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Veterinary Dermatology 2002, 13, 237 241

Comparison of two sampling techniques to assess quantity and

distribution of Malassezia yeasts on the skin of Basset Hounds

t4Blackwell Science, Ltd

E. BENSIGNOR,* F. JANKOWSKI, W. SEEWALD, F. TOUATI, M. DEVILLE and

J. GUILLOT
*Dermatology Referral Service, Veterinary Clinic, Paris, France Novartis Animal Health, Rueil Malmaison,
France Novartis Animal Health, Basel, Switzerland Unit de Parasitologie-Mycologie, Ecole Nationale
Vtrinaire dAlfort, Maisons-Alfort, France
(Received 4 October 2001; accepted 15 July 2002)

Abstract Cytological examination using the tape-strip technique and fungal culture using contact plates with
modified Dixons medium were compared to evaluate the carriage of Malassezia yeasts on four cutaneous sites
(left pinna, umbilical region, axilla and perianal area) in adult Basset Hounds. Twenty animals were included in
the study. High numbers of Malassezia were isolated from at least one area in 100% of the animals. The frequencies
of isolation and population sizes differed significantly according to anatomical location. They were greater on
the pinna, followed by the umbilical area, axilla and perianal area. Fungal culture was more sensitive than cytology for the isolation of Malassezia yeasts. Frequencies of isolation were greater using this method, but population
sizes were constantly smaller than with cytology.
Keywords: Basset Hounds, contact-plates, cytology, Malassezia, modified Dixons medium

INTRODUCTION
The genus Malassezia comprises lipophilic yeasts that
belong to the normal cutaneous microflora of most
warm-blooded vertebrates.1 These yeasts are now considered to be emerging and opportunistic pathogens
that may be responsible for various cutaneous diseases
in humans2,3 as well as in domestic and wild animals.4,5
In dogs, Malassezia yeasts are frequently associated
with ceruminous otitis externa and pruritic seborrhoeic skin disorders.6,7 The pathogenesis of Malassezia dermatitis in dogs remains unclear. However, as
Malassezia population densities in affected cutaneous
areas generally exceed those of healthy skin, proliferation of the yeasts seems to be a preliminary step to
Malassezia dermatitis.8,9 In recent years, various
studies have been conducted to assess Malassezia
populations from canine skin.810 Malassezia yeasts
have been isolated commonly from the anal sacs,
rectum, groin and ear canals of healthy dogs.810 It has
been shown that some breeds (e.g. Basset Hounds)
carry large amounts of Malassezia yeasts on their
skin, without any associated clinical signs.9 Quantifying
methods currently include cytological examinations
and fungal cultures. Cytological techniques include
impression methods using glass slides,10,11 cotton

Correspondence: Emmanuel Bensignor, Dermatology Referral

Service, Veterinary Clinic,17 bvd des Filles du Calvaire, 75003 Paris,
France. E-mail: emmanuel.bensignor@wanadoo.fr
Conflict of interest: this study was sponsored by Novartis Animal
Health.
2002 Blackwell Science Ltd

swabs, skin scrapings7,10 and tape-strip preparations.10,12 Mycological cultures may be obtained from
cotton swabs, squares of fitted carpets13 or directly
with contact plates.14 It has been shown that some of
these techniques may be less sensitive than others.1113
The aim of this study was to confirm the high
prevalence of Malassezia yeasts on Basset Hound
skin in France and to compare two sampling techniques to assess quantity and distribution of the yeasts:
fungal culture using contact plates with modified
Dixons medium and cytology using the tape-strip
technique.

MATERIALS AND METHODS

Dogs
Twenty Basset Hounds, aged between 1 and 9 years
(mean 4.7 years), were studied. Four were male and 16
female. The animals were all privately owned, living
in the same kennel in south-west France. All the dogs
were in good general health and did not present any
inflammatory skin disease, except a mild greasy seborrhoea, without any sign of skin inflammation, in 16
animals. No pruritus was noted and no history of skin
disease was reported.
The dogs had not received any medication nor
had they been bathed for 14 days prior to sample
collection.
Site preparation
For each animal, samples were collected from four
different anatomic areas: medial aspect of left pinna,
237

VDE_308.fm Page 238 Friday, September 20, 2002 1:08 PM

238

E. Bensignor et al.

umbilical region, axilla and perianal area. At each site,

hair was clipped using a N40 clipper blade over an
area of 100 cm2. Samples were obtained by the two
different techniques on adjacent areas of exposed skin.
Cytological examination
Population sizes and frequencies of detection of
Malassezia yeasts were determined by cytology using
the tape-strip technique. This technique has previously
been shown to be reproducible and more effective
than other cytological methods for sampling yeasts in
case of Malassezia dermatitis.12 Briefly, a piece of tape
(Scotch), was firmly applied to the skin and gently
removed (stripping). This was repeated three times to
ensure that a sufficient quantity of stratum corneum,
debris and material was sampled. Each sample was
stained using Diff Quik and placed on a glass slide
for microscopical examination. Ten random fields,
each of an area of 3.14 10 4 cm2, were examined
using the oil immersion objective (1000). Yeasts
were recognized by their typical microscopic morphology, as reported previously.14 The total number of
yeast organisms found in 10 random oil-immersion
fields was counted. If more than 100 yeasts were
found, the investigator did not count further (running
total of all fields counted). Counts per cm2 were
obtained by dividing the number of yeasts observed
by 3.14 103.
Mycological cultures
Population sizes and frequencies of isolation of Malassezia yeasts were determined using Rodac contact
plates of 5 cm diameter.15 The plates were filled with
18 mL of modified Dixons medium (3.6% malt extract,
0.6% peptone, 2.0% dessicated ox-bile, 1.0% Tween
40, 0.2% glycerol, 0.2% oleic acid, 1.2% agar, 0.5%
chloramphenicol, 0.5% cycloheximide, pH 6.0). The
medium was poured such as the meniscus protruded
above the edges of the lid. Plates were stored at 4 C
until used. The contact plates were applied and gently
pressed on each anatomical area for 10 s. They were
then incubated at 32 C for four days before counting.
Malassezia yeasts or any other fungal species were
identified by microscopic examination of a wet mount
from colonies, using lactophenol cotton blue stain. The
numbers of Malassezia colonies were counted until
100 by an investigator who was not aware of the site of
sampling. If more than 100 colonies were present on the
plate, the investigator did not count further. Counts
per cm2 were obtained by dividing the number of colonies observed after four days by the surface area of the
plate (19.6 cm2).

RESULTS
Cytological examination
Of 80 slides available for examination, only 76 were
readable. Four slides could not be read because the tapes
were damaged or poorly stained. Yeasts were detected
from at least one site in all 20 dogs (100%), but counts
were extremely variable according to the sampling site.
Yeast numbers were generally high on the inner surface
of the pinna, except for five dogs. For these animals,
yeast counts were also low on the other sites sampled.
Eleven samples were negative (five from the perianal
area, four from the axilla, two from the umbilical area).
The mean number of yeasts by site is represented in
Table 1. Yeast counts were higher on the pinna, followed
by umbilical area, axilla and perianal area (Table 2).
Mycological cultures
Yeasts were isolated from at least one site for all 20
dogs. All sites were positive, except in one case on the
umbilical area and in one case on the perianal region.
The mean number of yeasts by site is represented
in Table 1. Yeast counts were higher on the pinna,
followed by umbilical area, axilla and perianal area
(Table 2).
Comparison of the two sampling techniques
Malassezia yeasts were recovered from at least one site
for every dog of the study. Yeasts were identified statistically less frequently by cytological examination (65/
76 slides, 85.5%) than by fungal culture (78/80 plates,
97.5% or 74/76 contact plates, 97.4% if the four plates
corresponding to the four unreadable cytology samples
are omitted) (P = 0.0082 or 0.0172, respectively, for
Fishers exact test). Two anatomical sites presented a
positive cytological examination but a negative culture
(umbilical area of dog 7, and perianal area of dog 20).
In contrast, for 11 sites, the cytological examination
was negative, whereas fungal culture was positive, with
an average of 64.8 colonies (2100 colonies) (data not
shown). The correlation coefficient for cytological
results vs. culture results was 0.56 (P = 0.05).
When fungal culture was used, total colony counts
were constantly equal to or greater than the total
number of cells observed by cytological examination,
except for the two sites where fungal culture was negative (data not shown). However, if we consider the total
number of yeasts per cm2, the mean cell tape-strip
counts were > 100 times higher than the colony counts
by culture (data not shown).

DISCUSSION
Statistical analyses
A Fishers exact test was performed for the number
of positive results with each method. The correlation coefficient between the two methods was calculated after log transformation. The software program
used for calculations was , version 8.1 (Cary NC,
USA).
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 237 241

Even if yeast populations vary markedly between

different anatomical sites, Basset Hound skin proved
to be extensively colonized by Malassezia yeasts in
this study. These results are in accordance with original
observations from Basset Hounds in the UK.9 In the
present study, some dogs were mildly seborrhoeic, whereas

VDE_308.fm Page 239 Friday, September 20, 2002 1:08 PM

Quantity and distribution of Malassezia yeasts

239

Table 1. Yeast counts from 20 dogs using tape-strips and contact plates
Dog

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Age

7
8
7
7
1
4
4
4
4
4
4
3
3
3
3
2
2
2
2
2

Gender

Pinna

M
F
F
F
F
M
M
M
F
F
F
F
F
F
F
F
F
F
F
F
mean
Standard
deviation

Umbilical area

Axilla

Perianal area

TS

CP

TS

CP

TS

CP

TS

CP

2
75
97
> 100
> 100
> 100
> 100
> 100
> 100
50
> 100
60
15
50
> 100
5
15
20
80
40
65.45
37.58

100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
100
0.00

5
7
2
46
46
12
3
15
> 100
2
10
4
3
0
1
0
3
1
ND
11
14.26
24.81

10
> 100
77
> 100
> 100
> 100
0
60
> 100
23
> 100
> 100
100
> 100
33
85
> 100
> 100
> 100
> 100
79.40
34.32

0
10
0
7
7
80
2
2
ND
0
10
2
7
12
2
0
3
2
6
7
8.37
17.76

8
64
40
> 100
> 100
> 100
> 100
100
> 100
70
> 100
> 100
> 100
> 100
36
26
48
23
100
35
72.50
33.69

0
3
4
1
1
15
2
1
ND
2
10
0
0
ND
1
0
3
0
9
2
3.00
4.16

2
17
24
100
100
40
40
25
92
100
40
40
52
60
46
40
57
50
> 100
0
51.25
32.11

TS, mean number of yeasts derived from tape-strips; CP, mean number of colonies derived from contact plates; M, male; F, female; ND, not done.

Table 2. Frequency of detection and isolation, respectively, of

Malassezia yeasts on the cytological examination and mycological
culture of the skin of healthy Basset Hounds

Total
Pinna
Umbilical area
Axilla
Perianal area

Cytological
examination
(n = 76)

Mycological
culture
(n = 80)

65 (85.5)
20 (100)
17 (89.4)*
15 (78.9)*
13 (72.2)

78 (97.5)
20 (100)
19 (95)
20 (100)
19 (95)

*One cytology slide was not analysed. Two cytology slides were not
analysed.

others were not. These animals were living in a

kennel and it is not therefore surprising that many
of them presented a mild seborrhoea. As these few
scales were not associated with any inflammatory
lesions (no erythema, papules, wheals) or pruritus, it
is very unlikely that these animals were suffering from
Malassezia dermatitis, either clinical or subclinical.
Furthermore, in this study, no difference was noted
between normal dogs without scales and normal
dogs with scales regarding the number of yeasts (data
not shown).
These results may explain why dogs of this breed
are predisposed to Malassezia dermatitis.16,17 As suggested by Bond et al. the variability among breeds has
to be considered to establish normal reference range
values for cutaneous Malassezia population densities.9
For example, it has been demonstrated that it is difficult to recover Malassezia yeasts from the axilla and
groin areas of healthy pet dogs.8,10,15 This discrepancy
is of clinical relevance, as the criteria required to

diagnose Malassezia dermatitis in dogs are not precisely

known: the diagnosis is most often linked to the demonstration of elevated numbers of Malassezia on the
skin. Cytology showing > 35 yeasts per high-power
microscope field seems to be a reasonable limit in the
literature.16,1820 This may not always be relevant, as
the threshold population density is not determined in
dogs and probably varies depending on the breed, as
demonstrated here, but also on the virulence of the
strain, the area sampled and individual variations.
Even if the detection of Malassezia yeasts was possible on at least one site for every dog of this present
study, large differences in the intensity of the colonization were observed according to the anatomical area
sampled. The pinnae presented higher frequencies of
isolation and higher yeast counts than the abdomen,
axillae and perianal area. It is noteworthy that the perianal area was consistently less colonized than other
areas. In the study by Bond et al. the anal mucosa was
less colonized than other sites, both regarding frequencies of isolation and population sizes.9 It has been
postulated that anal mucosa could act as a reservoir
for yeasts in dogs, Malassezia yeasts being passively transferred to the skin by licking, as already demonstrated
for Staphylococcus intermedius.8
The factors enabling the development of abnormally
high cutaneous Malassezia populations in dogs remain
to be understood. As relatively low numbers of yeasts
were recovered from the axillary area, a site commonly
affected by Malassezia dermatitis, and because very
high numbers were isolated from normal pinnae without clinical signs, we can assume that factors other
than the absolute number of organisms are important
in the pathogenesis of yeast infection.10
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240

E. Bensignor et al.

The two sampling techniques studied here had

very different abilities to detect the yeasts. Cytology
appeared to be much less sensitive than fungal culture
considering frequencies of isolation of yeasts, contrary
to previous data from Bond et al.,15 but similarly to
another study by the same author.21 In our study, the
correlation coefficient for the two methods was only
0.54. We therefore consider that fungal culture should
be preferred in future studies aiming to quantitatively
assess yeast populations and/or the antifungal activity
of drugs. However, for large yeast populations, there
was a good correlation between the results of the
two techniques (i.e. when counts were high by culture,
they were also high by cytology), as demonstrated previously.9 Furthermore, the mean tape-strip counts of
yeast organisms per cm2 were much more elevated than
the mean colony counts established using the culture
method, as reported previously in the literature.10,15,21
The reasons for this difference are not known. Bond
et al. proposed that each colony on contact plates may
represent growth derived from a cluster of cells rather
than from just one yeast, thus underestimating population sizes.15 Another explanation could be that not
all yeasts adhere to the medium, or that the time of
contact of the plates on the skin was insufficient.15 Be
that as it may, our data suggest that cytology remains a
helpful technique for the routine diagnosis and follow
up of the cases of Malassezia dermatitis in dogs.
Nevertheless, for doubtful cases, when cytological examination is negative but a strong clinical suspicion of
Malassezia dermatitis exists, the clinician should
perform a fungal culture before eliminating this hypothesis. The detergent scrub technique may also be very
helpful and has been demonstrated to correlate well
with the results obtained by contact plates,21 but it is
mainly reserved for research purposes.

CONCLUSIONS
Malassezia yeasts were commonly isolated in high
numbers from the skin of healthy Basset Hounds living
in south-west France, by either cytology or fungal culture. These yeasts should definitively be considered as
part of the normal microflora of Basset Hound skin.
Cytology was less sensitive than fungal culture to isolate the yeasts except in cases of heavy colonization. As
fairly high numbers of Malassezia were isolated from
clinically normal dogs, factors other than the absolute
number of organisms probably play a role in the pathogenesis of Malassezia dermatitis.

REFERENCES
1. Midgley, G., Guho, E., Guillot, J. Diseases caused by
Malassezia species. In: Collier, L., ed. Topley and
Wilsons Microbiology and Microbial Infections, Vol. 4.
Medical Mycology, Part IV Superficial Basidiomycetous
Yeasts, 9th edn. London: Arnold, 1998; 20111.
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 237 241

2. Guho, E., Boekhout, T., Ashbee, H.R. et al. The role of

Malassezia species in the ecology of human skin and as
pathogens. Medical Mycology 1998; 36: 2209.
3. Marcon, M.J., Powell, D.A. Human infections due to
Malassezia spp. Clinical Microbiology Reviews 1992; 5:
10119.
4. Guillot, J., Bond, R. Malassezia pachydermatis a review.
Medical Mycology 1999; 37: 295306.
5. Guillot, J., Poujade, A., Boulouha, L. et al. Could Malassezia dermatitis be diagnosed in animals other than pet
carnivores? Veterinary Dermatology 2000; 11 (Suppl. 1):
38.
6. Mansfield, P.D., Boosinger, T.R., Attleberger, M.H.
Infectivity of Malassezia pachydermatis in the external
ear canal of dogs. Journal of the American Animal Hospital Association 1990; 26: 97100.
7. Dufait, R. Pityrosporum canis as the cause of canine
chronic dermatitis. Veterinary Medical Small Animal
Clinic 1983; 78: 10557.
8. Bond, R., Saijonmaa-Koulumies, L.E.M., Lloyd, D.H.
Population sizes and frequency of Malassezia pachydermatis at skin and mucosal sites on healthy dogs. Journal
of Small Animal Practice 1995; 36: 14750.
9. Bond, R., Lloyd, D.H. Skin and mucosal populations
of Malassezia pachydermatis in healthy and seborrheic Basset Hounds. Veterinary Dermatology 1997; 8:
1016.
10. Kennis, R.A., Rosser, E.J., Olivier, N.B. et al. Quantity
and distribution of Malassezia organisms on the skin of
clinically normal dogs. Journal of the American Veterinary Medical Association 1996; 208: 104851.
11. Plant, J.D., Rosenkrantz, W.S., Griffin, C.E. Factors
associated with a prevalence of high Malassezia pachydermatis numbers on dog skin. Journal of the American
Veterinary Medical Association 1992; 201: 87985.
12. Bensignor, E., Carlotti, D.N. Comparaison de diffrentes techniques cytologiques pour la mise en
vidence de Malassezia pachydermatis sur la peau du
chien. Pratique Mdicale et Chirurgicale de lAnimal de
Compagnie 1999; 34: 3341.
13. Guillot, J., Breugnot, C., De Barros, M. et al. Usefulness
of Dixons medium for quantitative culture of Malassezia
sp. from canine skin. Journal of Veterinary Diagnostic
Investigation 1998; 10: 3824.
14. Guillot, J., Guho, E., Lesourd, M. et al. Identification
of Malassezia yeasts: a practical approach. Journal de
Mycologie Mdicale 1996; 6: 10310.
15. Bond, R., Collin, N.S., Lloyd, D.H. Use of contact plates
for the quantitative culture of Malassezia pachydermatis
from canine skin. Journal of Small Animal Practice 1994;
35: 6872.
16. Scott, D.W., Miller, W.H., Griffin, C.E. Fungal skin
diseases. In: Muller and Kirks Small Animal Dermatology, 6th edn. Philadelphia: W.B. Saunders, 2000; 336
422.
17. Bond, R., Ferguson, E.A., Curtis, C.F. et al. Factors
associated with elevated cutaneous Malassezia pachydermatis populations in dogs with pruritic skin diseases.
Journal of Small Animal Practice 1996; 37: 1037.
18. Mason, K.V. Cutaneous Malassezia. In: Griffin, C.E.,
Kwochka, K.W., MacDonald, J.M., eds. Current Veterinary Dermatology, the Science and Art of Therapy. St.
Louis, MO: Mosby Year Book, 1993: 448.
19. Carlotti, D.N., Laffort-Dassot, C. Dermatite Malassezia pachydermatis. tude bibliographique et tude

VDE_308.fm Page 241 Friday, September 20, 2002 1:08 PM

Quantity and distribution of Malassezia yeasts

rtrospective de 12 cas gnraliss traits par des drivs
azols. Pratique Mdicale et Chirurgicale de lAnimal de
Compagnie 1996; 31: 297307.
20. Guagure, E., Prlaud, P. Etude rtrospective de 54 cas
de dermatite Malassezia chez le chien: rsultats
pidmiologiques, cliniques, cytologiques et histo-

241

pathologiques. Pratique Mdicale et Chirurgicale de

lAnimal de Compagnie 1996; 31: 30923.
21. Bond, R., Lloyd, D.H., Plummer, J. Evaluation of a
detergent scrub technique for the quantitation of Malassezia pachydermatis on canine skin. Research in Veterinary Science 1995; 58: 1337.

Rsum Nous avons compar lexamen cytologique par test la cellophane adhesive et la culture fongique avec
les boites de contact contenant du milieu de Dixon pour valuer le portage de levures Malassezia au niveau de
quatre zones (pavillon auriculaire gauche, ombilic, zone axillaire et zone prianale) chez des Basset Hound adultes. Vingt animaux ont t inclus dans cette tude. Des populations leves de Malassezia ont t isoles sur au
moins un site chez 100% des animaux. Les frequencies disolement et les populations taient significativement
diffrentes en fonction de la rgion prleve. Elles taient plus importantes sur le pavillon auriculaire, suivi par
la rgion ombilicale, la rgion axillaire et la rgion prianale. La culture fongique sest avre plus sensible que
la cytologie pour isoler les levures. Les frquences disolement taient plus leves avec cette mthode, mais les
populations taient systmatiquement plus faibles que par la cytologie.
Resumen El examen citolgico utilizando la tcnica de cinta adhesiva y el cultivo fngico utilizando placas de
contacto con medio Dixon modificado fueron comparados para evaluar la carga de levaduras de Malassezia en
cuatro reas cutneas (pabelln auricular izquierdo, regin umbilical, axila y rea perianal) de perros Basset
Hound adultos. Se incluyeron veinte animales en el estudio. Se aisl un elevado nmero de Malassezia en al menos
una zona en el 100% de los animales. Las frecuencias de aislamiento y el tamao de la poblacin diferan significativamente segn la localizacin anatmica. Fueron mayores en el pabelln auricular, seguidos por la regin
umbilical, axila y el rea perianal. El cultivo fngico fue ms sensible que la citologa para aislar las levaduras
Malassezia. Las frecuencias de aislamiento fueron mayores por este mtodo, pero el tamao de la poblacin fue
constantemente menor que con la citologa.
Zusammenfassung Zytologische Untersuchung von Tesaprparaten und Pilzkultur mittels Kontaktplatten mit
modifiziertem Dixons Medium wurden verglichen, um das Vorkommen von Malassezia-Hefen an vier kutanen
Stellen (linke Ohrmuschel, Nabelgegend, Achselhhle und Perianalgegend) bei erwachsenen Basset Hunden zu
bewerten. Die Studie umfasste zwanzig Tiere. Eine hohe Anzahl von Malassezia wurden bei allen Tieren an mindestens einer Stelle festgestellt. Die Isolationsfrequenzen und Populationsgrssen variierten je nach anatomischer
Lage signifikant. Sie waren am grssten an der Ohrmuschel, gefolgt von der Nabelgegend, Achselhhle und Perianalgegend. Pilzkultur war sensitiver als Zytologie in der Isolierung von Malassezia Hefen. Isolationshufigkeiten waren mit dieser Methode grsser, aber Populationsgrssen waren konstant kleiner als bei der Zytologie.

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 237241