by NGS.

studying bacterial phylogeny in various environments [32].

studies of new phyla [34].

38 Metagenomics for Gut Microbes

5. Flyway

4. Variable region of the 16S rRNA gene

For field samples, it is important to confirm that the specimens are derived from the desired avian host. Mitochondrial DNA (mtDNA) sequences from avian hosts are ideal for this purpose because they provide phylogenetic information and a high copy number in host cells. Kenzaka et al. [39] amplified avian host DNAs by PCR with primers L5216 (5´-ACTCTTRTT-TAAGGCTTTGAAGGC-3<sup>0</sup> ) and H6313 (5´-GGCCCATACCCCGRAAATG-3<sup>0</sup> ) targeting the NADH dehydrogenase subunit 2 (ND2) gene and determined the sequences to confirm the avian host feces [40]. The mtDNA sequences from a variety of avian species are available in DNA database (e.g., GenBank).

#### 6.2. Eurasian wigeon

The Eurasian wigeon (Mareca penelope or Anas penelope) breeds in the northernmost areas of Europe and Asia. The size of the wigeon is approximately 50 cm in length (Figure 1a). The global population is estimated approximately 2.8–3.3 million individuals [41]. The species is strongly migratory, undertaking significant cold weather movements of varying magnitudes. It leaves its breeding grounds in late summer to arrive in its wintering grounds across Europe and Asia in October and November. It lives primarily in lakes, rivers, and along coastlines and

Figure 1. Photographs of (a) Eurasian wigeon and (b) barn swallow.

prefers a location near water plants and land plants that it can eat. The number of observed individuals in Japan has been about 180,000 per year.

Kenzaka et al. collected fresh feces from the Eurasian wigeon that had fallen on plant surfaces along the southern coast of Lake Biwa (Japan) [39]. From this research, most fecal sample bacterial communities were dominated by the phyla Firmicutes (51.7%) and Proteobacteria (45.1%), composing an average of about 97% (Figure 2a). At the family level, on average, Enterobacteriaceae composition was 37.6%, Bacillaceae was 21.5%, Paenibacillaceae was 16.5%, Clostridiaceae was 7.5%, and Pseudomonadaceae was 6.3% (Figure 2b). Although there were individual differences, these families were the dominant groups in all samples collected.

Detected genera that have been reported in association with human pathogenicity are shown in Table 1. The genera Pantoea, Bacillus, Paenibacillus, Pseudomonas, Clostridium, Escherichia/ Shigella, Helicobacter, and Serratia were found at a rate of more than 0.1% of total sequences. On the other hand, the genus Campylobacter, which is present in various birds and known as causative bacteria of food poisoning [42], was detected but composed less than 0.1% of the bacterial community in 60% of the samples. Compositions for both the genus Listeria, a zoonotic infectious pathogen-causing listeriosis [43], and the genus Pasteurella, a pathogen of poultry cholera [5], were less than 0.1% in all of the samples.

> In Osaka Prefecture (Japan), Kenzaka et al. collected specimens of fresh swallow feces from under a mating pair's nest, made at the edge of a private house or artificial building [45]. Figure 2c shows the results of the bacterial community composition analysis (at the phylum level) found in swallow feces. Most fecal samples were dominated by Proteobacteria (72.1%), Firmicutes (15.9%), and Tenericutes (5.7%), composing on average about 94% of the bacterial community. Moreover, the proportion of Bacteroidetes, which is a human gut-dominant bacterial phylum, was about 0.4%. On the family level, Enterobacteriaceae composition was about 53.3% on average, Pseudomonadaceae was 13.6%, Mycoplasmataceae was 5.5%, Enterococcaceae was 4.8%, Streptococcaceae was 4.6%, Alcaligenaceae was 4.3%, Lactobacillaceae was 1.7%, and Coxiellaceae was ~1.3% (Figure 2d). Although there were individual differences, any of these

> Figure 2. Relative proportions of bacterial phylotypes in individual fecal samples of barn swallow shown at the

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(a) phylum level and (b) family level and of Eurasian wigeon at the (c) phylum level and (d) family level.

Table 1 shows the major genera with high abundance, namely, Pseudomonas spp., Escherichia/ Shigella spp., Enterobacter spp., Yersinia spp., Mycoplasma spp., Enterococcus spp., Achromobacter spp., Fusobacterium spp., and Serratia spp. All of these genera include species that are reported as

bacterial groups dominated more than 10% in all samples.

#### 6.3. Barn swallow

The barn swallow (Hirundo rustica) has a total length of about 17 cm, and its weight is about 18 g (Figure 1b). The global population is estimated more than 190 million individuals approximately. This species breeds in a wide range of climates and over a wide range of altitudes, preferring open country like farmland and near water and buildings that provide nesting sites. The barn swallow is primarily a rural species in Europe and North America, while in North Africa and Asia, it often breeds in towns and cities [44]. Many swallows migrate to Japan from Southeast Asia (i.e., Philippines, Malaysia, Indonesia, etc.) and breed near human-living environments, such as private houses and the eaves. Swallows mainly feed on insects. After breeding, they gather at river beds and reed borders, forming group roosts of 1000–10,000 of individuals, and then return to Southeast Asia in autumn. The number of observed individuals in Japan is estimated at several hundred thousand birds per year.

Public Health Implications of Intestinal Microbiota in Migratory Birds http://dx.doi.org/10.5772/intechopen.72456 41

prefers a location near water plants and land plants that it can eat. The number of observed

Kenzaka et al. collected fresh feces from the Eurasian wigeon that had fallen on plant surfaces along the southern coast of Lake Biwa (Japan) [39]. From this research, most fecal sample bacterial communities were dominated by the phyla Firmicutes (51.7%) and Proteobacteria (45.1%), composing an average of about 97% (Figure 2a). At the family level, on average, Enterobacteriaceae composition was 37.6%, Bacillaceae was 21.5%, Paenibacillaceae was 16.5%, Clostridiaceae was 7.5%, and Pseudomonadaceae was 6.3% (Figure 2b). Although there were individual differences, these families were the dominant groups in all samples collected.

Detected genera that have been reported in association with human pathogenicity are shown in Table 1. The genera Pantoea, Bacillus, Paenibacillus, Pseudomonas, Clostridium, Escherichia/ Shigella, Helicobacter, and Serratia were found at a rate of more than 0.1% of total sequences. On the other hand, the genus Campylobacter, which is present in various birds and known as causative bacteria of food poisoning [42], was detected but composed less than 0.1% of the bacterial community in 60% of the samples. Compositions for both the genus Listeria, a zoonotic infectious pathogen-causing listeriosis [43], and the genus Pasteurella, a pathogen of

The barn swallow (Hirundo rustica) has a total length of about 17 cm, and its weight is about 18 g (Figure 1b). The global population is estimated more than 190 million individuals approximately. This species breeds in a wide range of climates and over a wide range of altitudes, preferring open country like farmland and near water and buildings that provide nesting sites. The barn swallow is primarily a rural species in Europe and North America, while in North Africa and Asia, it often breeds in towns and cities [44]. Many swallows migrate to Japan from Southeast Asia (i.e., Philippines, Malaysia, Indonesia, etc.) and breed near human-living environments, such as private houses and the eaves. Swallows mainly feed on insects. After breeding, they gather at river beds and reed borders, forming group roosts of 1000–10,000 of individuals, and then return to Southeast Asia in autumn. The number of observed individuals

individuals in Japan has been about 180,000 per year.

Figure 1. Photographs of (a) Eurasian wigeon and (b) barn swallow.

poultry cholera [5], were less than 0.1% in all of the samples.

in Japan is estimated at several hundred thousand birds per year.

6.3. Barn swallow

40 Metagenomics for Gut Microbes

Figure 2. Relative proportions of bacterial phylotypes in individual fecal samples of barn swallow shown at the (a) phylum level and (b) family level and of Eurasian wigeon at the (c) phylum level and (d) family level.

In Osaka Prefecture (Japan), Kenzaka et al. collected specimens of fresh swallow feces from under a mating pair's nest, made at the edge of a private house or artificial building [45]. Figure 2c shows the results of the bacterial community composition analysis (at the phylum level) found in swallow feces. Most fecal samples were dominated by Proteobacteria (72.1%), Firmicutes (15.9%), and Tenericutes (5.7%), composing on average about 94% of the bacterial community. Moreover, the proportion of Bacteroidetes, which is a human gut-dominant bacterial phylum, was about 0.4%. On the family level, Enterobacteriaceae composition was about 53.3% on average, Pseudomonadaceae was 13.6%, Mycoplasmataceae was 5.5%, Enterococcaceae was 4.8%, Streptococcaceae was 4.6%, Alcaligenaceae was 4.3%, Lactobacillaceae was 1.7%, and Coxiellaceae was ~1.3% (Figure 2d). Although there were individual differences, any of these bacterial groups dominated more than 10% in all samples.

Table 1 shows the major genera with high abundance, namely, Pseudomonas spp., Escherichia/ Shigella spp., Enterobacter spp., Yersinia spp., Mycoplasma spp., Enterococcus spp., Achromobacter spp., Fusobacterium spp., and Serratia spp. All of these genera include species that are reported as


the genus Pseudomonas belonged to phylum Proteobacteria, and Arthrobacter belonged to Actinobacteria. Wang et al. compared the bacterial compositions between wild and artificially reared populations of bar-headed geese [48]. They found that Bacteroidetes was significantly

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They also reported on functional profiling and found that artificially reared bar-headed geese had more genes related to carbohydrate transport and metabolism, energy metabolism and

Ryu et al. examined intestinal microbiota of migrating shorebirds in Delaware Bay (Delaware, United States) on Atlantic flyway using a 16S rRNA clone library [50]. The authors collected the pellets from ruddy turnstones, red knots, and semipalmated sandpipers, which use the Atlantic flyway. The flyway route generally follows the Atlantic Coast of North America and

The ruddy turnstone (Arenaria interpres) is a small wading bird. The global population is estimated approximately 460,000–730,000 individuals [51]. The ruddy turnstone breeds in northern latitudes around the sea. A subspecies occurs in Northern Alaska and in Arctic Canada, Greenland, Northern Europe, and Northern Russia. In the America, the species winters on coastlines from Washington and Massachusetts southward to the southern tip of South America. The red knot (Calidris canutus) is a medium-sized shorebird. The global population is estimated approximately 891,000–979,000 individuals [52]. The species has an extremely large range, breeding from Alaska across the Arctic to Greenland and Northern Russia. It winters on the Atlantic and Pacific coasts of North and South America, as well as Northwestern Europe. The semipalmated sandpiper (Calidris pusilla) is a very small shorebird. The global population was estimated at about 2 million individuals in 2006 [52]. It is a common breeder in the Arctic and subarctic, from Far Eastern Siberia east across Alaska and Northern Canada to Baffin Island and Labrador. In the non-breeding season, the species uses coastal estuarine habitats, wintering on the Pacific coast from Mexico to Peru and on the Atlantic coast from the Yucatan and the West Indies south to central Argentina. At one particular staging site in Delaware Bay, thousands of these shorebirds aggregate every spring to refuel for their

Of about 4000 16S rRNA clone sequences analyzed from these shorebirds, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacterium (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). The high abundance of Firmicutes in shorebird excreta was consistent with other avian studies. At the genus level, three main genera, Bacillus spp., Catellicoccus spp., and Lysinibacillus spp., constituted about 60% of the total sequences. The relatively low abundance of phylum Bacteroidetes and genus Bacteroides in shorebird excreta also was consistent with other avian studies. Analysis of epsilonproteobacterium-specific 23S rRNA gene clone libraries showed that sequences were dominated by Campylobacter (82.3%) or Helicobacter (17.7%) spp. In particular, Campylobacter jejuni, C. coli, and C. lari are known to be pathogenic species causing human gastroenteritis worldwide. C. lari constituted about 30% of the total Epsilonproteobacteria clones, but the pathogenic species of C. jejuni and C. coli were not

more abundant in the artificially reared population compared to the wild population.

coenzyme transport, and metabolism, in general.

6.5. Shorebirds

the Appalachian Mountains.

migration to the Canadian Arctic.

detected in the feces of the three shorebird species.

Table 1. Relative proportion of OTUs belonged to representative genus in feces samples determined by 16S metagenomics sequencing.

pathogenic to humans. The genus Campylobacter was detected in some samples but at a rate of <0.1% in only 40% of the samples. The genera Pasteurella and Listeria composed of <0.1% in all samples. Also, Bacteroides spp., Bifidobacterium spp., and Prevotella spp., which are all commonly dominant in the human intestine [46, 47], comprised <0.1% in more than 90% of samples.

#### 6.4. Bar-headed goose

Wang et al. examined metagenomic profiling of gut microbial communities in both wild and artificially reared bar-headed geese in China [48]. The bar-headed goose (Anser indicus) breeds in the high plateaus of Central Asia in colonies of thousands near mountain lakes and winters in South Central Tibet and India. This species has been reported as migrating south from Tibet, Kazakhstan, Mongolia, and Russia, crossing the Himalayas [49].

The authors found that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the four most abundant phyla in the gut of bar-headed geese. In wild bar-headed geese, the predominant phylum was the Firmicutes, with an average relative abundance of 83.2%. The second most predominant bacterial lineage, constituting 11.8%, was identified as phylum Proteobacteria, followed by Actinobacteria and Bacteroidetes, accounting for 2.5 and 0.9%, respectively, of the relative abundance.

At the genus level, the sequences from the wild samples represented 106 genera. Four major genera (Streptococcus, Lactococcus, Bacillus, and Enterococcus) belonged to phylum Firmicutes,

the genus Pseudomonas belonged to phylum Proteobacteria, and Arthrobacter belonged to Actinobacteria. Wang et al. compared the bacterial compositions between wild and artificially reared populations of bar-headed geese [48]. They found that Bacteroidetes was significantly more abundant in the artificially reared population compared to the wild population.

They also reported on functional profiling and found that artificially reared bar-headed geese had more genes related to carbohydrate transport and metabolism, energy metabolism and coenzyme transport, and metabolism, in general.

#### 6.5. Shorebirds

pathogenic to humans. The genus Campylobacter was detected in some samples but at a rate of <0.1% in only 40% of the samples. The genera Pasteurella and Listeria composed of <0.1% in all samples. Also, Bacteroides spp., Bifidobacterium spp., and Prevotella spp., which are all commonly dominant in the human intestine [46, 47], comprised <0.1% in more than 90% of samples.

Table 1. Relative proportion of OTUs belonged to representative genus in feces samples determined by 16S

Genus Relative proportion (%)<sup>a</sup>

Pseudomonas spp. 33.2 <0.1 Escherichia/Shigella spp. 21.1 <0.1 Enterobacter spp. 16.5 <0.1 Yersinia spp. 6.1 17.7 Mycoplasma spp. 5.7 <0.1 Enterococcus spp. 3.1 13.4 Achromobacter spp. 0.4 <0.1 Fusobacterium spp. 0.1 0.2 Serratia spp. <0.1 11.2 Pantoea spp. <0.1 9.9 Bacillus spp. <0.1 9.2 Paenibacillus spp. <0.1 7.2 Clostridium spp. <0.1 4.8 Helicobacter spp. <0.1 0.8

Eurasian wigeon Barn swallow

Wang et al. examined metagenomic profiling of gut microbial communities in both wild and artificially reared bar-headed geese in China [48]. The bar-headed goose (Anser indicus) breeds in the high plateaus of Central Asia in colonies of thousands near mountain lakes and winters in South Central Tibet and India. This species has been reported as migrating south from Tibet,

The authors found that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the four most abundant phyla in the gut of bar-headed geese. In wild bar-headed geese, the predominant phylum was the Firmicutes, with an average relative abundance of 83.2%. The second most predominant bacterial lineage, constituting 11.8%, was identified as phylum Proteobacteria, followed by Actinobacteria and Bacteroidetes, accounting for 2.5 and 0.9%, respectively, of the

At the genus level, the sequences from the wild samples represented 106 genera. Four major genera (Streptococcus, Lactococcus, Bacillus, and Enterococcus) belonged to phylum Firmicutes,

Kazakhstan, Mongolia, and Russia, crossing the Himalayas [49].

6.4. Bar-headed goose

>0.1% of total OTUs.

metagenomics sequencing.

42 Metagenomics for Gut Microbes

a

relative abundance.

Ryu et al. examined intestinal microbiota of migrating shorebirds in Delaware Bay (Delaware, United States) on Atlantic flyway using a 16S rRNA clone library [50]. The authors collected the pellets from ruddy turnstones, red knots, and semipalmated sandpipers, which use the Atlantic flyway. The flyway route generally follows the Atlantic Coast of North America and the Appalachian Mountains.

The ruddy turnstone (Arenaria interpres) is a small wading bird. The global population is estimated approximately 460,000–730,000 individuals [51]. The ruddy turnstone breeds in northern latitudes around the sea. A subspecies occurs in Northern Alaska and in Arctic Canada, Greenland, Northern Europe, and Northern Russia. In the America, the species winters on coastlines from Washington and Massachusetts southward to the southern tip of South America. The red knot (Calidris canutus) is a medium-sized shorebird. The global population is estimated approximately 891,000–979,000 individuals [52]. The species has an extremely large range, breeding from Alaska across the Arctic to Greenland and Northern Russia. It winters on the Atlantic and Pacific coasts of North and South America, as well as Northwestern Europe. The semipalmated sandpiper (Calidris pusilla) is a very small shorebird. The global population was estimated at about 2 million individuals in 2006 [52]. It is a common breeder in the Arctic and subarctic, from Far Eastern Siberia east across Alaska and Northern Canada to Baffin Island and Labrador. In the non-breeding season, the species uses coastal estuarine habitats, wintering on the Pacific coast from Mexico to Peru and on the Atlantic coast from the Yucatan and the West Indies south to central Argentina. At one particular staging site in Delaware Bay, thousands of these shorebirds aggregate every spring to refuel for their migration to the Canadian Arctic.

Of about 4000 16S rRNA clone sequences analyzed from these shorebirds, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacterium (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). The high abundance of Firmicutes in shorebird excreta was consistent with other avian studies. At the genus level, three main genera, Bacillus spp., Catellicoccus spp., and Lysinibacillus spp., constituted about 60% of the total sequences. The relatively low abundance of phylum Bacteroidetes and genus Bacteroides in shorebird excreta also was consistent with other avian studies. Analysis of epsilonproteobacterium-specific 23S rRNA gene clone libraries showed that sequences were dominated by Campylobacter (82.3%) or Helicobacter (17.7%) spp. In particular, Campylobacter jejuni, C. coli, and C. lari are known to be pathogenic species causing human gastroenteritis worldwide. C. lari constituted about 30% of the total Epsilonproteobacteria clones, but the pathogenic species of C. jejuni and C. coli were not detected in the feces of the three shorebird species.

#### 6.6. Bacterial community composition in migratory and nonmigratory birds

Application of NGS for analyzing the intestinal flora of various animals, including humans, is rapidly increasing. In studies on nonmigratory birds, such as chickens, turkeys, ducks, and penguins, Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria are reported to be high at the phylum level in all birds [53–55]. In particular, Firmicutes was present in almost all bird samples, while Proteobacteria and Bacteroidetes were present in about 90% of samples. It has been reported that Tenericutes was present in about 60% of samples. In the swallow samples, it was characteristic that Proteobacteria occupied a high percentage of the community, 50% or more, but the proportions of phylum Bacteroidetes, represented by genera Bacteroides, Bifidobacterium, and Prevotella, which are widely present in human intestines, were low.

In the case of the Eurasian wigeon, it was characteristic that the proportions of Bacteroidetes, Actinobacteria, and Tenericutes were lower, which is different from other birds. Also, genera Bacteroides and Bifidobacterium, which were widely present in human intestine, were low although the genera which belonged to Firmicutes and Proteobacteria were high.

Figure 3 shows the relative proportions of bacterial phylotypes in intestinal microbial communities of the Eurasian wigeon, the barn swallow, other birds, and mammals registered in DNA database GenBank. Figure 4 shows the results of principal component analysis comparing the similarities between the intestinal microbial communities of the migratory birds with other birds and

mammals registered. It is highly likely that migratory birds may eat different foods from individual to individual, so differences across individuals are large compared to poultry; however, compared with other organisms (□, ■ in Figure 4), individual intestinal microbiota from the Eurasian wigeon (▼) and the swallow (○) were relatively similar. In particular, intestinal bacterial composition was found to be greatly different from mammals, such as swine, beef cattle, and dairy cattle (■). It seems that each intestinal bacterial community is formed by the food consumed, whether it is an insect

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For comprehensive analysis by NGS of eukaryotic parasite, 18S rRNA gene, 28S rRNA gene, or cytochrome c oxidase I (COX1) gene on mitochondrial DNA has been used. The universal primers can amplify species across a broad variety of taxa, making them a time- and costeffective alternative to group-specific primers. Using multiple markers may provide a broader taxonomic resolution of biological communities including diet. The diversity of sequences that can be detected by universal primers is often compromised by high concentrations of DNA templates of some groups. Moreover, up to 90% of the sequences obtained from NGS can be

meal, an herbivorous meal, an omnivorous meal, a carnivorous meal, and so on.

Figure 4. Principal component analysis of class abundance data from migratory birds and others.

7. Protozoa and fungi in migratory birds

Figure 3. Relative proportions of bacterial phylotypes shown at the class level in gut samples of migratory birds and others.

Figure 4. Principal component analysis of class abundance data from migratory birds and others.

mammals registered. It is highly likely that migratory birds may eat different foods from individual to individual, so differences across individuals are large compared to poultry; however, compared with other organisms (□, ■ in Figure 4), individual intestinal microbiota from the Eurasian wigeon (▼) and the swallow (○) were relatively similar. In particular, intestinal bacterial composition was found to be greatly different from mammals, such as swine, beef cattle, and dairy cattle (■). It seems that each intestinal bacterial community is formed by the food consumed, whether it is an insect meal, an herbivorous meal, an omnivorous meal, a carnivorous meal, and so on.

#### 7. Protozoa and fungi in migratory birds

6.6. Bacterial community composition in migratory and nonmigratory birds

44 Metagenomics for Gut Microbes

although the genera which belonged to Firmicutes and Proteobacteria were high.

Application of NGS for analyzing the intestinal flora of various animals, including humans, is rapidly increasing. In studies on nonmigratory birds, such as chickens, turkeys, ducks, and penguins, Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria are reported to be high at the phylum level in all birds [53–55]. In particular, Firmicutes was present in almost all bird samples, while Proteobacteria and Bacteroidetes were present in about 90% of samples. It has been reported that Tenericutes was present in about 60% of samples. In the swallow samples, it was characteristic that Proteobacteria occupied a high percentage of the community, 50% or more, but the proportions of phylum Bacteroidetes, represented by genera Bacteroides, Bifidobacterium, and Prevotella, which are widely present in human intestines, were low.

In the case of the Eurasian wigeon, it was characteristic that the proportions of Bacteroidetes, Actinobacteria, and Tenericutes were lower, which is different from other birds. Also, genera Bacteroides and Bifidobacterium, which were widely present in human intestine, were low

Figure 3 shows the relative proportions of bacterial phylotypes in intestinal microbial communities of the Eurasian wigeon, the barn swallow, other birds, and mammals registered in DNA database GenBank. Figure 4 shows the results of principal component analysis comparing the similarities between the intestinal microbial communities of the migratory birds with other birds and

Figure 3. Relative proportions of bacterial phylotypes shown at the class level in gut samples of migratory birds and

others.

For comprehensive analysis by NGS of eukaryotic parasite, 18S rRNA gene, 28S rRNA gene, or cytochrome c oxidase I (COX1) gene on mitochondrial DNA has been used. The universal primers can amplify species across a broad variety of taxa, making them a time- and costeffective alternative to group-specific primers. Using multiple markers may provide a broader taxonomic resolution of biological communities including diet. The diversity of sequences that can be detected by universal primers is often compromised by high concentrations of DNA templates of some groups. Moreover, up to 90% of the sequences obtained from NGS can be less-degraded host DNA [56]. If the DNA within the sample contains a small number of interesting sequences in relatively high concentrations of non-interesting sequences, less sequences are often not amplified. In this case, the use of annealing inhibiting primers which overlap with the 3<sup>0</sup> end of one of the universal primers is effective [57]. The inclusion of primers to block host DNA amplification can increase the number of nonhost sequences significantly.

Author details

References

Takehiko Kenzaka\* and Katsuji Tani

science.286.5448.2331

2007.9674363

24.2.299

mimet.2011.04.006

DOI: 10.1128/mBio.01098-14

\*Address all correspondence to: kenzat@osaka-ohtani.ac.jp Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan

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As fungi contained in the intestinal tract of seabirds, Blastocladiomycota, Chytridiomycota, Entomophthoromycotina, Ascomycota, Mucoromycotina, and Basidiomycota have been detected [58, 59]. Nebela spp., Alveolata, Stramenopila, Rhizaria, Amoebozoa, Excavata, Choanoflagellatea, Glaucophyta, Cryptophyceae, Chlorophyceae, Trebouxiophyceae, Ulvophyceae, Prasinophyceae, and Mamiellophyceae have been detected as protozoa contained in the intestinal tract of seabirds.
