Fungi are an integral part of the skin microbiota, and, as such, they play an important role in cutaneous homeostasis and atopic dermatitis.1,2 Notwithstanding, the exact nature of this role, and the interaction of the mycobiome with the microbiome in general, remain elusive and understudied.1–3 Fungal species, such as Cladosporium spp and Malassezia spp, can act both as an allergen and a pathogen.4–6 While there may be some data as to whether they are part of the core or normal flora,7,8 some authorities consider fungi as opportunist pathogens.9,10
In humans, Malassezia spp account for the most commonly recovered fungal family on the trunk and arms of adults.11,12 Given the diversity of available cutaneous microniches, this, very limited, spectrum of fungal species on the majority of the human body is not understood.1 Despite this apparent paucity of species on the skin of adult people, there is some significant body site variation.11,12 For example, the human foot harbors complex fungal communities composed of Aspergillus spp, Cryptococcus spp, Rhodotorula spp, and Epicoccum spp.13 Age is also a recognized factor, as children carry a much more diverse fungal flora than postpubescent adults.14
The canine cutaneous mycobiota, with the exception of Malassezia spp,6 also has received less attention than the bacterial biome, particularly with regard to next-generation sequencing. According to NCBI, as of March 2023, of the 12 studies investigating the canine cutaneous biome, which used 16S rRNA new technology, only 6 reported the fungal component.7,8,15–18 Species of fungi found on the skin of animals have been considered opportunistic9 with the 2 most consistently recovered genera from normal dogs being Alternaria spp and Cladosporium spp.7,8,15–18 Tang et al8 reported that 20% of the fungal isolates from their 172 normal dogs were Capnodiales spp (related to Cladosporium).19 The mycobiome of the skin of dogs shows very little site variation, with interdog variation being much more significant than body site.7,8,15
This study was undertaken to sample, in a quantitative manner, the skin of normal dogs to assess the density of the fungal constituents of the cutaneous mycobiome. A secondary study was undertaken to assess if gently scraping the skin before swabbing had an effect on the subsequent sample, as it was reported to do when used to sample the skin of dogs with dermatidides.20
Methods
Twenty dogs with no history of internal disease or skin disease were sampled. None of the dogs were receiving any medication, other than routine antiparasitics. All dogs were owned by members of the practice, or their families, all of whom gave informed, signed, consent.
To minimize contamination of the skin samples 2 steps were taken. First, owners were given an appointment, such that they could then be escorted through the waiting room without waiting. Second, examination tables were cleaned, before sampling, with disinfectants expected to denature any residual DNA (Virkon; VioVet).
Each dog was gently restrained in lateral recumbency by the owner and sampled twice on the same occasion, once on the left, the A sample, and once on the right, the B sample, of the umbilicus. Hair was not clipped.
Sterile, single-use gloves were worn. Swab samples were taken by using sterile, dry, DNA-free HydraFlock (catalog No. 25-3406-H; Puritan), which was used to rub the skin surface, in an agreed, standard manner within the confines of a brass metal guide, with an internal area of 3.5 cm2.
The guides were No. 9 door furniture letters. These were cold sterilized between patients, and samples, with Reprodis HLD4l. The A and B samples were taken by gently swabbing the skin 6 times vertically and then 6 swabs laterally. Swab strokes were to cover the height and width of the area within the guide. The B samples were taken as with the A samples, but only after a sterile spatula was used to gently scrape the surface of the skin. The scrapes were performed in a standard manner of 6 gentle scrapes vertically followed by 6 gentle scrapes laterally to cover the height and width with the guide.
Swab tips were placed into vials containing a sterile DNA preservative (DNA/RNA Shield; catalog No. R1108; Zymo Research Corp) and immediately frozen. Samples were shipped en masse to the MiDOG LLC testing facility.
Genomic DNA was purified using the ZymoBIOMICSTM-96 DNA kit (catalog No. 79 D4304; Zymo Research Corp). Sample library preparation and data analysis for fungal profiling were performed by MiDOG LLC, using a Quick-16S NGS Library Prep Kit (catalog No. D6400; Zymo Research Corp) with minor modifications. As internal controls to ensure the accuracy and cleanliness of the data generated, and to control for any potential contamination of the equipment, sequencing buffers, and other material, several negative controls were run for both the extraction process and the library preparation. These included an “extraction negative control,” which was the storage buffer (catalog No. R1100-50; DNA/RNA Shield), which was lysed, extracted, library prepped, and sequenced in parallel with experimental samples. Further, a “library preparation negative control” and a “no template control” for the library preparation were run. The workflow is automated using a Hamilton Star liquid handling robot (Hamilton Company) to minimize human error during the sampling process.
To control for contamination, both cellular and DNA mock communities were used as positive controls (ZymoBIOMICS Microbial Community Standard; catalog No. D6300 and D6305; Zymo Research Corp) to account for any bias in the workflow starting from the DNA extraction process. ZymoBIOMICS Microbial Community Standard (Zymo Research Corp) was used as a positive control to monitor the performance of all steps of the next-generation sequencing workflow including the bioinformatic analysis. Primer sequences targeted the internal transcribed spacer 2 region for mycobiome analysis as previously described.8 Libraries were sequenced using an Illumina HiSeq 1500 sequencer, for a sequencing depth of 7 to 8 million reads, generating at least 10,000 reads per sample. Reads were filtered through Dada2 (R package version 3.4). Taxonomy prediction was performed with Centrifuge21 combined with a custom reference database (version 24; Zymo Research) curated, in part, from draft or complete genomic sequences available from NCBI GenBank.
Phylotypes were computed as percent proportions based on the total number of sequences in each sample. The species-level resolution of the sequencing approach used here has previously been demonstrated by shotgun sequencing.8 Absolute microbial quantification was achieved using real-time PCR targeting the internal transcribed spacer 2 region.
Statistical Analysis
The statistical analysis was performed using Stata version 15.1.
The analysis made comparisons between the 2 sets of measurements. As both measurements were made on the same animals, this gave rise to paired data. Due to the skewed distribution of the outcome values, the Wilcoxon matched pairs test was used for the analysis. Due to the distribution of the outcome values, the median and IQR were used to summarize the responses for each set of measurements. The P value was set at .05.
Results
There were 9 males and 11 females, with a mean age of 4.9 years (range, 9 months to 12 years). There were 20 A samples and 20 B samples.
A total of 99 species of fungi were identified, 87 of which were found on 1 or 2 dogs only. Twelve species occurred on 3 or more of the dogs, and these are listed and ranked (Table 1). The 87 species found on 1 or 2 dogs are listed elsewhere (Supplementary Table S1).
The most prevalent fungi on the skin of 20 healthy dogs ranked, with median count and IQR.
| Species | Prevalence | Median (IQR) |
|---|---|---|
| Cladosporium sp | 20 | 397 (11.7, 1,524) |
| Vishniacozyma victoriae | 20 | 22.5 (6.41, 90.6) |
| Boeremia exigua | 17 | 57 (17.3, 140) |
| Alternaria sp | 15 | 11.9 (5, 34.2) |
| Pleosporales sp | 12 | 11.5 (4.8, 33.3) |
| Ascomycota sp | 11 | 25.2 (11.9, 75.8) |
| Alternia abundans-armoraciae | 11 | 57 (17.9, 140) |
| Fungi sp | 10 | 35 (14.5, 140) |
| Botrytis caroliniana-cinerea | 9 | 34 (15.1, 68) |
| Aureobasidium proteae-pullulans | 8 | 2.64 (2.91, 3.8) |
| Claviceps sp | 7 | 5 (2.3, 11) |
| Claviceps paspali | 6 | 11 (5.3, 19,5) |
| Fusarium sp | 5 | 4.9 (3.1, 21) |
| Sporobolynae roseus | 4 | 12.8 (4.8, 29.4) |
The median fungal count was 1.1 X 105 cells cm−2 (IQR, 27,561, 409,572). The median count from the A samples was 4.8 X 104 cells cm−2 (IQR, 29,245, 128,291), and the median fungal count from the B samples was 6.6 X 105 cells cm−2 (IQR, 29,668, 184,270). This difference was not significant (P = .31) The statistical analysis suggested that there was no difference between the A and B samples (P = .17).
There was no difference between males and females, between males and neutered males, or between females and neutered females. There was no significant difference between A or B samples for any of the taxa recovered.
Cladosporium spp were found on every dog (Table 1), and in every dog, it was the dominant isolate, accounting for over 73% of the total count, giving a median count of 3.97 X 102 cells cm−2 (IQR, 11.7, 1,524). Vishniacozyma victoria also was found on every dog, with a median count of 22.5 cells cm−2 (IQR, 6.41, 90.6). V victoria was typically 2 to 5% of the count, although in 8 dogs it accounted for over 7.5%. Boeremia exigua was found on 17 of the dogs with a median of 57 (IQR, 17.3, 140), and Alternaria sp was found on 15 with a median of 11.9 (IQR, 5, 34.2). Malassezia pachydermatis was found on 2. A graphical depiction of these results is shown (Figure 1).
A graphical representation of the 12 most prevalent fungi on the skin of 20 normal dogs. The dominance of Cladosporium spp is readily apparent.
Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.04.0071
Discussion
These results show that, in contrast to humans and in accord with other canine studies,7,8,16 the principal fungal species on healthy dogs was Cladosporium spp and not Malassezia pachydermatis. We found Malassezia pachydermatis on 2 dogs, and while this prevalence is a little higher than that found by Meason-Smith et al,7 Tang et al,8 or Chermprapai et al,15 it is of the same order as that reported for healthy dogs by Prado et al.22 The axilla and groin were noted to carry a lower number of malassezial yeast than other areas, such as the chin.23
Cladosporium spp were the most commonly found fungal species in 4 of the studies reporting the members of canine mycobiome,7,8,16 although in rare cases not all15 Cladosporium spp have been associated with severe disease in dogs.5,24 The genera are also considered a relevant allergen in some atopic dogs and, accordingly, are a component of both serum IgE and intradermal skin testing panels.4,6,25 We found this organism on every dog, on both A and B samples, rather suggesting it is not an opportunist.
Vishniacozyma victoriae (renamed from Cryptococcus victoriae) was found on all 20 dogs. Recently, Rush et al26 analyzed the concentrations of V victoriae in houses participating in the New York City Neighborhood Asthma and Allergy Study. V victoriae was significantly associated with the presence of a dog within the house. The inverse relationship of the V victoriae concentration within the house and the presence of asthma within the household occupants suggested a protective effect, either of the presence of the fungus, or a dog, or both.26 V victoriae also has been shown to induce inflammation in a mouse model of asthma.27 There is evidence to support the protective effect of a dog, or a member of its cutaneous microbiome, within the house, as it has been associated with a lower incidence of allergic diseases, such as food allergy and asthma,28–32 but the evidence a protective effect is not clear cut.33,34 Notwithstanding, our findings go some way to explaining the relationship between V victoriae and the presence of a dog in the house.26
Both Cladosporium spp and V victoriae appear to be members of the core cutaneous flora of the dog, but the classification of their exact status requires temporal studies.
Alternaria spp, much like Cladosporium spp, have been regularly recovered from the skin of dogs.7,14,15 However, in our study, Alternaria spp were only found on 70% dogs and in numbers an order of magnitude lower than that of Cladosporium spp. Alternaria spp have been associated with opportunistic mycoses in the skin of humans and the dog after cutaneous inoculation, after iatrogenic immunosuppression, or through blood dissemination in immune-suppressed patients.35,36
Also, we found Boeremia exigua on a large number of dogs (Table 1). B exigua is an environmental fungus that has been associated with fungal infections in a variety of plants.37 It must be regarded as a transient, perhaps a contaminant, although its recovery from 60% in dogs rather speaks against this.
The limitations of this study include extrapolating findings from 2 sampling sites on the ventral abdomen, notwithstanding the 20-dog sample size. The umbilical area was selected as the sample site to comply with ethical approval, as clipping was not required and only gentle manual restraint was envisaged. However, several studies5,6,13,38 have demonstrated that the individual dog factor has a greater influence on the beta diversity of cutaneous microbiota than the body site, and to the authors’ knowledge, this finding still stands. Diet, and the presence of cohabiting members in the household, have been shown to affect the cutaneous microbiome.39,40 Although these studies did not assess the mycobiome, there is no reason to think that they might not be pertinent. We have alluded above to the requirement for temporal studies, which might allow more confidence in stating the status of an organism as resident, transient, or contaminant.41 A 1-time study precludes this deduction.
We took steps to minimize DNA contamination at the time of sampling by minimizing transit time in the waiting room and using disinfection products on the consulting-room table anticipated to denature any residual DNA.42 Cusco et al38 provided the only 1 of the recent papers for a study, which used 16S rRNA new technology tools to investigate the canine biome, that performed an environmental control. However, we could not eliminate contamination from the home environment.
Another limitation of this study might be the use of flocked swabs to sample the mycobiome. Although these have been shown to be superior to simple cotton for the recovery of bacterial samples,43 there appears no evidence that they are superior, or inferior, for sampling the mycobiome. There was no statistical difference between A samples and B samples, suggesting that in healthy dogs there is no reason to scrape the skin before sampling for members of canine cutaneous mycobiome. This should simplify sample collection in future studies.
In summary, we have demonstrated that the cutaneous mycobiota on the abdomen of normal dogs has a median density of 1.1 X 105 cm−2. Cladosporium spp and Vishniacozyma victoriae could be considered part of the core mycobiome on the skin of healthy dogs although their status is, as yet, unknown. Prior skin scraping is not necessary for sampling the canine cutaneous mycobiome.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
Statistical Consultancy Ltd for performing statistical analysis.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Linnaeus Veterinary Limited supported the costs of Open Access Publication Charges. MiDOG provided swabs and paid for transportation of said swabs and the 16S rRNA analysis and the subsequent data analysis.
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