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. 2020 Aug 4;15(8):e0237108. doi: 10.1371/journal.pone.0237108

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

Seo-Jin Lee 1, Seongwoo Cho 1, Tae-Min La 1, Hong-Jae Lee 1, Joong-Bok Lee 1, Seung-Yong Park 1, Chang-Seon Song 1, In-Soo Choi 1, Sang-Won Lee 1,*
Editor: Arda Yildirim2
PMCID: PMC7402502  PMID: 32750076

Abstract

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 [5–7]. 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.

Materials and methods

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 CO2 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.

Results

Sequencing results

The cloaca, colon, and magnum samples of 11 SPF hens were analyzed. Subsequently, 6,707,244 raw reads (mean 209,601.375 ± 88,595.49) were obtained (Table 1). Following filtering, 1,315,288 reads (mean 41,102.75 ± 27,937) were obtained and classified into 1192 OTUs, which clustered at a 99% identity level. The raw sequence reads were deposited in the NCBI sequence read archive under BioProject accession number: PRJNA604381.

Table 1. Raw reads, filtered reads, and total OTUs of each sample.

Samples Raw reads filtered reads OTUs
Cloaca1 218949 27012 203
Cloaca2 214261 29918 146
Cloaca3 262902 37777 152
Cloaca4 258339 30567 154
Cloaca5 252877 34276 170
Cloaca6 303497 37132 98
Cloaca7 340755 37701 111
Cloaca8 434301 70203 207
Cloaca9 208477 21132 127
Cloaca10 253007 27500 201
Cloaca11 209453 22595 148
Colon1 230704 8928 143
Colon2 190807 9963 154
Colon3 151946 5281 103
Colon4 149177 6690 120
Colon5 185545 3502 81
Colon6 172814 8161 139
Colon7 195808 6609 126
Colon8 98102 3474 83
Colon9 175641 8161 141
Colon10 184051 7398 97
Colon11 212088 8556 125
Magnum1 110363 7933 107
Magnum2 68544 6684 335
Magnum3 106573 7876 123
Magnum4 60056 7039 204
Magnum5 84874 11503 132
Magnum6 315157 22100 188
Magnum7 431246 34927 235
Magnum8 181004 29282 343
Magnum9 193660 24836 183
Magnum11 252266 27712 243
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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).

Fig 1. Comparison of the Shannon index between the cloaca, colon, and magnum.

Fig 1

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Microbiota in the cloaca, colon, and magnum of SPF laying hens were analyzed via Shannon’s index. (A) Rarefaction curve for Shannon's index. The dark blue line represents the cloaca, the orange line represents the magnum, and the light (sky) blue line represents the colon. (B) Shannon's index for each group. Box plots show the quartiles, median, and extremities of the values.

However, this difference was not significant as indicated by the pairwise Kruskal–Wallis test for the Shannon index (Table 2).

Table 2. Pairwise Kruskal-Wallis tests for Shannon’s index of each group.

Group 1 Group 2 H p-value q-value
Cloaca Colon 2.588214 0.107662 0.161492
Cloaca Magnum 4.462810 0.034640 0.103921
Colon Magnum 0.714050 0.398103 0.398103
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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).

Fig 2. PCoA plot based on unweighted unifrac distance matrix.

Fig 2

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PCoA plots demonstrating unweighted unifrac distance among microbiota in the cloaca, colon, and magnum of laying hens. Red spheres represent the cloaca, blue spheres represent the colon, and yellow diamonds represent the magnum.

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).

Table 3. Pairwise PERMANOVA results based on unweighted unifrac distance matrix.

Group 1 Group 2 pseudo-F p-value q-value
Cloaca Colon 15.239907 0.001 0.001
Cloaca Magnum 7.236330 0.001 0.001
Colon Magnum 13.728121 0.001 0.001
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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).

Fig 3. PCoA plot based on weighted unifrac distance matrix.

Fig 3

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PCoA plots demonstrating weighted unifrac distance among microbiota in the cloaca, colon, and magnum of laying hens. Red spheres represent the cloaca, blue spheres represent the colon, and yellow diamonds represent the magnum.

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).

Table 4. Pairwise PERMANOVA results based on weighted unifrac distance matrix.

Group 1 Group 2 pseudo-F p-value q-value
Cloaca Colon 8.492881 0.003 0.0030
Cloaca Magnum 10.851457 0.001 0.0015
Colon Magnum 17.966760 0.001 0.0015
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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.

Fig 4. Relative frequency of ten of the most abundant common taxa among all groups at the genus level.

Fig 4

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Ten of the most abundant taxa, classified by different colors, are shown. Each bar indicates the relative frequencies of ten of the most abundant common taxa among all groups at genus level.

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).

Table 5. Core taxa* of each sampling group.

Group Taxa
Cloaca Actinomyces
Enterococcus
Lactobacillus
Unclassified Actinomycetales
Unclassified Gammaproteobacteria
Colon Bacteroides
Coprobacillus
Lactobacillus
Megamonas
Unclassified Firmicutes
Unclassified Bacteroidales
Unclassified Burkholderiales
Unclassified Clostridiales
Unclassified RF39
Unclassified Coriobacteriaceae
Unclassified Lachnospiraceae
Unclassified Rikenellaceae
Unclassified Ruminococcaceae
Unclassified Veillonellaceae
Magnum Bacteroides
Brevundimonas
Faecalibacterium
Flavobacterium
Lactobacillus
Megamonas
Methylobacterium
Pseudomonas
Rhodobacter
Unclassified Betaproteobacteria
Unclassified Actinomycetales
Unclassified Bacteroidales
Unclassified Burkholderiales
Unclassified Clostridiales
Unclassified Caulobacteraceae
Unclassified Enterobacteriaceae
Unclassified Lachnospiraceae
Unclassified Microbacteriaceae
Unclassified Moraxellaceae
Unclassified Ruminococcaceae
Unclassified Xanthomonadaceae
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* Genera detected in all samples in each group were considered as core genera.

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. 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).

Fig 5. The distribution of Lactobacillus spp. detected at each location.

Fig 5

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Detected Lactobacillus spp. at each location are indicated with different colors. Each bar indicates the relative detected frequencies of Lactobacillus spp. among all groups.

Differential abundance analysis

ANCOM was used to identify differentially abundant genera among the cloaca, colon and magnum. Gallibacterium, Enterococcus, Janthinobacterium, unclassified Gammaproteobacteria, Actinomyces, Helococcus, unclassified Pasteurellaceae, Stenotrophomonas, Morganella, and Comamonas were differentially abundant in cloaca. Unclassified Actinomycetales, unclassified Enterobacteriaceae, Acinetobacter, unclassified Xanthomonadaceae, and Corynebacterium were differentially abundant in the cloaca and the magnum compared with the colon. Flavobacterium, unclassified Rhodobacteraceae, Brevundimonas, unclassified Microbacteriaceae, unclassified Caulobacteraceae, unclassified Flavobacteriaceae, Propionibacterium, Methylobacterium, and Rhodobacter were differentially abundant in the magnum. Unclassified RF39, 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).

Fig 6. Common and unique phylotypes at the genus level among each group.

Fig 6

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Venn diagram demonstrating the number of common or unique phylotypes at the genus level among the groups. Phylotypes observed in each part were counted.

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).

Table 6. Contribution of each source to each sink.

Sink Colon Magnum Cloaca Unknown
Colon - 0.1029(0.0171) 0.0257(0.007) 0.8714(0.0176)
Magnum 0.0192(0.0074) - 0.0111(0.0054) 0.9697(0.0093)
Cloaca 0.0669(0.0138) 0.0809(0.0089) - 0.8522(0.0161)
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* Standard deviations are in parentheses.

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 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 SourceTracker2 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.

Supporting information

S1 Table. Percentile abundance between groups.

(XLSX)

Click here for additional data file. (11.1KB, xlsx)

Data Availability

All raw sequence reads files are available from the NCBI database (accession number: PRJNA604381).

Funding Statement

This paper was supported by Konkuk University in 2016 to SWL.

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Decision Letter 0

Arda Yildirim

19 May 2020

PONE-D-20-06110

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

PLOS ONE

Dear Dr Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I feel that the manuscript is dealing with a good topic but lacks in the quality of preparation. I agree with reviewers in particular, I would like to inform that I care about criticism of referee#3 more and please review the referee comments and make your peer revision.

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Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The study is well presented, I feel that the manuscript is dealing with a good topic but lacks in the quality of preparation. The main problem found in the manuscript is related to the some aspects of the methodology, poor discussion and typo errors or ambiguous phrases or sentences. It is necessary to improve the manuscript by examining the questions that need to be clarified in a way. Please be aware of the manuscript should be presented according to guidelines for authors of Plos One. For your guidance, you can check the reviewers' comments.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is presented in an intelligible fashion. It is written well and clear. The data provided, along with the figures and it's discussion support the conclusions in this manuscript. The authors have made all data underlying the findings in their manuscript fully available.

Reviewer #2: In terms of methodology, the work is correct. Although the purpose of the study, according to the authors, is clearly defined, i.e. „ To investigate similarities between the cloacal

microbiota and the microbiota of the reproductive and digestive systems, microbiota

inhabiting the colon, cloaca, and magnum of 34-week-old, specific-pathogen-free

(SPF) hens were analyzed….” , The authors did not clearly explain the scientific or application significance of the analyzes performed. This is another qualitative and quantitative analysis of the microbiota of selected sections of the gastrointestinal tract of hens of a specific age group. The novelty of the conducted research should be indicated in relation to the available data or the results of other authors known so far. The study designed in this way, and above all the way of discussing the results obtained in the discussion, significantly reduces the value of the manuscript.

Abstract:

line 15-16-I suggest rewriting this sentence

Line 18-22: The authors did not analyze reproductive system microbiota in comparative studies, so the purpose of the research is not entirely consistent with the analysis. Obviously, comparing the biota of the cloaca and the other two sections of the intestine, we can indirectly conclude but still only indirectly, so it would be necessary to verify the presentation of the goal

Introduction:

line 44: please explain what "products" mean

Line55-56: Since the cloaca is a combination of the few systems, by definition the composition of the microbiota from the material taken from the cloaca cannot be directly related to the microbiota of the digestive tract.

Materials and methods:

line 66: in line with the assumptions of written work, each abbreviation should be explained at least once

Line 66 and 70: please specify the maximum storage time

Line 62, 70, 109, 119 and others: how many samples have been tested? In one place Authors give 10 in another 11 chickens - please verify

Results

Line 205: It is interesting why the authors focused on Lactobacillus species analysis. This needs more clarification.

Discussion

Unfortunately, the discussion is mostly a repetition of the results. According to the discussion, the results obtained by the Authors here, were already confirmed by other Authors, therefore my question remains open: What is the novelty of the research? In the conclusion, the Authors only drew attention to the fact that the results obtained from cloaca (microbiota analysis) should be applied with great caution to the analysis of gastrointestinal biota - this was already known, so it is difficult to find new elements in the presented studies. Perhaps if the discussion were supplemented with a discussion of the importance of individual taxa shown in the study in relation to the impact on the physiological processes of birds in individual sections of the gastrointestinal tract would be much more valuable. In its present form, the manuscript is another study in the form of registering the presence / absence of specific taxa. In addition, the Authors did a more accurate species analysis of Lactobacillus but did not use these results to enrich the discussion in suitable way.

Reviewer #3: The purpose of the study is to determine whether in birds, the microflora in the cloaca is a good estimate of the microflora in the gut. The cloaca in birds serves as the only opening for the digestive, reproductive, and urinary tracts, all of which may have their own microbiome. If the microflora of the cloaca is a mixture of these three different organ systems, it may not be particularly representative of the digestive system. The study samples the microflora of 11 specific-pathogen-free (SPF) chickens in three different regions: the cloaca, the colon (digestive system), and the magnum (part of the oviduct, which is part of the reproductive system). The authors investigated the microflora by amplicon sequencing of the 16S rRNA gene. The authors found that the microflora differs between these three different regions using a number of community ecology statistics. They found that bacteria belonging to the Genus Lactobacillus were among the most common. To determine the community of Lactobacillus in greater detail, the authors used another 10 Korean commercial layer chicken carcasses and cultured Lactobacillus bacteria on MRS agar followed by species identification via MALDI-TOF spectrometry and 16S rRNA sequencing. This study found that different Lactobacillus species were dominant in the different regions.

One problem with the study is that methodological differences are often confounded with the main factors of interest. First example, the cloaca is sampled using a swab whereas the magnum and colon were sampled using a scalpel. So differences in the microflora between the cloaca and the other two regions (magnum and colon) are potentially confounded by the method of sampling the bacteria. Second example, the authors use two different types of chickens for the two different parts of their study: SPF chickens to determine the general bacterial community and Korean commercial layer chickens to determine the community of Lactobacillus bacteria. Different strains of chickens could have very different microflora, but this obvious possibility is not discussed. Third example, the microflora in the first part of the study was amplified via PCR whereas the microflora in the second part of the study was amplified via culturing on MRS agar. These different methods of microflora amplification (PCR versus culture) will change the microflora, which makes inference difficult. Fourth example, the authors used 16S rRNA gene sequencing in the first study and MALDI-TOF spectrometry in the second study to identify bacterial species. These different identification methods could also influence the composition of the bacterial community that was detected. Nowhere in the manuscript do the authors acknowledge or discuss how the use of all of these different methods could bias their results.

In summary, there are many differences between the first and second experiment: breed of chicken, method of microflora amplification (PCR versus culture), and bacterial identification method (16S rRNA sequencing verus MALDI-TOF spectrometry). All these differences undermine the rationale for combining these two experiments into a single study. PLOS ONE requires that study has a sound experimental design, which I do not find to be the case for this study. I believe that this manuscript would be better suited to a more specialized journal on poultry science.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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PLoS One. 2020 Aug 4;15(8):e0237108. doi: 10.1371/journal.pone.0237108.r002

Author response to Decision Letter 0

1 Jul 2020

Reviewer #2

Reviewer #2: In terms of methodology, the work is correct. Although the purpose of the study, according to the authors, is clearly defined, i.e. „ To investigate similarities between the cloacal

microbiota and the microbiota of the reproductive and digestive systems, microbiota

inhabiting the colon, cloaca, and magnum of 34-week-old, specific-pathogen-free

(SPF) hens were analyzed….” , The authors did not clearly explain the scientific or application significance of the analyzes performed. This is another qualitative and quantitative analysis of the microbiota of selected sections of the gastrointestinal tract of hens of a specific age group. The novelty of the conducted research should be indicated in relation to the available data or the results of other authors known so far. The study designed in this way, and above all the way of discussing the results obtained in the discussion, significantly reduces the value of the manuscript.

- Following reviewer’s suggestion, we rewrote the introduction section to clarify the aim of this study and revised the discussion section with reviewing of other previous studies.

Abstract:

line 15-16-I suggest rewriting this sentence

-We rewrote the sentence in line 15-16 on page 2

Line 18-22: The authors did not analyze reproductive system microbiota in comparative studies, so the purpose of the research is not entirely consistent with the analysis. Obviously, comparing the biota of the cloaca and the other two sections of the intestine, we can indirectly conclude but still only indirectly, so it would be necessary to verify the presentation of the goal-?

-We added a detailed description of the magnum in line 20 on page 2

Introduction:

line 44: please explain what "products" mean

-We rewrote the sentence to make clear in line 49-50 on page 4

Line55-56: Since the cloaca is a combination of the few systems, by definition the composition of the microbiota from the material taken from the cloaca cannot be directly related to the microbiota of the digestive tract.

-We added that results of the previous studies which compared microbiota in the digestive tract and cloaca swab in line 55-61 on page 4. In addition, we revised the aim of this study in line 61-64 on page 4-5

Materials and methods:

line 66: in line with the assumptions of written work, each abbreviation should be explained at least once

-We added explanation of the abbreviation in line 72 and 79 on page 5

Line 66 and 70: please specify the maximum storage time

-We added the maximum storage time in line 76 on page 5

Line 62, 70, 109, 119 and others: how many samples have been tested? In one place Authors give 10 in another 11 chickens - please verify-

-We used 11 SPF chickens for analyzing microbiota in each sampling site and used 10 Hy-Line brown commercial layer chickens to culture Lactobacillus spp. in each sampling site.

Results

Line 205: It is interesting why the authors focused on Lactobacillus species analysis. This needs more clarification.-

-We explained why we focused on Lactobacillus species in line 212-217 on page 13

Discussion

Unfortunately, the discussion is mostly a repetition of the results. According to the discussion, the results obtained by the Authors here, were already confirmed by other Authors, therefore my question remains open: What is the novelty of the research?

In the conclusion, the Authors only drew attention to the fact that the results obtained from cloaca (microbiota analysis) should be applied with great caution to the analysis of gastrointestinal biota - this was already known, so it is difficult to find new elements in the presented studies.

-We added the novelty and significance of this research in line 258-270 on page 15 and line 314-316 on page 17

Perhaps if the discussion were supplemented with a discussion of the importance of individual taxa shown in the study in relation to the impact on the physiological processes of birds in individual sections of the gastrointestinal tract would be much more valuable.

-We added physiological effect of dominant Lactobacillus species in line 287-293 on page 17

In its present form, the manuscript is another study in the form of registering the presence / absence of specific taxa. In addition, the Authors did a more accurate species analysis of Lactobacillus but did not use these results to enrich the discussion in suitable way.

-We added the discussion in line 295-302 on page 16

Reviewer #3

Reviewer #3: The purpose of the study is to determine whether in birds, the microflora in the cloaca is a good estimate of the microflora in the gut. The cloaca in birds serves as the only opening for the digestive, reproductive, and urinary tracts, all of which may have their own microbiome. If the microflora of the cloaca is a mixture of these three different organ systems, it may not be particularly representative of the digestive system. The study samples the microflora of 11 specific-pathogen-free (SPF) chickens in three different regions: the cloaca, the colon (digestive system), and the magnum (part of the oviduct, which is part of the reproductive system). The authors investigated the microflora by amplicon sequencing of the 16S rRNA gene. The authors found that the microflora differs between these three different regions using a number of community ecology statistics. They found that bacteria belonging to the Genus Lactobacillus were among the most common. To determine the community of Lactobacillus in greater detail, the authors used another 10 Korean commercial layer chicken carcasses and cultured Lactobacillus bacteria on MRS agar followed by species identification via MALDI-TOF spectrometry and 16S rRNA sequencing. This study found that different Lactobacillus species were dominant in the different regions. One problem with the study is that methodological differences are often confounded with the main factors of interest.

First example, the cloaca is sampled using a swab whereas the magnum and colon were sampled using a scalpel. So differences in the microflora between the cloaca and the other two regions (magnum and colon) are potentially confounded by the method of sampling the bacteria.

-Many previous studies measured microbiota in reproductive system, digestive tract using a scalpel and microbiota in cloaca using cotton swab. Therefore, we followed the same method with precedent studies. We added the possibility that various sampling methods can affect the study results in line 270-272 on page 15

Second example, the authors use two different types of chickens for the two different parts of their study: SPF chickens to determine the general bacterial community and Korean commercial layer chickens to determine the community of Lactobacillus bacteria. Different strains of chickens could have very different microflora, but this obvious possibility is not discussed.

-We added the controversy in discussion in line 281-286 on page 16

Third example, the microflora in the first part of the study was amplified via PCR whereas the microflora in the second part of the study was amplified via culturing on MRS agar. These different methods of microflora amplification (PCR versus culture) will change the microflora, which makes inference difficult.

-We explained why we should use culture method to determine exact species of Lactobacillus in all sampling sites. We added the sentence in the result section in line 212-217 on page 13

Fourth example, the authors used 16S rRNA gene sequencing in the first study and MALDI-TOF spectrometry in the second study to identify bacterial species. These different identification methods could also influence the composition of the bacterial community that was detected. Nowhere in the manuscript do the authors acknowledge or discuss how the use of all of these different methods could bias their results.

-The results which were deduced by MALDI-TOF spectrometry were consistent with those of the 16S rRNA metagenome sequencing in genus level. We added the sentence in discussion in line 281-286 on page 16

In summary, there are many differences between the first and second experiment: breed of chicken, method of microflora amplification (PCR versus culture), and bacterial identification method (16S rRNA sequencing verus MALDI-TOF spectrometry). All these differences undermine the rationale for combining these two experiments into a single study. PLOS ONE requires that study has a sound experimental design, which I do not find to be the case for this study. I believe that this manuscript would be better suited to a more specialized journal on poultry science.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (22.9KB, docx)

Decision Letter 1

Arda Yildirim

21 Jul 2020

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

PONE-D-20-06110R1

Dear Dr. Lee,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please make a small correction:

Lines 304-306: please correct and unify spelling „cloacal lactobacilli”

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript has improved significantly, and the authors have referred to all comments, therefore I recommend manuscript for publication.

Please make a small correction:

Lines 304-306: please correct and unify spelling „cloacal lactobacilli”

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Acceptance letter

Arda Yildirim

24 Jul 2020

PONE-D-20-06110R1

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

Dear Dr. Lee:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Arda Yildirim

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Percentile abundance between groups.

    (XLSX)

    Click here for additional data file. (11.1KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (22.9KB, docx)

    Data Availability Statement

    All raw sequence reads files are available from the NCBI database (accession number: PRJNA604381).

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