The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current ‘critically endangered’ status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.
Citation: Waite DW, Deines P, Taylor MW (2012) Gut Microbiome of the Critically Endangered New Zealand Parrot, the Kakapo (Strigops habroptilus). PLoS ONE 7(4): e35803. https://doi.org/10.1371/journal.pone.0035803
Editor: Jack Anthony Gilbert, Argonne National Laboratory, United States of America
Received: January 23, 2012; Accepted: March 22, 2012; Published: April 18, 2012
Copyright: © 2012 Waite et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from the Department of Conservation as well as a University of Auckland Faculty Research Development Fund (grant 9841 3626187) to MWT, Feodor Lynen Research Fellowship from the Alexander von Humboldt Foundation to PD, and a University of Auckland Doctoral Scholarship to DW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The kakapo (Strigops habroptilus) is one of the world's rarest bird species, with only 126 individuals remaining on two predator-free islands off New Zealand's south coast. Endemic to New Zealand, the kakapo possesses a range of behaviours and physiological characteristics that make it unique: it is the world's heaviest parrot, the only flightless parrot and the only parrot to carry out lek breeding . Due to a combination of infrequent mating, low clutch numbers, and poor defense against mammalian predators the kakapo has been pushed to the verge of extinction , , though intensive conservation efforts by New Zealand's Department of Conservation have recently reversed the decline in numbers. Research programs into kakapo ecology, nutrition and genetics are well established and a management program has been enacted with the aim of restoring the kakapo population in New Zealand. Such practices as confining birds to predator-free islands, supplementary feeding, breeding programs and constant human supervision of both newborn chicks and adults have had a marked effect on the kakapo population – from just 62 remaining individuals in 1991  to the current level. By contrast, the potentially important roles of symbiotic microorganisms in kakapo nutrition and pathogen defense remain unstudied, although positive bacterial influence on the gastrointestinal tract (GIT) was first observed in vertebrates almost 50 years ago .
The interactions between hosts and GIT-associated bacterial communities have been the subject of intense study in mammals, particularly humans , , with murine models often used to demonstrate causal links between microbes and aspects of host health . Among avians, microbial research has mainly focused on either pathogen detection, or effects on weight gain in broiler chickens . In the last decade, the study of microbial communities in the GIT of birds has become commonplace, with cultivation-dependent and -independent methods used to examine microbial presence and activity within avian gastrointestinal environments. The microbial communities associated with commercially farmed species such as turkey  and ostrich  have been investigated, as well as a range of wild birds, including parrots ,  and the South American hoatzin , , , and their roles in bird fitness extend far beyond involvement in digestion and nutrient uptake. For example, studies on the effect of feather-degrading bacteria on mate selection and breeding fitness have revealed novel mechanisms through which bacteria can influence the lifecycle of their host , .
Links between microbial community structure and increased energy harvest from food have been demonstrated for a wide range of organisms by a variety of indirect techniques , , , . In controlled murine models, these effects can be shown at a much more direct level, with gnotobiotic (germ-free) rodents used as controls in experiments that demonstrate the role of bacteria in regulating gene pathways in a range of organs , , , . Microbes isolated from a particular host gut have been shown to be highly adapted to the host environment with the community being shaped by host-specific factors in a range of organisms , , . Microbes transplanted into a new gnotobiotic host provide significantly reduced benefits to the new host . Stimulation of the host immune system by GIT microbes has also been recognized in response to both viral and bacterial challenge , , , and development of gut-associated lymphoid tissues is increased in conventionally raised mice compared to their germ-free counterparts , .
With such varied and important roles being influenced by microbes, the lack of an accurate baseline description of kakapo-associated microbes represents a major gap in our knowledge of kakapo biology. Identification of the indigenous microbial community would be of great value to conservation efforts by enabling identification of allochthonous – potentially pathogenic – microbes. The existing literature surrounding kakapo-associated bacteria has so far focused on detecting and responding to pathogen outbreaks. Such an event occurred in 2004, when three kakapo died from erysipelas within 72 hours of translocation. The birds had been checked for known pathogens , and erysipelas had not previously been observed in kakapo . While attacks from previously unidentified pathogens are unavoidable, this highlights an area in which molecular microbiology could play a key role in aiding kakapo recovery efforts, through the use of specific, high-sensitivity molecular probing techniques to detect pathogens before their numbers expand to levels that affect the bird.
Human interaction with wild birds can influence the composition of the GIT community , , and the potential for human impact on the kakapo GIT community is great (although unavoidable). In times of sickness, wild kakapo are taken into captivity and frequently treated with broad-spectrum antibiotics to combat pathogens. In captivity kakapo are fed a diet supplemented with fruit and pellets not available in the wild and hand-reared chicks are fed on bird formula exclusively until approximately 30 days of age . A better understanding of kakapo microbiology carries clear potential for aiding conservation of this endangered bird, yet there are also sound academic reasons for researching this area. The kakapo diet consists mainly of shoots and leaves, and there has been speculation that kakapo may utilize microbes in the foregut to ferment ingested plant material . While this process is common in ruminants (e.g. cattle and sheep) it is almost unknown among avians, with only the hoatzin known to use the foregut to facilitate fermentation . The hoatzin, sole member of the family Opisthocomidae, exploits a diverse microbial community in its enlarged crop to aid in digestion, utilising up to 40 bacterial phyla as well as archaea to ferment plant material in the crop . The kakapo has been suggested as a possible candidate for foregut fermentation due to its lack of a cecum, which is the primary site of hindgut fermentation , and its similar diet to the hoatzin.
The key aim of this study was to document the microbial community of the kakapo digestive tract in both newly hatched chicks and adults, using samples derived from both the fore- and hindgut to ensure maximum coverage of the GIT. 16 S rRNA gene analysis was used to identify bacteria at each sampling site, and the samples were compared to test for changes in community structure along the GIT. This study represents the first step in a wider investigation of the kakapo microbiome, with the ultimate goal of aiding conservation and management of this critically endangered bird.
Bacterial community composition within the kakapo gastrointestinal tract
Bacterial 161S rRNA gene amplification was successful for all samples, whereas no archaea were amplified from any samples. A total of 1007 clones yielded high-quality sequence that passed chimera checking. The phylum Firmicutes was present in all libraries, and Gammaproteobacteria present in all except one (Sass). Slight representation from Fusobacteria was seen in a single chick choana sample (Fig. 1). When sequence data were dereplicated into 99% OTUs it was revealed that most of the sequences belonged to only a few key OTUs, such as Haemophilus felis and Streptococcus pasteurianus (Fig. 2). A Chao1 diversity estimator for each clone library was calculated at the 99% OTU level, and in almost all cases the expected number of OTUs per library was close to the observed number. The remainder of the diversity in each library was split among several low-abundance OTUs. Phylogenetic trees of kakapo-associated Firmicutes and Gammaproteobacteria are shown in Figs. 3 and 4, respectively.
Phylum-level affiliation of 16 S rRNA gene sequences obtained from the kakapo GIT. Samples to the left of the dotted line represent clone libraries derived from juveniles, and samples on the right represent adult-derived sequences.
OTU-level affiliation of 16 S rRNA gene sequences obtained from the kakapo GIT. Values in this heatmap are scaled as a proportion of the total number of sequences per clone library. Observed numbers of OTUs at 99% sequence similarity are provided below the figure, as well as the estimated diversity for each library using the Chao1 estimator.
16 S rRNA gene-based phylogenetic analysis of Firmicutes recovered from kakapo samples. Solid junctions represent >90% bootstrap support, and hollow junctions >75%. Kakapo-derived sequences are in bold. Dashed lines indicate sequence length <1200 bp. Branch lengths were calculated using the maximum-likelihood method RAxML, using sequences >1200 bp in length, and short sequences were added subsequently using the Parsimony Interactive tool in ARB. Bootstrap values were calculated using maximum parsimony with 5000 resamplings. Scale bar, 10% sequence divergence.
16 S rRNA gene-based phylogenetic analysis of Gammaproteobacteria found within kakapo samples. Solid junctions represent >90% bootstrap support, and hollow junctions >75%. Kakapo-derived sequences are bolded. Dashed lines indicate sequence length <1200 bp. Branch lengths were calculated using the maximum-likelihood method RAxML, using sequences >1200 bp in length, and short sequences were added subsequently using the Parsimony Interactive tool in ARB. Bootstrap values were calculated using maximum parsimony with 5000 resamplings. Scale bar, 10% sequence divergence.
The extent of differences in bacterial community composition between samples was tested at the OTU level using an unweighted UniFrac analysis. Sequences obtained from kakapo chick, and adult faecal, samples were pooled according to sample type. Sequence data obtained from the single adult choana swab were not included in statistical comparison. Pairwise comparisons were made between sample types to test the null hypothesis that the bacterial community is homogeneous throughout the GIT. Significant differences in community structure were observed between the chick choana/crop (p<0.001) and crop/faeces (p = 0.002), but not between the choana/faeces. Between chick and adult faecal samples, no significant difference was seen. Given that Sass and Sirocco had been subject to considerable human intervention prior to sampling, their faecal samples were compared separately to the chick samples and wild adult sample (Millie). The faecal communities of Sass/Sirocco were significantly different from those of the wild adult (p = 0.002), but did not differ significantly from the chick samples. There was no significant difference between the wild adult and chick faecal samples.
Determination of Bacteroidetes and Archaea sensitivity
Bacteroidetes are common GIT-associated bacteria in many vertebrates, but were not detected in any of the kakapo samples. As certain DNA extraction techniques can lead to under-representation of Bacteroidetes in a sample , we tested whether our DNA extraction methods are able to detect the presence of Bacteroidetes within faecal and swab samples. Bacteroidetes DNA was successfully detected in all spiked faecal and swab samples (data not shown), down to approximately 0.15% of bacterial cell load, indicating that Bacteroidetes were not excluded by our DNA extraction protocols. Similarly, while Archaea were not detected in un-spiked kakapo faeces, the archaeal 16 S rRNA gene could be detected when swab and faecal samples were spiked with archaeal cells, down to approximately 0.4% of cell load.
This paper describes the first molecular examination of the bacterial communities within the kakapo GIT, and provides evidence that qualitative differences exist between sites sampled throughout the GIT. The kakapo GIT appears to harbor a low-diversity community of microbes, with essentially only two phyla detected, Gammaproteobacteria and Firmicutes. Microbes in the kakapo GIT are abundant, with both cultivation-based measurements and DAPI cell counts indicating a microbial cell density in the order of 1010 cells per gram of faecal material (data not shown), yet each sample is dominated by only a few genera, typically Haemophilus, Streptococcus, and Clostridium. As the Fusobacteria discovered were only in a single sample, and found in low abundance, it is possible that their presence represents some form of contamination which occurred during sampling or DNA extraction. The Fusobacteria-associated sequences were similar to isolates and clones of the genus Leptotrichia, a bacterium commonly found in the human oral cavity .
At the phylum level, bacterial diversity is well conserved among all chicks sampled, but within the adults there is large variation in terms of relative abundance of each phylum. This may be explained by a range of factors regarding the adults, such as the frequent handling of Sirocco and, to a lesser extent, Sass, or the age difference between Sass and Millie/Sirocco. The bird Sass died several weeks after the collection of faecal samples, but not due to pathogen-related illness, and had not been treated with antibiotics prior to sampling (which can disrupt the GIT community , ). Subject age has been linked to a shift in the bacterial gut community in humans ,  and mice , so it is conceivable that such a community change may be a natural phenomenon. While functional roles of the bacteria detected in this study can only be speculated upon, those bacteria encountered in the kakapo GIT correspond to genera commonly observed in other herbivores. In a study of the gut microbiota of deer it was recognized that Streptococcus played a role in degrading tannins ingested by the host animal, and many of the Streptococcus detected in the kakapo clone libraries were closely related to this species (Fig. 3, Streptococcus gallolyticus sub. macedonicus, AB563237) . Most of the bacterial genera detected throughout the kakapo GIT are known anaerobic fermenters, capable of converting sugars such as glucose and cellulose into acids such as acetate, which are utilized by the host. Members of the genus Clostridium are frequently identified as cellulolytic , , , , and have been found to increase in proportion within the herbivore gut in the absence of starch . The inability to detect Bacteroidetes in a parrot, using either 16 S rRNA-based techniques or cultivation, is not unique to our study , . In addition to playing roles in butyrate production and bile acid metabolism , , Bacteroidetes are well-characterized degraders of starch and cellulose in the gut , , , . Historically the kakapo have relied on a low-starch diet , which may have selected against Bacteroidetes colonization, as diet has been identified as one of the factors that shape gut microbiota , , , . While the DNA extraction method utilized in this study is capable of extracting detectable levels of DNA from Bacteroidetes comprising less than 1% of the community, it is conceivable that the inability to detect Bacteroidetes stems from low sequence counts compared to those obtained using next-generation sequencing technologies.
Given the endangered status of the kakapo, destructive sampling (via dissection) is not possible. As such, our analyses were limited to swab and faecal samples rather than direct tissue and gut content samples. Although surface swabs may not give a perfect representation of the local bacterial community, they have been previously applied in a range of avian study systems , , , ,  where dissection of the target organism was not feasible. There still exists the potential that mucosa-associated bacteria of the crop may not be recovered through the swabbing of live animals, indicating a potential blind spot in sampling. Nevertheless, it appears that swabbing of the kakapo crop is adequate for differentiating between microbial communities of the choana and crop, despite the fact that any probe into the crop risks potential contamination as the swab passes the choana. The use of faecal samples as a proxy for hindgut bacterial communities has been used extensively in a range of vertebrates, including humans and birds. While several studies have highlighted differences between bacterial recovery from mucosal biopsies and faecal samples , , this appears to be due to faecal samples containing not only mucosa-associated bacteria that have been shed into the faeces, but also bacteria that colonize the faecal substrate directly. Community data taken from faecal samples contains a reasonable representation of microbes within the hindgut, and differences in faecal microbiota (both at presence/absence and functional levels) have been shown to reflect differences in the intestinal tract of the host , , although it must be stressed that they do not provide an exact representation of microbial community structure and function within the intestine itself. Based on unweighted UniFrac analysis it appears that the faecal bacterial communities of adults and chick kakapo are not significantly different, which may indicate a vector for inoculation of kakapo chicks with their parents' microbiota. The lack of significant difference between choana and faecal communities in the chicks is not surprising considering the lifestyle of unfledged kakapo chicks. Essentially immobile, the chicks are unable to distance themselves from their own faeces. The chicks studied in this project have since been fledged and given the low population of kakapo and constant attention to the birds, these present an excellent opportunity for longitudinal studies throughout the lifespan of the birds.
Given the low-energy diet of the kakapo and its lack of cecum, it has been speculated that the kakapo may utilize microbially-mediated foregut fermentation to derive additional energy from its food. While this study was not targeted at confirming or rejecting the notion of kakapo foregut fermentation, the possibility that key microorganisms may be resident in the crop rather than hindgut was taken into consideration when planning this study. There are several frequently found bacterial phyla in the microbial community of foregut-fermenting mammals and the hoatzin, predominantly Firmicutes and Bacteroidetes, with representation from Verrucomicrobia, Actinobacteria and Spirochaetes commonly observed , . Methanogenic archaea are also commonly found in the rumen or crop of foregut fermenters , , , , , . With the exception of Firmicutes, none of the above-mentioned taxa were detected in the kakapo samples. In the hoatzin it has been shown that the microbial community of the foregut is similar to that of ruminants , but given the apparent absence of so many bacterial phyla in the kakapo crop it is unlikely that the kakapo shares this community structure and gut adaptation. Foregut fermentation is an adaptation to a diet rich in celluloses that the host cannot digest , but kakapo do not retain and digest cellulose in the manner seen in ruminants and the hoatzin, typically spitting away masticated plant material after extracting juices from the flesh , . Although merely speculative at this stage, it thus seems unlikely that kakapo perform foregut fermentation in the traditional manner.
One observation from this study that may prove to be of future concern is the high number of Pasteurellaceae-like sequences within the choana and crop swabs. Many of the sequences were clustered with bacterial genera such as Haemophilus, or with several non-cultivated clades commonly detected in the avian respiratory tract, which are frequently found as respiratory pathogens in vertebrates . It has been noted that certain Pasteurellaceae which were present in our libraries (Bisgaard Taxon 34, Bisgaard Taxon 44, Fig. 4) are frequently associated with respiratory disease in psittacine birds. Although not all bacterial species in these clades are causative agents of disease, their presence should be considered a warning, as they are often found in sick birds . During the 2011 breeding season several chicks were removed from the nest due to respiratory problems, although this did not cause long-term health issues in the birds (D. Eason, personal communication). While there is no data to imply a causal link between the observed Pasteurellaceae and the illness, pathogens do appear to have been introduced to the kakapo population previously through avian vectors .
In summary, we performed the first 16 S rRNA-based microbial analysis of the bacteria that inhabit the kakapo GIT. We have shown that the GIT is inhabited by a few key organisms, and that the community composition changes throughout the GIT. Our results also provided preliminary evidence that the human influence on kakapo lifestyle appears to cause a shift in these bacterial communities, although whether this has a positive, negative, or neutral effect on the bird remains unknown.
Materials and Methods
Samples were obtained from four kakapo on Codfish Island (46°47′S 167°38′E), off the coast of Stewart Island, New Zealand, during the nesting season, between 17th and 23rd April 2011. Two additional faecal samples had previously been collected from adult birds when they were brought to Auckland Zoo. A total of 13 samples were used in this analysis, collected from three unfledged chicks, and three adults. Samples of the upper GIT were taken by Department of Conservation staff using sterile rayon-tipped swabs (Copan, #170KS01), and samples were taken from the choana and crop of chicks, and choana of one adult. The choana is an opening in the roof of the mouth that joins to the nasal passage. Due to difficulties in restraining adults, crop samples could not be taken from adult birds. A faecal sample was collected from all six birds at the time swabbing was performed. As interference with nesting mothers can cause them to abandon the nest, samples from chick parents could not be obtained. Swabs were stored in sterile polypropylene tubes and kept on ice until they were frozen at the ranger hut on Codfish Island. Samples were shipped to The University of Auckland on dry ice, and stored at −20°C upon arrival.
Despite considerable efforts to standardize the DNA extraction procedure, it was necessary to use a different approach for extracting DNA from swab vs faecal samples due to unreliable DNA retrieval from hard-to-obtain swab samples and unwillingness to risk valuable samples. Genomic DNA was extracted from swabs using heat lysis. Swab tips were placed in a 1.5 mL cryotube containing 1 mL extraction buffer (20 mM EDTA, 0.1 M Tris (pH 8.0), 1% CTAB, 56 mM NaCl), briefly vortexed, then incubated at 94°C for 15 min in order to lyse both Gram-negative and Gram-positive cells , . The solution was cooled briefly on ice, 300 µL of chloroform/isoamyl alcohol (24∶1 ratio) was added and the solution mixed by inversion, then centrifuged at 13,000 rpm for 5 min at room temperature. The supernatant was transferred to a 2 mL microcentrifuge tube, to which 0.6 volume (vol) isopropanol and 0.1 vol 3 M sodium acetate (pH 5.2) were added. Samples were incubated overnight at −20°C then centrifuged at 13,000 rpm at 4°C for 30 min. The supernatant was discarded and the pellet washed twice with ice-cold 70% ethanol followed by centrifugation at 13,000 rpm at 4°C for 10 min. Samples were dried and suspended in 50 µL UltraPure water (Invitrogen).
Genomic DNA was extracted from kakapo faecal samples using a variation on an extraction protocol previously described . 100 mg of faeces were suspended in 1 mL of 70% ethanol with 200 mg of 0.1 mm zirconia/silica beads in a 1.5 mL cryotube. Samples were agitated using a FastPrep FP120 bead beater, at 5.5 ms−1 for 30 s, followed by centrifugation at 13,000 rpm for 5 min and removal of supernatant. 1 mL of extraction buffer was added to each tube in addition to 30 mg of polyvinylpolypyrrolidone (PVPP), before being agitated using the previous settings. Samples were then incubated at 65°C for 30 min, with gentle mixing every 10 min. Samples were centrifuged at 13,000 rpm for 5 min and the supernatant was transferred to a fresh 1.5 mL microcentrifuge tube containing 0.5 mL of chloroform/isoamyl alcohol solution (24∶1 ratio) and inverted to mix. Samples were centrifuged at 13,000 rpm for 5 min, before the supernatant (approx. 1 mL) was transferred to a 2 mL microcentrifuge tube containing 0.6 vol isopropanol and 0.1 vol 3 M sodium acetate (pH 5.2). Samples were mixed then incubated on ice for 1 h, then centrifuged at 13,000 rpm at 4°C for 1 min to remove any remaining sediment (presumed to be leftover SDS). The supernatant was transferred to a new microcentrifuge tube and centrifuged under the same conditions for 30 min. The supernatant was removed and the pellet washed twice using ice-cold 70% ethanol followed by 10 min centrifugation at 13,000 rpm, 4°C. The pellet was dried and resuspended in 50 µL UltraPure water (Invitrogen).
PCR and clone library construction
PCR was performed using the forward primer 616V and reverse primer 1492R, targeting Escherichia coli positions 8–27 and 1492–1513 respectively, to amplify bacterial 16 S rRNA genes, and 21F/958R for archaeal 16 S rRNA genes (Table 1). Reactions were conducted in 25 µL volumes, containing 20 mM Tris-HCl, 50 mM KCl (buffer), 1.5 mM MgCl2, 25 µM of each dNTP, 2.5 µM of each primer, 0.5 units Taq polymerase and 1.0 µL of extracted DNA template. Cycling conditions for the 616V/1492R primer pair were as follows: initial denaturing at 94°C for 5 min, 30 cycles of 94°C for 45 s, 57°C for 45 s and 72°C for 1.5 min, followed by a final elongation step at 72°C for 7 min. Cycling conditions for 21F/958R were described previously . In order to successfully amplify from faecal samples, the addition of 2% bovine serum albumin per tube was required . Cloning was performed using the P-GemT Easy Vector kit (Promega, Inc, Madison WI, USA) following the manufacturer's instructions. Approximately 96 clones from each of the 13 clone libraries were selected for sequencing (Macrogen Inc, Seoul, South Korea).
Sequences were analyzed using the Geneious software package  and low-quality data from the ends of each sequence removed. Chimeras were identified with the Pintail algorithm using the Mallard software package  and subsequently removed from the data set. Sequences were aligned via the SINA web aligner  and imported into ARB using the SILVA 108 database . Sequence data were divided into operational taxonomic units (OTU) of 99% sequence identity using mothur  and one sequence to represent each OTU per sample was used in tree construction. Sequences representing each OTU were submitted to the DDBJ/EMBL/GenBank databases under accession numbers JQ283115–Q283245, JQ302756, and JQ302757. Phylogenetic trees were constructed in ARB using the maximum likelihood method RAxML. Bootstrap values were calculated using 5000 parsimony replications. Unweighted UniFrac analyses were performed in mothur to statistically compare bacterial community composition among different sample types.
Determination of Bacteroidetes and Archaea sensitivity
A pure culture of Chryseobacterium formosense (phylum Bacteroidetes), originally isolated from wastewater, was obtained from a colleague and cultivated at the original isolation conditions (R2A broth, 28°C for 48 h, C. Brown, personal communication). C. formosense cells were added to samples of kakapo chick faeces at proportions down to 0.15% total bacterial cell load, calculated through enumeration of C. formosense via plating counts, and DAPI staining of faecal samples, then subjected to both extraction methods detailed previously. A fragment of the 16 S rRNA gene was amplified using the 341-GC/518R primer pair. Cycling conditions consisted of an initial denaturing step at 94°C for 5 min, followed by 25 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 30 s, then a final elongation step at 72°C for 5 min. The product was analyzed using denaturing gradient gel electrophoresis (DGGE) with a denaturing gradient of 40–70%. A positive control of pure C. formosense DNA was used as an indicator of a Bacteroidetes band in the gel pattern. A pure culture of Methanosarcina acetivorans (domain Archaea, strain DS2834) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and added to samples of kakapo chick faeces at proportions down to 0.4% total cell load. A fragment of the archaeal 16 S rRNA gene was amplified using the 21F/958R primer pair. Cycling conditions were described previously . Samples were visualised on a 1% agarose gel and analysed using the BioRad Gel Doc imaging system.
We gratefully thank Daryl Eason and Jo Ledington (Department of Conservation), and John Potter (Auckland Zoo) for provision of all kakapo samples, and Caroline Brown for supplying the required Bacteroidetes strain. We would also like to thank Ron Moorhouse and Deidre Vercoe (Department of Conservation), and Richard Jakob-Hoff (Auckland Zoo) for their endorsement and support of this project, plus Jacqueline Beggs and Mick Clout (University of Auckland) for useful discussions on this topic.
Conceived and designed the experiments: DWW PD MWT. Performed the experiments: DWW PD MWT. Analyzed the data: DWW PD MWT. Wrote the paper: DWW PD MWT.
- 1. Merton DV, Morris RB, Atkinson IAE (1984) Lek behaviour in a parrot: the Kakapo Strigops habroptilus of New Zealand. Ibis 126: 277–283.DV MertonRB MorrisIAE Atkinson1984Lek behaviour in a parrot: the Kakapo Strigops habroptilus of New Zealand.Ibis126277283
- 2. Lloyd BD, Powlesland RG (1994) The decline of kakapo Strigops habroptilus and attempts at conservation by translocation. Biol Conserv 69: 75–85.BD LloydRG Powlesland1994The decline of kakapo Strigops habroptilus and attempts at conservation by translocation.Biol Conserv697585
- 3. Houston D, Mcinnes K, Elliott G, Eason D, Moorhouse R, et al. (2007) The use of a nutritional supplement to improve egg production in the endangered kakapo. Biol Conserv 138: 248–255.D. HoustonK. McinnesG. ElliottD. EasonR. Moorhouse2007The use of a nutritional supplement to improve egg production in the endangered kakapo.Biol Conserv138248255
- 4. Elliott GP, Merton DV, Jansen PW (2001) Intensive management of a critically endangered species: the kakapo. Biol Conserv 99: 121–133.GP ElliottDV MertonPW Jansen2001Intensive management of a critically endangered species: the kakapo.Biol Conserv99121133
- 5. Dubos R, Schaedler RW (1964) The digestive tract as an ecosystem. Am J Med Sci 248: 267–272.R. DubosRW Schaedler1964The digestive tract as an ecosystem.Am J Med Sci248267272
- 6. Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI (2008) Worlds within worlds: evolution of the vertebrate gut microbiota. Nat Rev: Microbiol 6: 776–788.RE LeyCA LozuponeM. HamadyR. KnightJI Gordon2008Worlds within worlds: evolution of the vertebrate gut microbiota.Nat Rev: Microbiol6776788
- 7. Zoetendal EG, Rajilic-Stojanovic M, de Vos WM (2008) High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota. Gut 57: 1605–1615.EG ZoetendalM. Rajilic-StojanovicWM de Vos2008High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota.Gut5716051615
- 8. Yi P, Li L (2011) The germfree murine animal: An important animal model for research on the relationship between gut microbiota and the host. Vet Microbiol. P. YiL. Li2011The germfree murine animal: An important animal model for research on the relationship between gut microbiota and the host.Vet MicrobiolIn press. In press.
- 9. Yegani M, Korver DR (2008) Factors affecting intestinal health in poultry. Poultry Sci 87: 2052–2063.M. YeganiDR Korver2008Factors affecting intestinal health in poultry.Poultry Sci8720522063
- 10. Lu J, Domingo JS (2008) Turkey fecal microbial community structure and functional gene diversity revealed by 16 S rRNA gene and metagenomic sequences. J Microbiol 46: 469–477.J. LuJS Domingo2008Turkey fecal microbial community structure and functional gene diversity revealed by 16 S rRNA gene and metagenomic sequences.J Microbiol46469477
- 11. Matsui H, Kato Y, Chikaraishi T, Moritani M, Ban-Tokuda T, et al. (2010) Microbial diversity in ostrich ceca as revealed by 16 S ribosomal RNA gene clone library and detection of novel Fibrobacter species. Anaerobe 16: 83–93.H. MatsuiY. KatoT. ChikaraishiM. MoritaniT. Ban-Tokuda2010Microbial diversity in ostrich ceca as revealed by 16 S ribosomal RNA gene clone library and detection of novel Fibrobacter species.Anaerobe168393
- 12. Pacheco MA, Garcia-Amado MA, Bosque C, Dominguez-Bello MG (2004) Bacteria in the crop of the seed-eating Green-Rumped Parrotlet. Condor 106: 139–143.MA PachecoMA Garcia-AmadoC. BosqueMG Dominguez-Bello2004Bacteria in the crop of the seed-eating Green-Rumped Parrotlet.Condor106139143
- 13. Xenoulis PG, Gray PL, Brightsmith D, Palculict B, Hoppes S, et al. (2010) Molecular characterization of the cloacal microbiota of wild and captive parrots. Vet Microbiol 146: 320–325.PG XenoulisPL GrayD. BrightsmithB. PalculictS. Hoppes2010Molecular characterization of the cloacal microbiota of wild and captive parrots.Vet Microbiol146320325
- 14. Godoy-Vitorino F, Ley RE, Gao Z, Pei Z, Ortiz-Zuazaga H, et al. (2008) Bacterial community in the crop of the hoatzin, a neotropical folivorous flying bird. Appl Environ Microb 74: 5905–5912.F. Godoy-VitorinoRE LeyZ. GaoZ. PeiH. Ortiz-Zuazaga2008Bacterial community in the crop of the hoatzin, a neotropical folivorous flying bird.Appl Environ Microb7459055912
- 15. Godoy-Vitorino F, Goldfarb KC, Brodie EL, Garcia-Amado MA, Michelangeli F, et al. (2010) Developmental microbial ecology of the crop of the folivorous hoatzin. ISME J 4: 611–620.F. Godoy-VitorinoKC GoldfarbEL BrodieMA Garcia-AmadoF. Michelangeli2010Developmental microbial ecology of the crop of the folivorous hoatzin.ISME J4611620
- 16. Godoy-Vitorino F, Goldfarb KC, Karaoz U, Leal S, Garcia-Amado MA, et al. (2012) Comparative analyses of foregut and hindgut bacterial communities in hoatzins and cows. ISME J 6: 531–541.F. Godoy-VitorinoKC GoldfarbU. KaraozS. LealMA Garcia-Amado2012Comparative analyses of foregut and hindgut bacterial communities in hoatzins and cows.ISME J6531541
- 17. Shawkey MD, Pillai SR, Hill GE, Siefferman LM, Roberts SR (2007) Bacteria as an agent for change in structural plumage color: correlational and experimental evidence. Am Nat 169: S112–S121.MD ShawkeySR PillaiGE HillLM SieffermanSR Roberts2007Bacteria as an agent for change in structural plumage color: correlational and experimental evidence.Am Nat169S112S121
- 18. Burt EH, Schroeder MR, Smith LA, Sroka JE, McGraw KJ (2011) Colourful parrot feathers resist bacterial degradation. Biol Letters 7: 214–216.EH BurtMR SchroederLA SmithJE SrokaKJ McGraw2011Colourful parrot feathers resist bacterial degradation.Biol Letters7214216
- 19. Gill SR, Pop M, DeBoy RT, Eckburg PB, Turnbaugh PJ, et al. (2006) Metagenomic Analysis of the Human Distal Gut Microbiome. Science 312: 1355–1359.SR GillM. PopRT DeBoyPB EckburgPJ Turnbaugh2006Metagenomic Analysis of the Human Distal Gut Microbiome.Science31213551359
- 20. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027–1031.PJ TurnbaughRE LeyMA MahowaldV. MagriniER MardisJI Gordon2006An obesity-associated gut microbiome with increased capacity for energy harvest.Nature44410271031
- 21. Ohkuma M (2008) Symbioses of flagellates and prokaryotes in the gut of lower termites. Trends Microbiol 16: 345–352.M. Ohkuma2008Symbioses of flagellates and prokaryotes in the gut of lower termites.Trends Microbiol16345352
- 22. Torok VA, Ophel-Keller K, Loo M, Hughes RJ (2008) Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl Environ Microb 74: 783–791.VA TorokK. Ophel-KellerM. LooRJ Hughes2008Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism.Appl Environ Microb74783791
- 23. Stappenbeck TS, Hooper LV, Gordon JI (2002) Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci USA 99: 15451–15455.TS StappenbeckLV HooperJI Gordon2002Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.Proc Natl Acad Sci USA991545115455
- 24. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, et al. (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 101: 15718–15723.F. BackhedH. DingT. WangLV HooperGY Koh2004The gut microbiota as an environmental factor that regulates fat storage.Proc Natl Acad Sci USA1011571815723
- 25. Meinl W, Sczensy S, Brigelius-Flohe R, Blaut M, Glatt H (2008) Impact of gut microbiota on intestinal and hepatic levels of phase 2 xenobiotic-metabolizing enzymes in the rat. Drug Metab Dispos 37: 1179–1186.W. MeinlS. SczensyR. Brigelius-FloheM. BlautH. Glatt2008Impact of gut microbiota on intestinal and hepatic levels of phase 2 xenobiotic-metabolizing enzymes in the rat.Drug Metab Dispos3711791186
- 26. Bjorkholm B, Bok CM, Lundin A, Rafter J, Hibberd ML, et al. (2009) Intestinal microbiota regulate xenobiotic metabolism in the liver. PLoS ONE 4: e6958.B. BjorkholmCM BokA. LundinJ. RafterML Hibberd2009Intestinal microbiota regulate xenobiotic metabolism in the liver.PLoS ONE4e6958
- 27. van der Wielen PWJJ, Keuzenkamp DA, Lipman LJA, van Knapen F, Biesterveld S (2002) Spatial and temportal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb Ecol 44: 286–293.PWJJ van der WielenDA KeuzenkampLJA LipmanF. van KnapenS. Biesterveld2002Spatial and temportal variation of the intestinal bacterial community in commercially raised broiler chickens during growth.Microb Ecol44286293
- 28. Khachatryan ZA, Ktsoyan ZA, Manukyan GP, Kelly D, Ghazaryan KA, et al. (2008) Predominant role of host genetics in controlling the composition of gut microbiota. PLoS ONE 3: e3064.ZA KhachatryanZA KtsoyanGP ManukyanD. KellyKA Ghazaryan2008Predominant role of host genetics in controlling the composition of gut microbiota.PLoS ONE3e3064
- 29. Vaishnava S, Yamamoto M, Severson KM, Ruhn KA, Yu X, et al. (2011) The antibacterial lectin RegIIIγ promotes the spatial segregation of microbiota and host in the intestine. Science 334: 255–258.S. VaishnavaM. YamamotoKM SeversonKA RuhnX. Yu2011The antibacterial lectin RegIIIγ promotes the spatial segregation of microbiota and host in the intestine.Science334255258
- 30. Brisbin JT, Gong J, Sharif S (2008) Interactions between commensal bacteria and the gut-associated immune system of the chicken. Anim Health Res Rev 9: 101–110.JT BrisbinJ. GongS. Sharif2008Interactions between commensal bacteria and the gut-associated immune system of the chicken.Anim Health Res Rev9101110
- 31. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, et al. (2009) Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139: 485–498.II IvanovK. AtarashiN. ManelEL BrodieT. Shima2009Induction of intestinal Th17 cells by segmented filamentous bacteria.Cell139485498
- 32. Ichinohe T, Pang IK, Kumamoto Y, Peaper DR, Ho JH, et al. (2011) Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc Natl Acad Sci USA 108: 5354–5359.T. IchinoheIK PangY. KumamotoDR PeaperJH Ho2011Microbiota regulates immune defense against respiratory tract influenza A virus infection.Proc Natl Acad Sci USA10853545359
- 33. Hudson JA, Luckey TD (1964) Bacteria induced morphologic changes. P Soc Exp Biol Med 116: 628–631.JA HudsonTD Luckey1964Bacteria induced morphologic changes.P Soc Exp Biol Med116628631
- 34. Hooijkaas H, Benner R, Pleasants JR, Wostmann BS (1984) Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered antigen-free diet. Eur J Immunol 14: 1127–1130.H. HooijkaasR. BennerJR PleasantsBS Wostmann1984Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered antigen-free diet.Eur J Immunol1411271130
- 35. Brangenberg N, McInnes C, Connolly JH, Rogers LE (2003) Absence of Salmonella and Campylobacter species in fecal and cloacal swab samples from kakapo (Strigops habroptilus) on Codfish Island, New Zealand. J Avian Med Surg 17: 203–205.N. BrangenbergC. McInnesJH ConnollyLE Rogers2003Absence of Salmonella and Campylobacter species in fecal and cloacal swab samples from kakapo (Strigops habroptilus) on Codfish Island, New Zealand.J Avian Med Surg17203205
- 36. Gartrell BD, Alley MR, Mack H, Donald J, McInnes K, et al. (2005) Erysipelas in the critically endangered kakapo (Strigops habroptilus). Avian Pathol 34: 383–387.BD GartrellMR AlleyH. MackJ. DonaldK. McInnes2005Erysipelas in the critically endangered kakapo (Strigops habroptilus).Avian Pathol34383387
- 37. Janiga M, Sedlarova A, Rigg R, Novotna M (2007) Patterns of prevalence among bacterial communities of alpine accentors (Prunella collaris) in the Tatra Mountains. J Ornithol 148: 135–143.M. JanigaA. SedlarovaR. RiggM. Novotna2007Patterns of prevalence among bacterial communities of alpine accentors (Prunella collaris) in the Tatra Mountains.J Ornithol148135143
- 38. Eason D, Moorhouse R (2006) Hand-rearing kakapo (Strigops habroptilus), 1997–2005. Notornis 53: 116–125.D. EasonR. Moorhouse2006Hand-rearing kakapo (Strigops habroptilus), 1997–2005.Notornis53116125
- 39. Morton ES (1978) Avian arboreal folivores: why not? In: Montgomery GG, editor. The ecology of arboreal folivores. Washington, D.C.: Smithsonian Institution Press. pp. 123–130.ES Morton1978Avian arboreal folivores: why not?GG MontgomeryThe ecology of arboreal folivoresWashington, D.C.Smithsonian Institution Press123130
- 40. Grajal A, Strahl SD, Parra R, Dominguez MG, Neher A (1989) Foregut fermentation in the hoatzin, a neotropical leaf-eating bird. Science 245: 1236–1238.A. GrajalSD StrahlR. ParraMG DominguezA. Neher1989Foregut fermentation in the hoatzin, a neotropical leaf-eating bird.Science24512361238
- 41. Clench MH, Mathias JR (1995) The avian cecum: a review. Wilson Bull 107: 93–121.MH ClenchJR Mathias1995The avian cecum: a review.Wilson Bull10793121
- 42. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim van Dillen PME, et al. (1990) Rapid and simple method for purification of nucleic acid. J Clin Microbiol 28: 495–503.R. BoomCJA SolMMM SalimansCL JansenPME Wertheim van Dillen1990Rapid and simple method for purification of nucleic acid.J Clin Microbiol28495503
- 43. Kribe ERK, Olsen I (2008) Leptotrichia species in human infections. Anaerobe 14: 131–137.ERK KribeI. Olsen2008Leptotrichia species in human infections.Anaerobe14131137
- 44. O'Hara AM, Shanahan F (2006) The gut flora as a forgotten organ. EMBO Rep 7: 688–693.AM O'HaraF. Shanahan2006The gut flora as a forgotten organ.EMBO Rep7688693
- 45. Dethlefsen L, Huse S, Sogin ML, Relman DA (2008) The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16 S rRNA sequencing. PLoS Biol 6: e280.L. DethlefsenS. HuseML SoginDA Relman2008The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16 S rRNA sequencing.PLoS Biol6e280
- 46. Hopkins MJ, Sharp R, Macfarlane GT (2001) Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16 S rRNA abundance, and community cellular fatty acid profiles. Gut 48: 198–205.MJ HopkinsR. SharpGT Macfarlane2001Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16 S rRNA abundance, and community cellular fatty acid profiles.Gut48198205
- 47. Rajilic-Stojanovic M, Heilig HGHJ, Molenaar D, Kajander K, Surakka A, et al. (2009) Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults. Environ Microbiol 11: 1736–1751.M. Rajilic-StojanovicHGHJ HeiligD. MolenaarK. KajanderA. Surakka2009Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults.Environ Microbiol1117361751
- 48. Vaahtovuo J, Toivanen P, Eerola E (2001) Study of murine faecal microflora by cellular fatty acid analysis; effect of age and mouse strain. Antonie Leeuwenhoek 80: 35–42.J. VaahtovuoP. ToivanenE. Eerola2001Study of murine faecal microflora by cellular fatty acid analysis; effect of age and mouse strain.Antonie Leeuwenhoek803542
- 49. Hiura T, Hashidoko Y, Kobayashi Y, Tahara S (2010) Effective degradation of tannic acid by immobilized rumen microbes of a sika deer (Cervus nippon yesoensis) in winter. Anim Feed Sci Tech 155: 1–8.T. HiuraY. HashidokoY. KobayashiS. Tahara2010Effective degradation of tannic acid by immobilized rumen microbes of a sika deer (Cervus nippon yesoensis) in winter.Anim Feed Sci Tech15518
- 50. Shoham Y, Lamed R, Bayer EA (1999) The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends Microbiol 7: 275–280.Y. ShohamR. LamedEA Bayer1999The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides.Trends Microbiol7275280
- 51. Warnick TA, Methe BA, Leschine SB (2002) Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil. Int J Syst Evol Micr 52: 1155–1160.TA WarnickBA MetheSB Leschine2002Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil.Int J Syst Evol Micr5211551160
- 52. Varel VH, Pond WG (1992) Characteristics of a New Cellulolytic Clostridium sp. Isolated from Pig Intestinal Tract. Appl Environ Microb 58: 1645–1649.VH VarelWG Pond1992Characteristics of a New Cellulolytic Clostridium sp. Isolated from Pig Intestinal Tract.Appl Environ Microb5816451649
- 53. Sabathe F, Belaich A, Soucaille P (2002) Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum. FEMS Microbiol Lett 217: 15–22.F. SabatheA. BelaichP. Soucaille2002Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum.FEMS Microbiol Lett2171522
- 54. Laure R, Yu Z, Parisi VA, Egan AR, Morrison M (2005) Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencin. Environ Microbiol 7: 530–543.R. LaureZ. YuVA ParisiAR EganM. Morrison2005Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencin.Environ Microbiol7530543
- 55. Kim YS, Milner JA (2007) Dietary modulation of colon cancer risk. J Nutr 137: 2576–2579.YS KimJA Milner2007Dietary modulation of colon cancer risk.J Nutr13725762579
- 56. Thomas F, Hehemann J, Rebuffet E, Czjzek M, Michel G (2011) Environmental and gut Bacteroidetes: the food connection. Front Microbiol 2: F. ThomasJ. HehemannE. RebuffetM. CzjzekG. Michel2011Environmental and gut Bacteroidetes: the food connection.Front Microbiol2
- 57. Dongowski G, Lorenz A, Anger H (2000) Degradation of pectins with different degrees of esterigication by Bacteroides thetaiotaomicron isolated from human gut flora. Appl Environ Microb 66: 1321–1327.G. DongowskiA. LorenzH. Anger2000Degradation of pectins with different degrees of esterigication by Bacteroides thetaiotaomicron isolated from human gut flora.Appl Environ Microb6613211327
- 58. Chassard C, Goumy V, Leclerc M, Del'homme C, Bernalier-Donadille A (2007) Characterization of the xylan-degrading microbial community from human faeces. FEMS Microbiol Ecol 61: 121–131.C. ChassardV. GoumyM. LeclercC. Del'hommeA. Bernalier-Donadille2007Characterization of the xylan-degrading microbial community from human faeces.FEMS Microbiol Ecol61121131
- 59. Martens EC, Koropatkin NM, Smith TJ, Gordon JI (2009) Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm. J Biol Chem 284: 24673–24677.EC MartensNM KoropatkinTJ SmithJI Gordon2009Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm.J Biol Chem2842467324677
- 60. Bolam DN, Sonnenburg JL (2011) Mechanistic insight into polysaccharide use within the intestinal microbiota. Gut Microbes 2: 86–90.DN BolamJL Sonnenburg2011Mechanistic insight into polysaccharide use within the intestinal microbiota.Gut Microbes28690
- 61. Horrocks M, Salter J, Braggins J, Nichol S, Moorhouse R, et al. (2008) Plant microfossil analysis of coprolites of the critically endangered kakapo (Strigops habroptilus) parrot from New Zealand. Rev Palaeobot Palyno 149: 229–245.M. HorrocksJ. SalterJ. BragginsS. NicholR. Moorhouse2008Plant microfossil analysis of coprolites of the critically endangered kakapo (Strigops habroptilus) parrot from New Zealand.Rev Palaeobot Palyno149229245
- 62. Finegold SM, Attebery HR, Sutter VL (1974) Effect of diet on human fecal flora: comparison of Japanese and American diets. Am J Clin Nutr 27: 1456–1469.SM FinegoldHR AtteberyVL Sutter1974Effect of diet on human fecal flora: comparison of Japanese and American diets.Am J Clin Nutr2714561469
- 63. Hehemann J, Correc G, Barbeyron T, Helbert W, Czjzek M, et al. (2010) Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464: 908–914.J. HehemannG. CorrecT. BarbeyronW. HelbertM. Czjzek2010Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota.Nature464908914
- 64. Martinez I, Kim J, Duffy PR, Schlegel VL, Walter J (2010) Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects. PLoS ONE 5: e15046.I. MartinezJ. KimPR DuffyVL SchlegelJ. Walter2010Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects.PLoS ONE5e15046
- 65. Shanks OC, Kelty CA, Archibeque S, Jenkins M, Newton RJ, et al. (2011) Community structures of fecal cacteria in cattle from different animal feeding operation. Appl Environ Microb 77: 2992–3001.OC ShanksCA KeltyS. ArchibequeM. JenkinsRJ Newton2011Community structures of fecal cacteria in cattle from different animal feeding operation.Appl Environ Microb7729923001
- 66. Moreno J, Briones V, Merino S, Ballesteros C, Sanz JJ, et al. (2003) Beneficial effects of cloacal bacteria on growth and fledging size in Nestling Pied Flycatchers (Fidecula hypoleuca) in Spain. Auk 120: 784–790.J. MorenoV. BrionesS. MerinoC. BallesterosJJ Sanz2003Beneficial effects of cloacal bacteria on growth and fledging size in Nestling Pied Flycatchers (Fidecula hypoleuca) in Spain.Auk120784790
- 67. Blanco G, Lemus JA, Grande J (2006) Faecal bacteria associated with different diets of wintering red kites: influence of livestock carcass dumps in microflora alteration and pathogen acquisition. J Appl Ecol 43: 990–998.G. BlancoJA LemusJ. Grande2006Faecal bacteria associated with different diets of wintering red kites: influence of livestock carcass dumps in microflora alteration and pathogen acquisition.J Appl Ecol43990998
- 68. Klomp JE, Murphy MT, Smith SB, McKay JE, Ferrera I, et al. (2008) Cloacal microbial communities of female spotted towhees Pipilo maculatus: microgeographic variation and individual sources of variability. J Avian Biol 39: 530–538.JE KlompMT MurphySB SmithJE McKayI. Ferrera2008Cloacal microbial communities of female spotted towhees Pipilo maculatus: microgeographic variation and individual sources of variability.J Avian Biol39530538
- 69. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, et al. (2005) Diversity of the human intestinal microbial flora. Science 308: 1635–1638.PB EckburgEM BikCN BernsteinE. PurdomL. Dethlefsen2005Diversity of the human intestinal microbial flora.Science30816351638
- 70. Savage DC (1977) Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 31: 107–133.DC Savage1977Microbial ecology of the gastrointestinal tract.Annu Rev Microbiol31107133
- 71. Ley RE, Hamady M, Lozupone C, Turnbaugh P, Ramey RR, et al. (2008) Evolution of mammals and their gut microbes. Science 320: 1647–1651.RE LeyM. HamadyC. LozuponeP. TurnbaughRR Ramey2008Evolution of mammals and their gut microbes.Science32016471651
- 72. Tokura M, Chagan I, Ushida K, Kojima Y (1999) Phylogenetic study of methanogens associated with rumen ciliates. Curr Microbiol 39: 123–128.M. TokuraI. ChaganK. UshidaY. Kojima1999Phylogenetic study of methanogens associated with rumen ciliates.Curr Microbiol39123128
- 73. Tajima K, Nagamine T, Matsui H, Nakamura M, Aminov RI (2001) Phylogenetic analysis of archaeal 16 S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol Lett 200: 67–72.K. TajimaT. NagamineH. MatsuiM. NakamuraRI Aminov2001Phylogenetic analysis of archaeal 16 S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens.FEMS Microbiol Lett2006772
- 74. Irbis C, Ushida K (2004) Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa. J Gen Appl Microbiol 50: 203–212.C. IrbisK. Ushida2004Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa.J Gen Appl Microbiol50203212
- 75. Shin EC, Choi BR, Lim WJ, Hong SY, An CL, et al. (2004) Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16 S rDNA sequence. Anaerobe 10: 313–319.EC ShinBR ChoiWJ LimSY HongCL An2004Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16 S rDNA sequence.Anaerobe10313319
- 76. Yu Z, Garcia-Gonzalez R, Schanbacher FL, Morrison M (2008) Evaluations of different hypervariable regions of archaeal 16 S rRNA genes in profiling of methanogens by archaea-specific PCR and denaturing gradient gel electrophoresis. Appl Environ Microb 74: 889–893.Z. YuR. Garcia-GonzalezFL SchanbacherM. Morrison2008Evaluations of different hypervariable regions of archaeal 16 S rRNA genes in profiling of methanogens by archaea-specific PCR and denaturing gradient gel electrophoresis.Appl Environ Microb74889893
- 77. Russel JB, Rychlik JL (2001) Factors that alter rumen microbial ecology. Science 292: 1119–1122.JB RusselJL Rychlik2001Factors that alter rumen microbial ecology.Science29211191122
- 78. Oliver WRB (1955) New Zealand Birds. Wellington: A.H. & A.W. Reed. 641 p.WRB Oliver1955New Zealand BirdsWellingtonA.H. & A.W. Reed641
- 79. Christensen H, Foster G, Christense JP, Pennycott T, Olsen JE, et al. (2003) Phylogenetic analysis by 16 S rDNA gene sequence comparison of avian taxa of Bisgaard and characterization and description of two new taxa of Pasteurellaceae. J Appl Microbiol 95: 354–363.H. ChristensenG. FosterJP ChristenseT. PennycottJE Olsen2003Phylogenetic analysis by 16 S rDNA gene sequence comparison of avian taxa of Bisgaard and characterization and description of two new taxa of Pasteurellaceae.J Appl Microbiol95354363
- 80. Gregersen RH, Neubauer C, Christensen H, Korczak B, Bojesen AM, et al. (2010) Characterization of Pasteurellaceae-like bacteria isolated from clinically affected psittacine birds. J Appl Microbiol 108: 1235–1243.RH GregersenC. NeubauerH. ChristensenB. KorczakAM Bojesen2010Characterization of Pasteurellaceae-like bacteria isolated from clinically affected psittacine birds.J Appl Microbiol10812351243
- 81. Sadeghi HMM, Najafabadi AJ, Abedi D, Dehkordi AJ (2008) Identification of an isolate of Pseudomonas aeroginosa desposited in PTCC as a PHA producer strains: comparison of three different bacterial genomic DNA extraction methods. J Biol Sci 8: 826–830.HMM SadeghiAJ NajafabadiD. AbediAJ Dehkordi2008Identification of an isolate of Pseudomonas aeroginosa desposited in PTCC as a PHA producer strains: comparison of three different bacterial genomic DNA extraction methods.J Biol Sci8826830
- 82. Peterson A, Bisgaard M, Christensen H (2010) Real-time PCR detection of Enterococcus faecalis associated with amyloid arthropathy. Lett Appl Microbiol 51: 61–64.A. PetersonM. BisgaardH. Christensen2010Real-time PCR detection of Enterococcus faecalis associated with amyloid arthropathy.Lett Appl Microbiol516164
- 83. Costa R, Gomes NCM, Milling A, Smalla K (2004) An optimized protocol for simultaneous extraction of DNA and RNA from soils. Braz J Microbiol 35: 230–234.R. CostaNCM GomesA. MillingK. Smalla2004An optimized protocol for simultaneous extraction of DNA and RNA from soils.Braz J Microbiol35230234
- 84. Webster NS, Negri AP, Munro MMHG, Battershill CN (2004) Diverse microbial communities inhabit Antarctic sponges. Environ Microbiol 6: 288–300.NS WebsterAP NegriMMHG MunroCN Battershill2004Diverse microbial communities inhabit Antarctic sponges.Environ Microbiol6288300
- 85. Wintzingerode FV, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21: 213–229.FV WintzingerodeUB GobelE. Stackebrandt1997Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis.FEMS Microbiol Rev21213229
- 86. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, et al. (2010) AJ DrummondB. AshtonS. BuxtonM. CheungA. Cooper2010Geneious. 5.5 ed. Available from http://www.geneious.com. Geneious. 5.5 ed. Available from http://www.geneious.com.
- 87. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2006) New screening software shows that most recent large 16 S rRNA gene clone libraries contain chimeras. Appl Environ Microb 72: 5734–5741.KE AshelfordNA ChuzhanovaJC FryAJ JonesAJ Weightman2006New screening software shows that most recent large 16 S rRNA gene clone libraries contain chimeras.Appl Environ Microb7257345741
- 88. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, et al. (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35: 7188–7196.E. PruesseC. QuastK. KnittelBM FuchsW. Ludwig2007SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB.Nucleic Acids Res3571887196
- 89. Ludwig W, Strunk O, Westram R, Richter L, Meier H, et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32: 1363–1371.W. LudwigO. StrunkR. WestramL. RichterH. Meier2004ARB: a software environment for sequence data.Nucleic Acids Res3213631371
- 90. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microb 75: 7537–7540.PD SchlossSL WestcottT. RyabinJR HallM. Hartmann2009Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.Appl Environ Microb7575377540
- 91. Spring S, Lins U, Amann R, Schleifer K, Ferreira LCS, et al. (1998) Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes. Arch Microbiol 169: 136–147.S. SpringU. LinsR. AmannK. SchleiferLCS Ferreira1998Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes.Arch Microbiol169136147
- 92. Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microb 64: 3472–3730.MF PolzCM Cavanaugh1998Bias in template-to-product ratios in multitemplate PCR.Appl Environ Microb6434723730
- 93. DeLong EF (1992) Archaea in costal marine environments. Proc Natl Acad Sci USA 89: 5685–5689.EF DeLong1992Archaea in costal marine environments.Proc Natl Acad Sci USA8956855689
- 94. Ishii K, Fukui M (2001) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl Environ Microb 67: 3753–3755.K. IshiiM. Fukui2001Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR.Appl Environ Microb6737533755
- 95. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16 S rRNA. Appl Environ Microb 59: 695–700.G. MuyzerEC de WaalAG Uitterlinden1993Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16 S rRNA.Appl Environ Microb59695700