**4. Intestinal microbiota of adult and young barn swallows**

The barn swallow (*Hirundo rustica*) is about 17 cm in length and is widely distributed across the temperate and cold regions of Africa, the Eurasian continent, the northern end of Australia, and North America. Their global population is estimated at more than 190 million individuals [35]. Several swallows migrate to Japan from Southeast Asia (i.e., the Philippines, Malaysia, and Indonesia) and make their nests by mixing mud with plant pieces, feathers, and other such items near human living environments, such as the eaves of a house. While flying in the sky, the barn swallows catch insects to eat. After breeding, they return to Southeast Asia in the autumn season. The number of observed individuals in Japan is estimated at several hundred thousand birds per year.

For the study on the intestinal microbial communities of barn swallow, fecal specimens of the adult and young barn swallows were collected from the nest on the university campus and from various nests around the Osaka prefecture, Japan. The NGS analysis was performed on the V4 region of the 16S rRNA gene.

Diversity indices of intestinal bacterial communities in barn swallows were determined for each of the sampling locations (**Table 1**). The diversity index of young birds was found to be lower than that of adult birds. Similar results were reported from studies with swallows in Europe [36].

The stability of intestinal bacterial communities in wild birds has not yet been studied. We collected feces over a period of time from the same nest within the university campus (OOU 20) and compared changes in the intestinal bacterial communities between adult and young birds. The day when the birds were born was set as day 0, that before the birth as day –N, and that after the birth as day +N (where, N = 1, 2, 3, …; **Figure 1**). Fecal samples in negative and positive value days represent ones of parent and young birds, respectively.

Adult samples were dominated by *Corynebacteriaceae*, *Halomonadaceae*, and *Pseudomonadaceae* at the family level before the hatch. In contrast, young samples were dominated by *Enterococcaceae*, *Mycoplasmataceae*, and *Enterobacteriaceae* (**Figure 1a**).

**Figure 1b** depicts the similarity in the intestinal bacterial community at the family level by principal component analysis (PCA). The intestinal bacterial communities of the adult birds changed greatly over time, but the profiles of adult and young birds remained similar before and after hatching, and then the young birds showed a great change. The adult birds brought insects and the likes in the nest to feed the young ones; therefore, it seems that the intestinal bacterial community was temporarily similar because of the similar diets. Similar results were reported from studies with swallows in Europe [36].

To examine the extent of change in the intestinal bacterial community of the barn swallows living in the same nest, the change was compared with that in other nests. The sampling sites OOU17 and OOU20 are nests located about 50 m apart.


#### **Table 1.**

*Alpha diversity of fecal microbiota in adult and young barn swallow.*

*Dissemination of Intestinal Microbiota by Migratory Birds across Geographical Borders DOI: http://dx.doi.org/10.5772/intechopen.82707*

#### **Figure 1.**

*Change in the bacterial community composition of fecal samples of barn swallows living in the same nest. (a) Relative portions of bacterial phylotypes in averaged fecal samples at the family level (b) Principle component analysis of family abundance data. Fecal samples in negative and positive value days represent ones of parent and young birds, respectively.*

The sampling sites Wakayama were about 50 km away from the sites OOU20 and OOU17 and consisted of a plurality of nests at a distance of 100 m. As compared with these adult and young birds, no such characteristics were observed in the groups of adult or young birds or in specific nests (**Figure 2**). As seen in the figure regarding the change in the bacterial community at OOU 20, the extent of difference in the bacterial community over a period of time in the same nest was found to exceed the extent of the difference among different nests.

In order to verify whether there were similarities in the bacterial communities for each collection area, PCA was performed on those from the adult birds (**Figure 3a** and **b**). We examined four areas (northern Osaka, southern Osaka, northern Wakayama, and others); these areas were about 50 km apart and beyond the activity range of the insects that served as the swallows' diets. The distribution of the samples in the same nest at OOU20 is shown in gray in **Figure 3a**. The extent of difference in the bacterial community in the same nest surpassed that

**Figure 2.** *Principal component analysis of class abundance data from adult and young barn swallows.*

**Figure 3.**

*Similarity analysis of 16S rRNA gene data from adult barn swallows. (a) Sampling sites around Osaka, Japan. (b) Principal component analysis of class abundance data.*

among different nests. No relationship was found between the bacterial community composition and the geographical area in the fecal samples. Since the intestinal tract of swallows is short, it was speculated that the intestinal microbial community might be influenced by the daily diet and may accordingly change greatly in only a couple of days.
