Comparison of microbiota in the cloaca, colon, and magnum of layer chicken

Anatomically terminal parts of the urinary, reproductive, and digestive systems of birds all connect to the cloaca. As the feces drain through the cloaca in chickens, the cloacal bacteria were previously believed to represent those of the digestive system. To investigate similarities between the cloacal microbiota and the microbiota of the digestive and reproductive systems, microbiota inhabiting the colon, cloaca, and magnum, which is a portion of the chicken oviduct of 34-week-old, specific-pathogen-free hens were analyzed using a 16S rRNA metagenomic approach using the Ion torrent sequencer and the Qiime2 bioinformatics platform. Beta diversity via unweighted and weighted unifrac analyses revealed that the cloacal microbiota was significantly different from those in the colon and the magnum. Unweighted unifrac revealed that the cloacal microbiota was distal from the microbiota in the colon than from the microbiota in the magnum, whereas weighted unifrac revealed that the cloacal microbiota was located further away from the microbiota in the magnum than from the microbiota inhabiting the colon. Pseudomonas spp. were the most abundant in the cloaca, whereas Lactobacillus spp. and Flavobacterium spp. were the most abundant species in the colon and the magnum. The present results indicate that the cloaca contains a mixed population of bacteria, derived from the reproductive, urinary, and digestive systems, particularly in egg-laying hens. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken’s cloacal microbiota should be performed using chickens of different ages and types.


Introduction
Avian gut microbiota displays certain features. First, avian gut microbiota aid in protecting host birds from pathogens and contribute to the development of the immune system of the hosts [1]. Second, antibiotics administered to these birds may affect the gut microbiota depending on the dose of the antibiotic used and the age of the birds [2]. Third, avian gut microbiota are saccharolytic rather than cellulolytic and help degrade polysaccharides contained in poultry feed [3]. Finally, gut microbes may be affected by the body temperature of their avian host [4]. The most abundant bacterial genus in chicken gut varied depending on the type of sample and measuring techniques for bacterial population used in previous studies. Studies using gut contents showed that the most abundant bacterial genus in chicken gut was Clostridium [5][6][7]. The most abundant bacterial genus in chicken feces was Bacteroides in lean chickens, but Clostridium in fat chickens [8]. Another study showed that the most abundant bacterial genus in chicken feces was Escherichia except unclassified genus [9], while the other study showed that the most abundant bacterial genus in chicken feces was Lactobacillus [10]. A Study used cloacal swabs showed that the most abundant bacterial genus in cloaca of broilers was Lactobacillus [11]. Usually feces were collected to study the gut microbiota, because collecting feces is non-invasive. However, cloacal swab was preferred for collecting individual samples from birds. Recently, gut microbiota of juvenile ostriches was compared with those of feces and cloaca. In the study, cloacal microbiota was far different to microbiota in colon and feces [12,13]. In contrast to this study, some of microbiota in cloaca of turkey were matched to microbiota in intestine in genus level [14]. These results raised the question of whether cloacal microbiota can represent the intestinal microbiota in chicken. Therefore, this study aimed to compare cloacal microbiota with those in colon and magnum, a part of oviduct in SPF laying hens.

Sample collections
Eleven 34-week-old SPF laying chickens were used in this study. All experimental procedures were approved by the institutional animal care and use committee of Konkuk University (approval number KU17103-1). Cloacae were swabbed using the CLASSIQ swabs (Coppan, Murrieta, CA, USA), which were then suspended in 2 ml phosphate-buffered saline (PBS). The suspended samples were stored at -20˚C until DNA extraction for a day. Birds were euthanized using CO 2 gas and the magnum in the oviducts and colons were aseptically harvested. Mucosal area of the magnum and colon were scraped using the back of a scalpel and suspended in 1 ml of PBS and stored at -20˚C until DNA extraction for a day. Ten 30-week-old Hy-Line brown commercial layer chicken carcasses were used for the isolation of Lactobacillus spp. from the cloaca, colon, and magnum. Each location was swabbed with the CLASSIQ swab and the swab was streaked on De Man, Rogosa and Sharpe agar (MRS) agar. Streaked MRS agars were incubated in 37˚C for 48 h. Species of all grown colonies were identified via Matrix-assisted laser desorption/ionization and time-of-flight (MALDI-TOF) spectrometry and species of colonies not identified via MALDI-TOF were identified via 16S rRNA sequencing with 357F and 926R primers.

Extraction of DNA and sequencing
Bacterial DNA was extracted in 1 ml of PBS using the DNeasy blood and tissue kit (Qiagen, Manchester, UK). Amplification of V2, V3, V4, V6-V7, and V9 regions of the 16S rRNA was conducted using primer sets from the Ion 16S Metagenomics kit (Thermo Fisher Scientific, Waltham, MA, USA). The Ion S5 XL sequencer and the Ion 530 chip were used for sequencing.

Sequence analysis
A Qiime2 platform [15] was used for metagenome analysis via the Greengenes database (13_8 release) as the 16s rRNA gene reference [16]. The first 15 bases of all reads were removed, each sequence was truncated at position 150, and reads below the phred quality score 15 were filtered using DADA2 [17]. Chimeric sequences were detected via vsearch [18] and removed.
Operational taxonomic units (OTUs) were constructed with filtered sequences using a 99% identity option. The OTUs were classified with a Naive Bayes classifier [19]. Sampling depth was set up to 3000 feature counts for diversity metrics and alpha rarefaction. One magnum sample was excluded because it showed very different microbial components compared to the other magnum samples. Alpha diversity was measured using the Shannon index for non-phylogenetic alpha diversity metric [20]. Beta diversity was measured using unweighted unifrac [21] and weighted unifrac [22] for phylogenetic beta diversity. The Emperor tool was used to visualize principal coordinates analysis (PCoA) plots [23]. To evaluate associations among microbiota in the cloaca, colon and magnum, the pairwise permutational multivariate analysis of variance (PERMANOVA) statistic was used and p-values were produced with 999 permutation tests. Relative frequencies of taxa for each group were displayed in bar plots. Differentially abundant taxa of each group were identified via analysis of microbiome composition (ANCOM) [24]. A SourceTracker2 [25] was used to calculate the contribution of microbiota in the colon and magnum to microbiota in the cloaca.

Sequencing results
The cloaca, colon, and magnum samples of 11 SPF hens were analyzed. Subsequently

Alpha diversity and beta diversity analysis
Alpha diversity of microbiota in the cloaca, colon, and magnum of 11 SPF hens were analyzed via the Shannon index, which is used to measure the non-phylogenetic alpha diversity metric. The Shannon index of microbiota in the cloaca was lower than those in the colon and magnum (Fig 1). However, this difference was not significant as indicated by the pairwise Kruskal-Wallis test for the Shannon index (Table 2).
Beta-diversity analysis using an unweighted unifrac metric was performed to analyze distance among the microbiota in the cloaca, colon, and magnum. Microbiota in the cloaca, colon, and magnum were grouped separately on the PCoA plot (Fig 2).
In the pairwise PERMANOVA, the cloaca, colon, and magnum showed statistically significant differences in microbial composition, furthermore the microbiota in the cloaca and colon were farther apart than the microbiota in the cloaca and the magnum (Table 3).
Beta-diversity analysis using a weighted unifrac metric was also performed to analyze distance among the microbiota in the cloaca, colon, and magnum. Microbiota in the cloaca, colon, and magnum were grouped separately on the PCoA plot (Fig 3).
Pairwise PERMANOVA showed that the cloaca, colon, and magnum showed statistically significant differences in microbial composition, furthermore the microbiota in the cloaca and magnum were farther apart than the microbiota in the cloaca and colon (Table 4).

Taxonomic analysis
The relative taxa abundance plots at the genus level show the 20 most abundant taxa in the three groups. The most abundant genus in the cloaca was Pseudomonas, followed by Gallibacterium, Lactobacillus, Bacteroides, and unclassified Actinomycetales. The most abundant genus in the colon was Lactobacillus, followed by Bacteroides, unclassified Bacteroidales, unclassified Lachnospiraceae, and Faecalibacterium. The most abundant genus in the magnum was Flavobacterium, followed by Lactobacillus, unclassified Moraxellaceae, Pseudomonas, and Megamonas. To perform a taxonomic analysis of the shared microbiota in the cloaca, colon, and magnum, a sample each was pooled from one group respectively. Relative common taxa abundance plots at the genus level show the 10 most abundant taxa in the 3 groups (Fig 4). Lactobacillus spp. was the most abundant common taxa among each group.
The most abundant common genus in the cloaca was Pseudomonas, followed by Lactobacillus, unclassified Burkholderiales, Megamonas, and unclassified Lachnospiraceae. The most abundant common genus in the colon was Lactobacillus, followed by Bacteroides, Faecalibacterium, unclassified Bacteroidales, and unclassified Lachnospiraceae. The most abundant common genus in the magnum was Lactobacillus, followed by Pseudomonas, Megamonas, unclassified Lachnospiraceae, and Faecalibacterium. The most abundant common genus among all groups was Lactobacillus, followed by Pseudomonas, Megamonas, Bacteroides, and unclassified Lachnospiraceae. There were 5 core taxa in the cloaca, 15 core taxa in the colon, and 20 core taxa in the magnum (Table 5).

PLOS ONE
Comparison of microbiota in the cloaca, colon, and magnum of layer chicken

Detection of Lactobacillus spp. at each location
Lactobacillus spp. was the most common genus among each group. However, since the sequencing results of metagenomic analysis using 16S rRNA amplicon usually are not accurate enough to determine the correct bacterial species, we could not say the detected Lactobacilli were the same species or not. Therefore, additionally the dominant species of Lactobacillus spp. inhabiting each sampling site were investigated using culture technique. Lactobacillus spp.

PLOS ONE
Comparison of microbiota in the cloaca, colon, and magnum of layer chicken from each location were identified via MALDI-TOF spectrometry and 16s rRNA sequencing. Eleven Lactobacillus spp. were detected in the cloaca, 5 in the colon, and 5 in the magnum. Lactobacillus reuteri was the most dominant Lactobacillus sp. in the cloaca and colon, and Lactobacillus vaginalis was the most dominant Lactobacillus sp. in the magnum (Fig 5).

Differential abundance analysis
ANCOM was used to identify differentially abundant genera among the cloaca, colon and magnum.

PLOS ONE
Comparison of microbiota in the cloaca, colon, and magnum of layer chicken unclassified Coriobacteriaceae, and unclassified Bacteroidales were differentially abundant in the colon (S1 Table). At the genus level, 56 genera were common to the cloaca, colon, and magnum (Fig 6).

PLOS ONE
Comparison of microbiota in the cloaca, colon, and magnum of layer chicken

Origin of microbiota in chicken cloaca
The SourceTracker 2 was used to analyze the origin of the microbiota in the cloaca and each sample from one group was pooled. When the cloaca was assigned as the sink, 0.0669 of microbiota in the colon and 0.0809 of microbiota in the magnum contributed to the microbiota in the cloaca, whereas the highest contribution (0.8714) to the microbiota in the cloaca was from an unknown source (Table 6).

Discussion
With the development of sequencing technology, research on gut microbiota is becoming active, and new roles of microorganisms in the intestine have been revealed [26]. Using a

PLOS ONE
Comparison of microbiota in the cloaca, colon, and magnum of layer chicken

PLOS ONE
suitable sample for the study of gut microbiota is a very important factor in obtaining valuable results. Cloacal swab is a non-invasive and multiple sampling method for the same individual for the study of poultry intestinal microbiota [13]. Anatomically, cloaca is connected to the end of the digestive system, however in case of a hen, it also connects to the urinary and reproductive systems [13], so there was a question of whether the microbiota of cloaca can represent gut microbiota. In this study, we compared and analyzed microbiota present in the colon, oviduct, and cloaca of laying hens to assess whether it is possible to study the intestinal microbiota of laying hens using cloacal swabs. The results of this study indicated that the cloacal microbiota was significantly different from those in the colon and the magnum in the beta diversity analysis. Since colon and magnum samples were taken with scalpel and cloaca samples with swab, there may be a possibility that the microbiota may be different due to the difference in sampling method. Results of beta diversity analysis were slightly different between unweighted unifrac and weighted unifrac. Unweighted unifrac is a qualitative measure that does not consider the relative abundance of taxa, whereas weighted unifrac is a quantitative measure that considers the relative abundance of taxa [22]. In relative taxa abundance, the most abundant common genus in the cloaca was Pseudomonas, while the most abundant common genus in the colon and magnum was Lactobacillus. The cloaca is more aerobic than the colon and the magnum [27], and Pseudomonas is an aerobic bacteria [28] that may easily colonize the cloaca compared to the colon and the magnum. The most abundant common genus among all different sites was Lactobacillus. We used SPF white leghorn chickens to perform 16S rRNA metagenome analysis, while the Hy-Line brown commercial chickens were used in order to culture Lactobacillus spp. in all sampling sites. Although it is possible that different Lactobacillus spp. present in different breeds of chicken, culture results were consistent with those of the 16S rRNA metagenome analysis as all sampling sties contained Lactobacillus spp. Lactobacillus reuteri was the most dominant Lactobacillus spp. in the cloaca and colon, while Lactobacillus vaginalis was the most dominant Lactobacillus spp. in the magnum. Lactobacillus reuteri is an inhabitant in gastrointestinal tract in mammal and bird. Administration of Lactobacillus reuteri could improve growth of chickens having avian growth depression [29] and protect chickens from Salmonella Enteritidis challenge infection [30]. Unfortunately, role of Lactobacillus vaginalis in chicken has never been studied before. Lactobacillus gasseri were observed in magnum and colon in this study. Lactobacillus gasseri has been reported that it can produce lactocillin [31] and bacteriocin which have antimicrobial activity [32]. A small number of Lactobacillus spp. abundance have been linked to the development of bacterial vaginosis in human [33,34]. According to our previous research [35], very few Lactobacillus spp. were present in the oviduct of unmatured pullets. Laying hen's oviduct can be more easily infected by external bacteria than unmatured pullets, which may be one of the reasons that Lactobacilli increase in the oviduct of laying hens. Probably in the oviduct of chicken, Lactobacilli can protect the host against pathogenic bacterial infections. Since different Lactobacillus spp. were present in the intestine and oviduct of laying hens, there is a possibility that variety Lactobacillus spp. may protect the host from different species of bacterial pathogens in different body sites. Cloacal Lactobacillus spp. probably formed by the mixed population of Lactobacilli derived from the magnum and colon, and some Lactobacillus spp., which were absent in both of the magnum and colon. It can be assumed that cloacal lactobacilli are derived from not only the magnum and colon but also an unknown source (i.e., the environment). When the Source-Tracker2 was used to find the original sources of the cloacal microbiota, the highest contribution (0.8714) was from an unknown source. Thus, in summation, although the colon and magnum contributed some species to the cloaca, overall, the microorganisms originating from the colon and the magnum were few. In conclusion, microbiota in the cloaca do not represent the microbiota in the digestive tract in egg laying chicken. Most notably, the SourceTracker2 results showed that the cloacal microbiota largely came from an unknown source, which is most likely an outside source from the ambient aerobic environment rather than from the digestive or reproductive track. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken's cloacal microbiota should be performed using chickens of different ages and types.