3. Methodology

Waterfowls such as ducks, geese, coots, and cormorants can play a role in the environmental dissemination of Giardia cysts and Cryptosporidium oocysts [4]. Migratory birds are also thought to be one of the mechanisms responsible for the wide geographic distribution of various human

Organized and periodic surveillance methods of bacterial pathogens are not as efficient as those for the highly pathogenic avian influenza. Avian cholera, caused by Pasteurella multocida, occurs sporadically in various parts of the world, including North America and Europe, where migratory birds are also implicated as a vector [5]. Many human enteric pathogenic bacteria have been isolated from wild birds [6]. Additionally, studies reported that antibiotic-resistant bacteria travel long distances through migratory birds [7]. Therefore, migratory birds carry a wide range of viruses, bacteria, protozoa, fungi, and other microorganisms that may be

Attempts have been made to clarify the microbiota contained within migratory birds' feces using new culture-independent genetic-based methods such as next-generation sequencing (NGS). Research on the spreading of bacterial populations over the vast distances has led to the elucidation of the roles of migratory birds regarding human health risks. Further studies of the spatial and temporal distribution of pathogenic bacteria in wild birds will enhance the assessment of their roles, thereby enabling the prediction of potential outbreaks based on migratory patterns. NGS is useful for the understanding of bacterial diversity and the discovery of novel bacteria [9]. This review considers the potential role of wild birds in the transmission of intestinal microbiota and our current knowledge of microbiota associated with

The majority of information regarding enteropathogens in wild birds has been ascertained by applying traditional microbiological techniques [10–12]. Research on the fecal flora of wild birds in a few well-studied species has focused on the prevalence of enteropathogenic bacteria that are most likely zoonotic. Representative species that are often found in diseased wild birds include Salmonella enterica serotypes Typhimurium and Enteritidis, Escherichia coli, Campylobacter jejuni, Pseudomonas aeruginosa, Pasteurella multocida, Clostridium botulinum, and Listeria monocytogenes. The carrier birds are often diverse species, including house sparrows, brown-headed cowbirds, white-throated sparrows, tufted ducks, crows, pheasants, pigeons, hook-bills, finches, free-living

Many classical methods such as microscopy, culture, or serology have been used to detect and isolate pathogenic protozoa or fungi from excreta of migratory birds. Candida albicans and Candida tropicalis, which can become pathogenic in immunocompromised individuals, have been isolated from excreta of migratory gulls and geese [13]. Toxoplasma gondii has been isolated from many migratory species including ducks and raptors. Eimeria spp., which cause severe intestinal coccidiosis, are distributed by geese and diving ducks. These infections are mainly caused by oral ingestion of oocysts excreted from feces. The oocysts of Cryptosporidium baileyi, an intracellular enteric coccidian parasite that can cause gastrointestinal and respiratory

pathogenic protozoans.

36 Metagenomics for Gut Microbes

transmitted to humans [8].

migratory birds using NGS technologies.

2. Intestinal microbiota in wild birds

flamingos, quails, pheasants, red grouse, and waterfowl [1, 6, 8].

Traditionally, studies on microbial community composition have been based on culturedependent methods [17]. When classifying and identifying bacteria, a combination of simple methods has helped to distinguish bacteria based on morphology, dyeability, and biochemical properties. Since automated devices have been developed, it is possible to assure the quality of microbial-based data in pharmaceutical fields and diagnostics fields as standards. When in need of more specific classification, sequence decoding for a specific gene or DNA fingerprinting is carried out. When determining bacterial species, it is a standard practice to analyze the 16S rRNA gene.

However, traditional culture methods underestimate bacterial populations because many environmental bacteria cannot yet to be cultivated by conventional laboratory techniques [18, 19]. Traditional microbiological methods of detection, enumeration, and identification using culture methods are often time-consuming and labor-intensive. These practical considerations often limit the extent to which microbiological tests are routinely applied to community analysis.

Various approaches have been developed to identify microorganisms in natural samples without the requirement for laboratory cultivation [20–23]. Since the latter half of the 1990s, direct sequencing approaches for 16S ribosomal RNA gene amplicon have been popularized. In these methodologies, DNA is extracted directly from the sample without culturing the bacteria. Target DNA is amplified by polymerase chain reaction (PCR) using a universal primer set targeting a conserved region of the 16S rRNA gene or a genus-specific primer, and then sequencing follows to identify the bacterial community members. Because the amplicons are mixed molecules derived from numerous complex bacterial species, PCR products should be separated using denaturing gradient gel electrophoresis or a clone library method in combination [24, 25]. With these methods, the number of bacterial clones that can be screened at one time is limited from several tens to thousands and proves to be labor-intensive.

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].

of the world's 10,000 bird species are long-distance migrants [35]. The bar-tailed godwit, Limosa lapponica, undertakes one of the avian world's most extraordinary migratory journeys. Recent research revealed that some individuals had made nonstop flights over 11,000 km, the longest

Public Health Implications of Intestinal Microbiota in Migratory Birds

http://dx.doi.org/10.5772/intechopen.72456

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Many species migrate along broadly similar, well-established routes, known as flyways. Recent research has identified nine such pathways: the East Atlantic, the Mediterranean/Black Sea, the West Asia/East Africa, the Central Asia, the East Asia/Australasia, and four flyways in the Americas [37]. The most common pattern involves flying north in the spring to breed in the temperate or Arctic summer and then returning south in the fall to warmer wintering grounds. Migration is often annual and is linked closely with the cyclic pattern of the seasons. Migration is most evident among birds, which have a highly efficient means for traveling swiftly over

The East Asia/Australasia flyway extends from Siberia and North America to the southern limits of Australia and New Zealand. It encompasses large parts of East Asia, all of Southeast Asia, and includes eastern India and the Andaman and Nicobar Islands. The scale of avian movements along the flyway is awesome, with over 50 million migratory birds using the route annually [38]. Bacterial community compositions in migratory birds from the East Asia/Aus-

A comprehensive analysis of the bacterial community structure in migratory birds using

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-

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

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

) and H6313 (5´-GGCCCATACCCCGRAAATG-3<sup>0</sup>

) targeting the

continuous journey that has ever been recorded for a bird [36].

tralasia flyway are described in the section below.

culture-independent methods is introduced below.

6.1. Confirmation of avian host

TAAGGCTTTGAAGGC-3<sup>0</sup>

6.2. Eurasian wigeon

DNA database (e.g., GenBank).

6. Bacterial community composition in migratory birds

long distances.

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 bacteria based on the taxonomic information obtained by NGS.
