Colonisation status of Malassezia pachydermatis on the hair and in the hair follicle of healthy beagle dogs

Research in Veterinary Science 2000, 68, 291–293 doi:10.1053/rvsc.2000.0377, available online at http://www.idealibrary.com on

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Colonisation status of Malassezia pachydermatis on the hair and in the hair follicle of healthy beagle dogs R. BOND*, A. I. LAMPORT AND D. H. LLOYD Dermatology Unit, Department of Small Animal Medicine and Surgery, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK SUMMARY Hair and hair follicle carriage of Malassezia pachydermatis was studied in 12 healthy beagle dogs. The yeast was isolated from hair clipped from the lip region at 13 sites in nine dogs but was less frequently recovered from the interdigital spaces on the forefeet and from two sites on the trunk. Population sizes at the lip were significantly greater (P < 0·01) than those at other sites. Skin biopsy specimens were obtained from the same sites and epidermal and follicular tissues dissected following immersion in 1 M CaBr2. Epidermal carriage of M. pachydermatis was identified in nine biopsy specimens taken from five dogs. Hair follicle carriage was identified in five skin specimens (four foot, one lip) from three dogs. This study indicates that M. pachydermatis is readily recovered from the distal hair in healthy dogs and that hair follicle carriage is infrequent or that populations are low at that site.

THE microflora of healthy mammalian skin exists in the superficial layers of the stratum corneum, in the pilosebaceous units and on the hair. In scanning electron microscopical studies of human and bovine skin, microcolonies of bacteria have been observed in the superficial layers of the stratum corneum with the majority being located at hair follicle ostia (Montes and Wilborn 1969, Lloyd et al 1979, Malcolm and Hughes 1980). Follicular dissection techniques have been applied to investigate further microbial colonisation of skin. The isolation of sebaceous glands and hair follicles from human skin biopsy specimens using dissection techniques can be enhanced by immersion of the specimen in solutions containing anions such as chloride and bromide which split the epidermal–dermal junction at the level of the lamina lucida (Kellum 1966, Puhvel et al 1975, Skerrow and Skerrow 1985, Harvey et al 1993). Harvey et al (1993) modified Puhvel’s technique by using cyanoacrylate adhesive to attach the epidermal side of the biopsy to glass slides to aid the dissection process and showed that the hair follicles of dogs harboured Staphylococcus intermedius, micrococci and Propionibacterium acnes (Harvey and Lloyd 1994, 1995). Although the distal hair shaft would appear to represent a relatively hostile environment for cutaneous microorganisms, bacteria have been readily recovered from this location on dogs in a number of studies (Hearst 1967, White et al 1983, Allaker et al 1992, Harvey and Lloyd 1994, 1995). The yeast Malassezia pachydermatis is a normal inhabitant and opportunistic pathogen of canine skin and mucosae (Gustafson 1955, Dufait, 1983, Hajsig et al 1985, Larsson et

*Corresponding author

0034-5288/00/030291 + 03 $35.00/0

al 1988, Bond et al 1995, Bond and Lloyd 1997). The hair coat of the dog has been shown to harbour M. pachydermatis (Dufait 1985) but investigations comparing carriage of the yeast on hair at different sites on healthy dogs have not been reported. Similarly, follicular carriage of M. pachydermatis does not appear to have been investigated in the dog, although Malassezia spp. have rarely been observed in the follicles in histopathological sections (Mason and Stewart 1993). Preliminary studies were performed in vitro to evaluate the suitability of calcium bromide and selected adhesives for the quantitation of M. pachydermatis in canine skin biopsy specimens (Bond 1996). The cyanoacrylate adhesive used by Harvey et al (Superglue, Locktite) inhibited the growth of three strains of M. pachydermatis when grown on modified Dixon’s agar plates, whereas an epoxy resin (Araldite Rapid Adhesive, Ciba-Geigy Plastics) had no such inhibitory effect (Bond 1996). Aerobic bacteria and fungi were not isolated when freshly extruded epoxy resin was cultured on blood and modified Dixon’s agar (Bond 1996). The viability of six M. pachydermatis strains was not adversely affected by suspension in 1 M calcium bromide plus epoxy resin over a 2-hour period (Bond 1996). Although it is possible that the effects of CaBr2 and epoxy resin on the yeast may be different in the presence of canine skin, the results of these in-vitro studies suggested that those substances could be used in skin dissection techniques designed to isolate M. pachydermatis. The purpose of the present study was to quantitate carriage of M. pachydermatis on the hair and in the hair follicles at a range of anatomical sites in healthy beagle dogs. Twelve healthy male beagles aged 2 years were sampled 15 minutes after euthanasia; these dogs had been humanely destroyed by the intravenous injection of sodium pentobarbitone for reasons not connected with this study. Hair samples were obtained by clipping the distal one-half of the hair from areas on left and right sides of the body at four sites, © 2000 Harcourt Publishers Ltd

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R. Bond, A. I. Lamport, D. H. Lloyd

each of 3 cm2 area, using scissors which were flamed in alcohol before use at each site. The sites sampled were the haired skin of the lower lip, 3 cm rostral to the commissures; the dorsal interdigital skin between digits III and IV on both fore feet; the haired skin of the axillae just caudal to the elbow; and the dorsal skin at the level of the thoracolumbar junction, approximately 6 cm from the midline. Hair samples were placed in sterile plastic bijou bottles which had been previously weighed. The bijou bottles were then re-weighed and 2-ml aliquots of wash fluid were added to each bijou bottle and shaken manually for 30 seconds. Estimates of the numbers of viable M. pachydermatis and bacteria were obtained by spread plating 25-µl aliquots onto paired quadrants of agar plates within 40 minutes of collection of the hair. M. pachydermatis was cultured aerobically on modified Dixon’s agar (Bond et al 1994) at 32°C for 3 days. Skin specimens were obtained from the same sites, aseptically, using a 6-mm biopsy punch (Stiefel Laboratories), which was flamed in alcohol between sites. The hair at each site was removed using scissors. Each skin specimen was placed, epidermal side down, on a blob of freshly prepared epoxy resin (Araldite Rapid, Ciba-Geigy Plastics) mounted on a glass coverslip which had been sterilised in alcohol, using aseptic technique. The skin specimens were held in sterile petri dishes until the resin had hardened (5 minutes), and the coverslip and attached specimen were immersed in sterile 1 M CaBr2. After 2 hours incubation at room temperature, each skin specimen was dissected using scissors and a dissecting microscope. The subcutis was removed, and the dermal tissues containing the hair follicles were dissected free, leaving a circular disc of tissue consisting of the epidermis and small remaining fragments of superficial dermis. Each follicular and epidermal tissue specimen was ground in a sterile glass tissue grinder (Jencons) containing 1 ml of wash fluid for 60 seconds. Aliquots were immediately spread-plated as described above. Counts of bacteria were also determined by incubating aliquots aerobically on blood agar at 37°C for 2 days in order to ensure that viable bacteria could be recovered from the tissue specimens and that the follicular microflora could be harvested from the follicular infundibula. Colony counts were expressed as log10(cfu.specimen–1 + 1). M. pachydermatis and bacterial population sizes at the four sites sampled were compared using the Kruskal Wallis one way analysis of variance; subanalysis was performed using the Mann-Whitney U test with the P value corrected using the method of Bonferroni (Altman 1991). M. pachydermatis was isolated from 11 out of the 12 beagles studied. The yeast was isolated from the hair of the lip region at 13 sites in nine dogs but was less frequently recovered from the other sites (Table 1). Population sizes of M. pachydermatis at the lip were significantly greater (P < 0·01) than those at the other sites; hair populations at the other sites were not significantly different (Table 2). Despite incubation in the CaBr2 solution, considerable sharp dissection was required to separate the follicular and epidermal tissues in the skin specimens. Hair follicle carriage of M. pachydermatis was identified in five skin specimens from three dogs; the yeast was isolated from hair follicles from the fore foot in four cases and from the lip region in one case (Table 1). At four of these sites,

TABLE 1: Frequency of isolation (%) of M. pachydermatis from distal hair samples and epidermal and follicular portions of skin biopsy specimens from 12 healthy beagles Hair Site

Lip Fore foot Axilla Dorsum

Epidermis

Follicles

Dogs (n = 12)

Sites (n = 24)

Dogs

Sites

Dogs

Sites

9 (75) 4 (33) 1 (8) 4 (33)

13 (54) 4 (17) 1 (4) 4 (17)

3 (25) 3 (25) 0 (0) 1 (8)

4 (17) 4 (17) 0 (0) 1 (4)

1 (8) 3 (25) 0 (0) 0 (0)

1 (4) 4 (17) 0 (0) 0 (0)

M. pachydermatis was also isolated from the epidermal specimen and the overlying distal hair; epidermal and follicular carriage without hair carriage was identified at the remaining site. Epidermal carriage of M. pachydermatis was identified in nine biopsy specimens taken from five dogs. In three dogs, M. pachydermatis was isolated from the overlying hair, and the yeast was isolated from the epidermal sample only at one site. In the remaining dog, the yeast was isolated from the epidermal and follicular specimens but not from the hair at the same site. M. pachydermatis was isolated from the hair but not the epidermal or follicular specimens at 14 sites. M. pachydermatis populations at the four sites studied did not vary significantly (P > 0·05) in both the epidermal and follicular portions of the skin specimens (Table 2). Coagulase-negative staphylococci were frequently isolated in moderate numbers from both epidermal and follicular tissues whereas CPS were less often recovered (Tables 3 and 4). Coagulase-positive staphylococci were not isolated from either the epidermal or follicular parts of the dorsal skin biopsies. It is clear that M. pachydermatis can be recovered from the canine hair coat, although there are regional differences in its distribution. Its frequent isolation from hair samples from the lip accords with the results obtained when this site was sampled using contact plates in a group of 40 healthy dogs (Bond et al 1995). It is possible that the high M. pachydermatis counts at this site reflect contamination with saliva from the oral cavity, although relatively low rates of oral colonisation have been reported previously (Bond et al 1995). An alternative hypothesis is that saliva, drinking water, and a degree of occlusion from the upper lip influences the microclimate in favour of the yeast. Kellum (1966) was able to dissect entire pilosebaceous units from human skin following the immersion of biopsy specimens in CaCl2. However, follicular tissues could not be readily separated from the epidermis in the canine skin specimens used in the present study. This was probably due to TABLE 2: Population sizes of M. pachydermatis from hair (log10[cfu·g–1 + ]) and epidermal and follicular portions of skin biopsy specimens (log10[cfu·specimen–1 + 1]) from 12 healthy beagles Hair Site Lip Fore foot Axilla Dorsum

Epidermis

Follicles

Median

Range

Median

Range

Median

Range

3·63* 0 0 0

0–5·83 0–5·18 0–3·35 0–3·26

0 0 0 0

0–2·51 0–3·05 0–0 0–1·32

0 0 0 0

0–1·32 0–2·90 0–0 0–0

Comparison with hair populations at all other sites, *P < 0·01

Carriage of M. pachydermatis in dogs TABLE 3: Frequency of isolation, per cent, of staphylococci from epidermal and follicular portions of skin specimens from 12 healthy beagles Epidermis Site Lip Fore foot Axilla Dorsum

Follicles

CPS

CNS

CPS

CNS

58 (46) 25 (17) 8 (4) 0 (0)

75 (54) 75 (58) 75 (54) 67 (54)

33 (25) 25 (12) 8 (4) 0 (0)

83 (67) 92 (83) 58 (33) 58 (38)

Figures in parentheses indicate frequency of isolation from each site (n = 24) CNS, coagulase-negative staphylococci; CPS, coagulase-positive staphylococci

TABLE 4: Population sizes of staphylococci from epidermal and follicular portions of skin specimens (median log10[cfu.specimen–1 + 1], range) from 12 healthy beagles Epidermis Site Lip Fore foot Axilla Dorsum CNS,

Follicles

CPS

CNS

0 (0–3·7) 0 (0–4·5) 0 (0–1·3) 0 (0)

1·3 (0–3·8) 1·3 (0–3·2) 1·3 (0–2·8) 1·3 (0–2·9)

CPS

0 (0–3·2) 0 (0–2·5) 0 (0–1·3) 0 (0)

CNS

1·8 (0–3·8) 2·0 (0–2·4) 0 (0–1·8) 0 (0–2·1)

coagulase-negative staphylococci; CPS, coagulase-positive staphylococci

the compound nature of the canine hair follicle where the numerous interdigitating secondary follicles prevented the clean removal of the surrounding dermal tissues. However, the recovery of substantial populations of viable bacteria, particularly CNS, from the follicular portion of the specimens indicates that the technique did enable the quantitation of the microbial flora in this niche. Assuming that the validation studies described are a reasonable model for the situation in vivo, then the rare isolation of M. pachydermatis from the follicular tissues indicates that this site was either infrequently colonised or that the yeast was present in low numbers. These results are in accordance with histopathological studies of dogs with dermatitis and elevated cutaneous M. pachydermatis populations. Yeast cells are often observed in the epidermal stratum corneum in such cases but are rarely observed in the follicles (Mason and Evans 1991, Mason and Stewart 1993), and it seems likely that follicular populations would be more frequently observed in such cases if the deeper parts of the pilosebaceous units were often colonised. ACKNOWLEDGEMENTS The authors thank Dr K Simpson for technical assistance.

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Accepted January 19, 2000