research article characterization of the fungal microbiome...

Hindawi Publishing CorporationVeterinary Medicine InternationalVolume 2013, Article ID 658373, 8 pageshttp://dx.doi.org/10.1155/2013/658373

Research ArticleCharacterization of the Fungal Microbiome (Mycobiome)in Fecal Samples from Dogs

M. Lauren Foster,1 Scot E. Dowd,2 Christine Stephenson,1

Jörg M. Steiner,1 and Jan S. Suchodolski1

1 Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine & Biomedical Sciences,Texas A&M University, College Station, TX 77843, USA

2Molecular Research DNA Laboratory, Shallowater, TX 79363, USA

Correspondence should be addressed to Jan S. Suchodolski; [email protected]

Received 21 December 2012; Revised 4 April 2013; Accepted 5 April 2013

Academic Editor: Lorraine M. Sordillo

Copyright © 2013 M. Lauren Foster et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The prevalence and phylogenetic description of fungal organisms and their role as part of the intestinal ecosystem have not yet beenstudied extensively in dogs.This study evaluated the fungal microbiome of 19 dogs (12 healthy dogs and 7 dogs with acute diarrhea)using fungal tag-encoded FLX-Titanium amplicon pyrosequencing. Five distinct fungal phyla were identified, with Ascomycota(medians: 97.9% of obtained sequences in healthy dogs and 98.2% in diseased dogs) and Basidiomycota (median 1.0% in healthydogs and median 0.5% in diseased dogs) being the most abundant fungal phyla. A total of 219 fungal genera were identified acrossall 19 dogs with a median (range) of 28 (4–69) genera per sample.Candidawas the most abundant genus found in both the diseaseddogs (median: 1.9%, range: 0.2%–38.5% of sequences) and healthy dogs (median: 5.2%, range: 0.0%–63.1% of sequences). Candidanatalensis was the most frequently identified species. No significant differences were observed in the relative proportions of fungalcommunities between healthy and diseased dogs. In conclusion, fecal samples of healthy dogs and dogs with acute diarrhea harborvarious fungal genera, and their role in gastrointestinal health and disease warrants further studies.

1. Introduction

Recent molecular-phylogenetic studies have revealed diversemicrobial communities in the canine gastrointestinal (GI)tract and have emphasized the importance of the intestinalmicrobiota for gastrointestinal health [1]. The intestinalmicrobiota plays a vital role in the health of the GI tract, par-ticipates in the development of the host immune system, andalso provides protection from invading pathogens [2]. Severalstudies have characterized the bacterial communities in thecanine GI tract in health and disease [3–5]. However, limitedinformation is available about the prevalence and classifica-tion of other members of the intestinal microbiome, such asfungal organisms. Previous studies that have described fungalorganisms in the canine GI tract have either used culturebased methods [6, 7], have utilized molecular-phylogeneticmethods on pooled intestinal samples [8, 9], or have analyzed

only a limited number of fungal sequences [10]. Studies inhumans have suggested that the fungal microbiomemay playa role in chronic GI disorders [11, 12]. Therefore, a moredetailed description of the fungal microbiome (mycobiome)is needed to better understand the role of fungi in the GI tractof healthy dogs and dogs with GI disease.

The aim of this study was to describe the fungal commu-nities present in fecal samples obtained from healthy dogsand dogs with acute, nonhemorrhagic diarrhea using high-throughput 18S rRNA gene pyrosequencing.

2. Materials and Methods

2.1. Fecal Samples. Naturally passed fecal samples were col-lected from a total of 19 privately owned dogs (12 healthydogs and 7 dogs with acute diarrhea). All dogs, healthy and

2 Veterinary Medicine International

diseased, lived in Texas at the time of sample collection.The dogs of the healthy dog group had a median weightof 16.5 kg with a range of 2.6–35.0 kg and a median age of5.6 years with a range of 2.0–15.0 years. The dogs of thediseased dog group had a median weight of 16.0 kg with arange of 2.5–28.0 kg and a median age of 7.0 years with arange of 1.0–15.0 years. The healthy dogs were owned bystudents and staff of Texas A&M University. At the timeof sample collection, all 12 healthy dogs (Table 1) were freefrom any clinical signs of disease. A complete blood countand serum biochemistry profile were analyzed at the TexasVeterinary Medical Diagnostic Laboratory (College Station,TX). To exclude subclinical gastrointestinal or pancreatic dis-ease in the healthy dogs, serum concentrations of cobalaminand folate (Immulite 2000, Siemens Healthcare Diagnos-tics Inc., Deerfield, IL, USA), pancreatic lipase immunore-activity (cPLI; Spec cPL ELISA kit, IDEXX Laboratories,Westbrook, ME, USA), and trypsin-like immunoreactivity(cTLI; I-RIA kit, Siemens Medical Solution Diagnostics, LosAngeles, CA, USA) were measured at the GastrointestinalLaboratory at Texas A&M University. Fecal samples fromall dogs in the diseased and healthy groups were analyzedfor Clostridium perfringens and Clostridium difficile usingan enzyme-linked immunosorbent assay kit (C. perfringensEnterotoxin Test and C. difficile Enterotoxin Test, TechLab,Inc., Blacksburg, VA, USA), Giardia and Cryptosporidiumusing an indirect fluorescent antibody test kit (Merifluor,Meridian Bioscience Inc., Cincinnati, OH), Campylobacterspp. using PCR, and other pathogens using a routine fecalflotation.

One fecal sample per animal was collected immediatelyafter natural defecation, stored at 4∘C before and duringtransport to the laboratory (within 24 hours of samplecollection), and subsequently stored frozen at −80∘C untilDNA extraction.

For the diseased dogs, leftover fecal samples that weresubmitted from veterinary hospitals in the College Sta-tion/Houston area for fecal pathogen analysis unrelated tothe current study were analyzed. Questionnaires were sentto submitting veterinarians to enquire about the clinicalsigns of gastrointestinal disease, the duration, and the finaldiagnosis. Furthermore, to ensure that samples were handledin a similar fashion as the samples from the healthy dogs,information was obtained about the collection, storage, andshipping of samples to the laboratory. Only samples that werehandled in a similar fashion to the samples from healthydogs were analyzed. Leftover fecal samples for the diseaseddogs (Table 1) were chosen based on a presenting complaintof acute, uncomplicated, nonhemorrhagic diarrhea (definedas <72 hours of onset) that resolved with symptomatictreatment.

At the time of sample collection, none of the evaluatedhealthy and diseased dogs were receiving any medicationsexpected to alter the gut microbiota (i.e., antibiotics) andwere vaccinated and dewormed regularly. All dogs were fedcommercial diets and no diet change was reported within the3-week period prior to sample collection.

This study was approved by the Clinical Research ReviewCommittee of Texas A&M University (CRRC#07-38).

2.2. DNA Extraction. Genomic DNA was extracted fromeach fecal sample using a bead-beating technique followed byphenol-chloroform-isoamyl alcohol extraction as describedpreviously [13].

2.3. 18S rRNA Gene Pyrosequencing for Fungal Organisms(fTEFAP). Tag-encoded FLX-Titanium amplicon pyrose-quencing (fTEFAP) and data processing for fungal organismswere performed as described previously [8, 14] with panfun-gal primers forward funSSUF-TGGAGGGCAAGTCTGGTGand reverse funSSUR-TCGGCATAGTTTATGGTTAAG.

The raw data from fTEFAP was screened and trimmedbased on quality scores (nominal phred20), binned intoindividual sample collections, and then depleted of anychimeras using B2C2 (http://www.researchandtesting.com/B2C2.html) [15]. The sequences were then compared againsta curated fungal sequence database as reported previously[8, 14]. Fungal sequences were grouped into operationaltaxonomic units (OTUs) based on identity scores to known18S fungal sequences; >97% of identity were reported atthe species level, between 95% and 97% at the genus level,between 90% and 95% at the family level, and between 80%and 90% at the order level.

2.4. Statistical Analyses. The pyrosequencing results wereexpressed as a percentage of the total fungal communityin each dog. Only taxa that were present in at least 50%of dogs (either healthy or diseased) were included inthe analysis. All percentage data was tested for normalityusing the Kolmogorov-Smirnov normality test. As the datawas found to be nonparametric, a Mann-Whitney 𝑈 testwas used to compare the percentages of fungal organismsbetween the healthy and the diseased dogs at the phylum,class, order, family, and genus levels. All statistical analyseswere conducted using a commercially available statisticalsoftware program (Prism 5, GraphPad, San Diego, CA,USA). Significance level was set at 𝑃 < 0.05 for allcomparisons.

To visualize differences in the relative abundance offungal genera in individual samples as a heat map, a doubledendrogram was generated using multivariate hierarchicalclustering methods based upon Furthest Neighbor metricwith Euclidean distances in NCSS 2007 (NCSS, Kaysville,Utah) [16]. The sequencing coverage for each sample wascalculated according to Good using the formula [1 − (𝑛/𝑁)]× 100, where 𝑛 is the number of unique sequences and𝑁 is the total number of sequences obtained for eachsample.

3. Results

3.1. Animals. The results of the serum biochemistry profile,complete blood count, serum concentrations of cobalamin,folate, cPLI, and cTLI did not reveal abnormalities in thehealthy group. Review of the clinical records of the dogs withacute diarrhea showed that all dogs recovered uneventfullywith symptomatic therapy. Results of fecal examination

Veterinary Medicine International 3

Table 1: Dogs enrolled into this study.

ID Health status Age Breed Sex Weight (kg)H1 Healthy 4.2 Shih-tzu fs 7.0H2 Healthy 4.0 English Bulldog fs 20.7H3 Healthy 15.0 Chihuahua fs 2.6H4 Healthy 2.0 Golden Retriever mn 35.0H5 Healthy 7.8 Mixed breed mn 30.4H6 Healthy 4.0 Dachshund fs 6.0H7 Healthy 9.6 Mixed breed fs 32.1H8 Healthy 10.0 Mixed breed mn 28.0H9 Healthy 3.5 Miniature Schnauzer mn 9.5H10 Healthy 5.2 Terrier Mix mn 27.7H11 Healthy 5.9 Brussels Griffon mn 6.4H12 Healthy 9.2 Beagle mn 12.4D1 Diseased 1.5 Labrador Retriever fs 28.0D2 Diseased 11.0 Mixed Breed fs 14.5D3 Diseased 15.0 Cocker Spaniel mn 16.0D4 Diseased 7.0 Chihuahua fs 2.5D5 Diseased 2.3 King Charles fs 6.0D6 Diseased 14.0 Golden Retriever fs 24.8D7 Diseased 1.0 Labrador mix mn 18.0m: male intact; f: female intact; mn: male neutered; fs: female spayed.

(i.e., fecal flotation, fecal analyses for C. perfringens, C.difficile, Cryptosporidium, and Giardia, and PCR analysis forCampylobacter spp.) on all dogs did not reveal the presenceof any specific enteropathogen.

3.2. Pyrosequencing for Fungal Organisms. A total of 57,179sequences (median 2800, range 840–6000 per sample) ofgood quality were obtained. To allow for equal sequencingdepth across all samples, 840 randomly selected sequenceswere analyzed per sample, as described previously [16]. Atotal of five phyla were identified. Of these, Ascomycotaand Basidiomycota were found in ≥50% of dogs in bothgroups of diseased and healthy dogs. The remaining phyla,Chytridiomycota, Neocallimastigomycota, and Microsporidia,were found in ≤50% of dogs in both groups. A total of 219fungal genera were identified across all 19 dogs in this studywith a median of 28 genera per dog and a range of 4–69genera per dog. There were a median of 32 genera (range 10–55) per dog in the healthy group and a median of 18 genera(range 4–69) per dog in the diseased group.The mean (±SD)coverage was 0.956 (±0.0256) based on a sequencing depth of840 sequences per samples.

No significant differences (𝑃 > 0.05) of the percentageof fungal organisms at the phylum, class, order, family, andgenus levels were found between the healthy and diseaseddogs.There were no significant differences in age (𝑃 = 0.983)or weight (𝑃 = 0.577) between the healthy and diseasedgroups. Table 2 summarizes the relative percentages in termsof median and range for the most abundant fungal groupson the various phylogenetic levels based on pyrosequencingresults (phylum, class, order, and genus levels). Only groups

that were present in at least 50% of either healthy or diseaseddogs are shown.

Members ofAscomycotawere found in all 19 dogs and thiswas the most abundant fungal phylum in both the healthydogs and the diseased dogs with a median of 97.90% anda range of 63.2%–100.0% and a median of 91.4% and arange of 91.4%–100.0% of all fungal sequences, respectively.Basidiomycotawas the second most abundant fungal phylumwith a median of 1.0% and a range of 0.0–36.8% for thehealthy group and a median of 0.5% and a range of 0.0%–7.8% for the diseased group.Basidiomycotawas found in eighthealthy dogs and five diseased dogs.

Within Ascomycota, classes in either the healthy orthe diseased groups consisted of Dothideomycetes, Saccha-romycetes, Eurotiomycetes, Taphrinomycetes, and Sordari-omycetes, with Dothideomycetes being the most abundantclass (median: 34.0%, range: 0.3%–74.4% for healthy dogs;median: 28.8%, range: 19.1%–90.9% for diseased dogs). Themost prominent orders in Dothideomycetes were Pleosporales(median: 16.1%, range: 0.0%–50.9% for healthy dogs and amedian of 15.0% and a range of 0.0%–81.4% for diseased dogs)and Capnodiales (median: 11.5%, range: <0.1%–36.0% forhealthy dogs; median: 8.7%, range: 0.8%–81.8% for diseaseddogs). Pleosporales was the most diverse of the two orders,containing four genera and eight species.

Within Basidiomycota, the predominant classes wereAgaricomycetes andUstilaginomycetes, with the latter consist-ing almost exclusively the orders Ustilaginales and the genusUstilaginaceae.

Table 3 shows several species in the major fungal groupsidentified in the dog feces using pyrosequencing, as wellas some species considered to have the potential to be


Table2:Re

lativepercentageso

fthe

mosta

bund

antfun

galg

roup

sonthevario

usph

ylogeneticlevelsbasedon

pyrosequ

encing

.Thelette

rsin

parenthesis

indicate

phylogeneticlevels(p:

phylum

,c:class,o:order,f:fam

ily,g:genus).

Taxono

my

Range(minim

um%–m

axim

um%)a

ndmedians

(%)

Num

bero

fdogsh

arbo

ringtaxa

Health

yrange

Health

ymedian

Dise

ased

range

Dise

ased

median

Mann-Whitney𝑃value

Health

yDise

ased

Ascomycota(

p)63.20–

100.00

97.90

91.40–

100.00

98.20

0.58

127

Catenu

lostrom

a(g)

0.00–31.4

18.23

0.09–4

8.36

7.09

0.83

117

Dothideom

ycetes(c)

0.26–74.38

34.03

19.07–90.91

28.82

0.29

127

Capn

odiales

(o)

0.04–36.00

11.45

0.84–81.8

08.65

0.97

127

Davidiellaceae

(f)

0.00–29.0

40.13

0.00–6

3.64

0.06

0.79

74

Cladosporiu

m(g)

0.00–29.0

40.13

0.00–6

3.64

0.06

0.79

74

Schizothyriaceae

(f)

0.04–33.96

8.55

0.14–51.6

48.42

0.83

127

Pleosporales(c)

0.00–50.88

16.09

0.00–81.3

814.96

0.80

116

Phom

a(g)

0.00–8.27

1.28

0.00–9.09

1.86

0.83

95

Massarin

aceae(f)

0.00–5.26

0.47

0.00–2.54

0.80

0.83

95

Keissler

iella(g)

0.00–5.26

0.47

0.00–2.54

0.80

0.83

95

Phaeosphaeria

ceae

(f)

0.00–9.22

0.06

0.00–9.73

0.00

0.82

63

Teratosphaeria(g)

0.00–7.14

0.32

0.00–3.27

0.26

0.90

86

Pleosporaceae(f)

0.00–4

3.86

6.09

0.00–80.59

6.90

0.97

106

Cochliobolus(g)

0.00–21.0

50.09

0.00–72.07

0.60

0.64

85

Phaeosphaeria

(g)

0.00–9.22

0.06

0.00–9.73

0.00

0.82

63

Pyrenophora(g)

0.00–11.9

20.05

0.00–7.09

0.26

0.93

74

Bipolaris

(g)

0.00–21.0

52.39

0.00–6

.54

1.41

0.55

96

Shira

iaceae

(f)

0.00–5.82

0.02

0.00–4

.36

0.00

0.89

63

Shira

ia(g)

0.00–5.82

0.02

0.00–4

.36

0.00

0.89

63

Dothideales(o)

0.00–10.71

1.71

0.04–9.09

0.44

0.97

97

Dothioraceae(f)

0.00–10.71

1.71

0.04–9.09

0.44

0.97

97

Aureobasidium

(g)

0.00–10.7

1.71

0.05–9.09

0.44

0.97

97

Saccharomycetes(c)

0.19–9

9.40

10.94

0.35–4

2.66

6.87

0.58

127

Saccharomycetales(o)

0.18–9

9.40

10.94

0.34–4

2.66

6.87

0.58

127

Wick

erhamom

ycetaceae(f)

0.00–6

3.08

5.20

0.19–38.46

1.86

0.53

117

Saccharomycetaceae(f)

0.00–9

9.40.67

0.00–14.91

0.14

0.73

94

Saccharomyces(g)

0.00–7.14

0.00

0.00–4

.190.06

0.52

54

Cand

ida(g)

0.00–6

3.08

5.20

0.19–38.46

1.86

0.53

117

Eurotio

mycetes(c)

0.04–14.7

4.16

0.00–4

3.9

4.90

0.97

126

Eurotia

les(o)

0.04–14.29

3.00

0.00–10.64

4.89

0.97

126

Trich

ocom

aceae(f)

0.04–14.29

3.00

0.00–10.64

4.89

0.90

126

Aspergillus

(g)

0.00–7.28

0.42

0.00–6

.65

0.53

0.86

74

Penicilliu

m(g)

0.00–14.29

2.09

0.00–5.81

1.53

0.74

116

Taphrin

omycetes(c)

0.00–30.53

4.49

0.00–11.7

65.05

0.61

116

Taphrin

ales(o)

0.00–30.53

4.49

0.00–11.7

65.05

0.61

116

Taphrin

aceae(f)

0.00–30.53

3.69

0.00–11.7

65.05

0.74

116

Sordariomycetes(o)

0.00–52.32

9.84

0.00–6

1.46

10.37

0.97

115

Phom

atospora

(g)

0.00–17.9

11.6

70.00–4

3.13

0.66

0.97

104

Meliolales(o)

0.00–17.9

11.6

70.00–4

3.13

0.66

0.97

104


Table2:Con

tinued.

Taxono

my

Range(minim

um%–m

axim

um%)a

ndmedians

(%)

Num

bero

fdogsh

arbo

ringtaxa

Health

yrange

Health

ymedian

Dise

ased

range

Dise

ased

median

Mann-Whitney𝑃value

Health

yDise

ased

Xylaria

les(o)

0.00–1.55

0.00

0.00–5.59

0.44

0.17

44

Xylaria

ceae

(f)

0.00–1.55

0.00

0.00–5.59

0.25

0.17

34

Sordariales

(o)

0.00–17.8

90.03

0.00–0

.44

0.00

0.54

62

Hypocreales(o)

0.00–30.53

4.49

0.00–9.09

2.71

0.10

115

Ophiocordycipita

ceae

(f)

0.00–32.58

3.73

0.00–9.09

1.99

0.33

115

Hypocreaceae(f)

0.00–17.33

0.03

0.00–0

.66

0.00

0.49

63

Hypocrea(g)

0.00–17.33

0.03

0.00–0

.66

0.00

0.49

63

Myrotheciu

m(g)

0.00–32.58

3.25

0.00–9.09

1.10

0.47

105

Clavicipitaceae

(f)

0.00–1.86

0.21

0.00–0

.66

0.00

0.36

83

Cordycipita

ceae

(f)

0.00–0

.61

0.00

0.00–0

.09

0.00

0.51

31

Engyodontiu

m(g)

0.00–3.57

0.11

0.00–0

.69

0.00

0.24

62

Papu

losaceae

(f)

0.00–17.9

1.67

0.00–4

3.10

0.67

0.97

126

Basid

iomycota(

p)0.00–36.75

0.97

0.00–7.76

0.54

0.97

85

Agarico

mycetes(c)

0.00–3.31

0.00

0.00–4

.88

0.27

0.23

55

Ustilaginom

ycetes(c)

0.00–33.44

0.15

0.00–2.88

0.00

0.12

71

Ustilaginales(o)

0.00–24.83

0.05

0.00–0

.66

0.00

0.13

61

Ustilaginaceae(f)

0.00–24.83

0.05

0.00–0

.66

0.00

0.13

61


Amou

nt

72.07

5.29

1.33

0.20

0.00H1 H2H12 D6 D7 H5 H9H10 D6 H11 D2 H6 H8 D4 H3 H7 D5 D3 H4

CandidaCatenulostromaPhomatosporaCochliobolusCladosporiumMyrotheciumBipolarisHypocreaPyrenophoraPhomaPhaeosphaeriaAspergillusPenicilliumSaccharomycesKeissleriellaShiraiaTeratosphaeriaEngyodontiumAureobasidium

Figure 1: Dual hierarchal dendrogram based upon the predominant fungal genera. The clustering is based upon Furthest Neighbor metricwith Euclidean distances. The heat map represents the relative percentages of the most abundant fungal genera identified in each sample (H= healthy, D = diseased).

pathogenic. The sequences were blasted against the NCBIdatabase.The accession number, the similarity to their closestneighbor in the NCBI, and the number of healthy anddiseased dogs harboring these species are summarized in thistable. Figure 1 shows the differences in the relative abundanceof the fungal genera in each dog displayed as a heat map.

4. Discussion

In this study, 454-pyrosequencing of the fungal 18S rRNAgene was utilized to characterize the fungal microbiomepresent in canine feces. Fecal samples from a total of 19 dogswere individually evaluated. Seven dogs were classified asdiseased based on the presence of acute nonhemorrhagicdiarrhea, and the remaining 12 were classified as healthy. Inthe current study, no significant differences in the relativeproportions of fungal groups were observed between thediseased and healthy dogs. The most abundant phylum incanine feces was Ascomycota (median: 97.9%, range: 63.2%–100.0% for healthy and median: 98.2%, range: 91.4%–100.0%

for diseased dogs). The most abundant classes includedDothideomycetes and Saccharomycetes,withCandida found tobe the most abundant genus.

Previous studies using either culture-based or molecularmethods have provided some information about the fungalmicrobiome present in the GI tract of dogs [6–9, 17]. Basedon cultivation studies, Mentula et al. observed a higherprevalence of yeast in the lumen of the jejunum comparedto the feces of healthy dogs (27% versus 5%, resp.) [7].Benno et al. reported the presence of yeasts and molds inthe stomach, ileum, colon, and rectum in 2 of 8 healthyBeagle dogs [6]. However, no further specifics regarding thephylogenetic classification of these organisms were reportedin these two studies. Using a panfungal PCR, Suchodolski etal. reported that 60% of healthy dogs and 76% of dogs withchronic enteropathies were positive for fungal DNA in smallintestinal brush samples [10]. All 51 phylotypes identified bySuchodolski et al. were members of the phyla Ascomycota (32phylotypes) or Basidiomycota (19 phylotypes) [10]. Handl etal. analyzed pooled fecal samples obtained from 12 healthy


Table 3: Fungal species identified in feces from dogs using pyrosequencing. The accession number and similarity to their closest relative inthe NCBI database and the number of healthy and diseased dogs harboring the species are summarized.

Fungal species Accession no. to closest relative Similarity Healthy dogs Diseased dogsCatenulostroma abietis FJ267703 99 11 7Bipolaris eleusines DQ337382 99 0 1Bipolaris sorokiniana DQ337383 99 8 6Candida albicans AF114470 99 0 1Candida austromarina AB013560 99 3 1Candida castellii AY497752 92 2 1Candida glycerinogenes AY584809 99 2 0Candida homilentoma AB018166 99 1 0Candida khmerensis AB158655 98 1 0Candida mesenterica AB013552 99 1 0Candida natalensis AB013541 99 10 6Candida neerlandica EF120593 99 2 0Candida zeylanoides EU590665 99 1 0Aspergillus flavipes AB002061 98 0 1Aspergillus niger JX112703 99 4 3Aspergillus ochraceus AB008405 99 2 1Aspergillus penicillioides AB002078 99 4 0Aspergillus terreus JN639854 99 5 2Penicillium brevicompactum AF548082 99 4 0Penicillium charlesii FJ430768 99 4 1Penicillium commune EU263609 99 2 1Penicillium coprobium FJ430772 99 1 0Penicillium janthinellum AB293968 100 7 4Penicillium tardum AF245233 99 4 1Penicillium verruculosum AF510496 100 4 2Myrothecium cinctum AJ301996 99 6 5Myrothecium gramineum FJ825369 99 8 3Myrothecium leucotrichum AJ301992 99 4 2Cryptococcus gastricus DQ645513 98 2 0Cryptococcus surugaensis AB100440 97 1 0

dogs using 18S rRNA gene pyrosequencing and found thatAscomycota comprised 99% of the fungal sequences, withSaccharomycetes found to be the most abundant class at85% and Candida found to be the most abundant genus[8]. Similarly, using a shotgun DNA sequencing approach,Swanson et al. also identified Ascomycota and Basidiomycotaas the most abundant phyla in pooled fecal samples of 6healthy dogs [9]. However, the overall abundance of fungalsequences was low (0.01% of the canine metagenome) andonly 3 distinct phylotypes were identified, most likely due toan insufficient sequencing depth [9].

Our results are in general agreement with the previouslycited studies. By analyzing the fungal 18S rRNA gene in indi-vidual fecal samples, we identified a higher number of fungalOTUs (at 97% similarity; OTU

97) compared to a previous

study that used a metagenomic approach based on DNAshotgun sequencing of fecal samples andwhich reported only3 OTUs [9]. Also, our study revealed a higher number ofOTUs than reported for pooled fecal samples (i.e., 33OTU

97).

This study also suggests that the fecal mycobiome harbors

more fungal OTUs compared to the small intestine, as onestudy examining the small intestinal fungal microbiomereported that 79% of dogs positive for fungal DNA harboredonly one unique phylotype [10].However, the species richnessof fungal organisms appears to be lower compared to thebacterial richness, as several hundred bacterial OTUs havebeen reported [8] in fecal samples of dogs.

While this study provided insight into the diversity ofthe fungal microbiome in healthy dogs and dogs with acutediarrhea, some limitations need to be noted. The methodsemployed did not allow quantitative determination of thefungal abundance. Previous metagenomic studies performedon canine fecal samples suggest that fungal sequences makeup a small proportion of the total fecal microbiota with0.3% of obtained sequences [9]. Quantitative studies utilizingfluorescence in situ hybridization (FISH) of fungal small-subunit rRNA probes estimated the abundance of fungi as<2% in fecal samples from mice [18] and <0.03% in fecalsamples from humans [11]. Future studies require the use ofa more quantitative enumeration technique such as FISH to


quantify the abundance of fungi in feces of dogs. The currentstudy was limited in terms of the number of animals and thecomparison of a healthy community to a community withonly one disease type, and these results were obtained fromfecal samples only, not biopsies. The current cost of high-throughput sequencing prohibited the use of a larger samplesize.

In conclusion, the current study provides informationabout the fungal microbiome present in canine feces fromboth healthy dogs and dogs with acute, nonhemorrhagicdiarrhea. The results provide a baseline for future studiesevaluating the fungal microbiome in dogs with variousgastrointestinal disorders.

Acknowledgment

This paper was presented in part as an abstract at the Forumof the American College of Veterinary Internal Medicine,Anaheim, California, June 2010.

References

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