Veterinary Dermatology 2003, 14, 305 312

A recombinant 31.5 kDa keratinase and a crude exo-antigen from

Microsporum canis fail to protect against a homologous
experimental infection in guinea pigs

Blackwell Publishing Ltd.

FRDRIC F. DESCAMPS,* FRDRIC BROUTA, SANDY M. VER MOUT,* CORINNE

WILLAME,* BERTRAND J. LOSSON* and BERNARD R. MIGNON*
*Department of Infectious & Parasitic Diseases, Faculty of Veterinary Medicine, University of Lige, B-43 Sart
Tilman, 4000 Lige, Belgium
UCB, Braine-lAlleud, Belgium
(Received 10 March 2003; Accepted 21 March 2003)

Abstract A Microsporum canis recombinant 31.5 kDa keratinase and a M. canis crude exo-antigen were tested
as vaccines in an experimental infection model in guinea pigs. Animals were vaccinated subcutaneously three
times at two-week intervals with either the keratinase, the exo-antigen or the adjuvant alone. Cutaneous challenge
was performed blindly. Both humoral and cellular-specific immune responses to M. canis antigens were evaluated
every 14 days, while a blind evaluation of clinical lesion development and fungal persistency in skin were monitored weekly. Vaccination induced very high and significant (P < 0.01) antibody responses towards both antigens.
High cell-mediated immune responses to both immunogens were also induced by vaccination. After challenge,
however, scores reflecting the severity of dermatophytic lesions did not differ significantly between vaccinated and
control groups at any time after challenge. These results suggest that, in the guinea pig, the induction of specific
immune responses against the M. canis-secreted antigens used in this study are not protective against challenge
exposure.
Keywords: dermatophyte, keratinase, Microsporum canis, subtilisin-like serine protease, vaccine.

INTRODUCTION
Microsporum canis is the main agent of dermatophytosis in dogs and cats.1 Control of this fungal dermatosis
is problematic for several reasons.2 First, both local and
systemic antifungal treatments are generally recommended, although very few appropriate drugs are licensed
for use in cats.3 Recommended topical therapies in the
cat are lime sulfur and enilconazole, while systemic
drugs comprise griseofulvin and itraconazole.35 Recent
studies suggest that lufenuron, a chitin synthase inhibitor used in the prevention and control of flea infestations, may be useful in the treatment and control of feline
dermatophytosis.6,7 However, further double-blinded
studies are clearly needed in order to assess the real
benefit of this highly safe drug. Among the recommended products, only lime sulfur and griseofulvin are
generally approved for use in cats, but they are not available everywhere, particularly in some European countries. Griseofulvin can be toxic and teratogenic.5 Control
of feline dermatophytosis often requires a long-lasting
treatment whose application, topical at least, may reveal
difficult to perform. Haircoat shearing is recommended,
at least when large lesions are observed.3 Finally, fungal
Correspondence: Bernard Mignon, Department of Infectious &
Parasitic Diseases, Faculty of Veterinary Medicine, University of
Lige, B-43 Sart Tilman, 4000 Lige, Belgium. E-mail:
bmignon@ulg.ac.be
2003 European Society of Veterinary Dermatology

spores may persist for up to 18 months in the environment and a thorough disinfection of the premises is
required.8 Consequently, feline dermatophytosis is particularly difficult, time-consuming and expensive to
treat. Also, the cat is considered to be the main source
of M. canis infections in man, which are on the increase
in some European countries.1,9 Cats can be asymptomatically infected and this enhances the risk for human
infection as such animals discreetly shed large amounts
of infective material in to the environment.10,11 In this
context, the development of a specific feline immunoprophylaxis would be of great benefit.
Experimental inactivated vaccines in cats have poor
efficacy, and attenuated ones may still produce active
lesions at the injection site.1214 The prophylactic efficacy of commercial products licensed for cats has not
been scientifically reported. Under these conditions, the
use of a safe subunit vaccine would be advantageous.
The 31.5 kDa keratinolytic subtilisin-like serine protease (SUB3), a M. canis potential virulence factor,
may be a useful vaccine candidate.15,16 Indeed, it is one
of the major in vitro secreted proteins of this fungus
and its expression has been demonstrated in vivo in naturally infected cats and experimentally infected guinea
pigs.1517 Most important, SUB3 was shown to induce
a cell-mediated immune response (CMI) in infected
guinea pigs.17 CMI is believed to play a major role in
both the elimination of the dermatophyte and in protection against re-infection.18,19 Recently, SUB3 was
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F. F. Descamps et al.

produced as a recombinant protease (r-SUB3) in Pichia

pastoris and was also shown to induce CMI in infected
guinea pigs.20 Another promising vaccine candidate is
a M. canis crude exo-antigen. This antigen is obtained
from supernatant of M. canis grown in a cat keratincontaining medium and is composed of at least 15 proteins, including SUB3.15 This antigen has been shown
to induce a very intense CMI in experimentally infected
guinea pigs.17
The purpose of this study was to assess the protective
efficacy of both r-SUB3 and M. canis exo-antigen used
as immunogens in a guinea pig experimental infection
model. This is the first report of an attempt at vaccination against a dermatophytic infection using a fully
characterized antigen.

MATERIALS AND METHODS

Animals
Twenty-one dermatophyte-free, 3-month-old, female
guinea pigs (Hartley strain, B&K Universal Ltd,
Humberside, UK) were used. During the entire study,
control and vaccinated animals were housed in separate cages, each containing a maximum of two guinea
pigs. Animal experiments were approved by the local
ethics committee (University of Lige).
Fungal strain and growth conditions
Microsporum canis strain IHEM 15221 (Brussels, Belgium),
previously isolated from a Woods lamp examinationpositive dermatophytic cat, was used to produce crude
exo-antigen and for the experimental infection.21
To isolate the exo-antigen, the fungus was grown for
12 days at 27 C in a liquid minimal medium containing cat keratin as described previously, whereas the
experimental infection was performed with infective
material grown for 10 days on Sabouraud agar.15
Vaccine preparation
The recombinant 31.5 kDa keratinolytic subtilisinlike serine protease was purified by cation-exchange
chromatography from the supernatant of a transformed
P. pastoris colony.20
Crude exo-antigen was obtained from M. canis culture supernatant by ultrafiltration on Amicon YM 10,
as described by Mignon et al.22
Both r-SUB3 and exo-antigen were dialysed against
0.01 phosphate-buffered saline (PBS, pH 7.4), diluted
in 400 L PBS and added to 200 L of Freunds complete or incomplete adjuvant for the first and the two
subsequent immunizations, respectively.
The protein concentrations of the antigens were
determined using the method described by Bradford.23
Experimental vaccination, challenge infection and blood
sampling
Guinea pigs were divided into four groups. Group 1
contained six animals vaccinated with r-SUB3. Group 2
contained six animals vaccinated with exo-antigen.

Group 3 contained six control guinea pigs injected with

the adjuvant diluted in PBS, and group 4 contained three
nonvaccinated, noninfected animals used as scarification lesion controls (see below). Subcutaneous vaccination was performed at 14-day intervals using three
injection sites each time. Animals from groups 1, 2 and
3 were injected three times (on study day 70, 56 and
42) with either 50 g of r-SUB3, 100 g of exo-antigen
or adjuvant alone, respectively. The allocation of experiment animals was randomized.
Six weeks after the last vaccination (study day 0), guinea
pigs from groups 13 were infected with M. canis, essentially as described by Van Cutsem.24 Briefly, the inoculum
comprising mycelia and spores collected from cultures
on Sabouraud agar slopes was suspended in a honey/
water (1:2 v/v) mixture and applied on a 15-cm2 -back
skin surface that had been previously clipped and scarified. Each guinea pig was inoculated with 2 105 cfu.
Negative controls from group 4 were exposed to the same
procedure except that the inoculum did not contain
any fungus. On days 70 and 56 and from day 0 to day
56 post infection (PI), blood samples were collected at
14-day intervals by intracardiac puncture performed
under general anaesthesia. These samples were used to
assess the cellular and antibody immune response raised
against r-SUB3 and the exo-antigen.
Clinical and mycological follow-up
Animal infection was monitored weekly by a single examiner using clinical and mycological criteria, essentially
as described previously.20 This investigator was not
aware of the guinea pig status (infected or not). A global score was calculated for each guinea pig by adding
both clinical and mycological scores. A mean global score
was then calculated for each group of animals.
Enzyme-linked immunosorbent assay
Serum samples were stored at 20 C until use and all
assays were performed in one batch at the end of the
study period. The enzyme-linked immunosorbent assay
(ELISA) was essentially the same for exo-antigen and
r-SUB3. Antigens, reference antisera and rabbit antiguineapig immunoglobulin (Ig; Dako, Glostrup, Denmark)
were appropriately diluted after standard checkerboard
titration (data available upon request). All washes were
repeated four times. Polystyrene microtitre plates (Maxisorp F16, Nunc, Roskilde, Denmark) were coated for
1 h at 37 C with 100 L per well of either 20 g mL1
r-SUB3 or 20 g mL1 exo-antigen solution in PBS. Odd
rows were sensitized with the antigen, whereas even rows
remained free of antigen as control wells. After washing with PBS, plates were saturated for 1 h at 37 C
with saturation buffer (PBS containing 1% w/v bovine
serum albumin; BSA, Sigma, Munich, Germany) and
then washed with PBS containing 0.05% v/v Tween 20
(PBS-T). Serum samples were diluted 1:100 in dilution
buffer (saturation buffer containing 0.05% v/v Tween 20).
One hundred microlitre serum samples were added for
1 h at 37 C to two antigen-coated and two control
wells. After washing with PBS-T, 100 L of horseradish

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Experimental vaccine to Microsporum canis

peroxidase-conjugated rabbit antiguinea-pig Igs diluted
1:20 000 in dilution buffer were added for 1 h at 37 C.
The plates were washed with PBS-T and peroxidase
activity was then revealed by the addition of 100 L of
a solution containing tetramethylbenzidin and hydrogen
peroxide (Coris BioConcept, Namur, Belgium). The reaction was stopped after 7 min at room temperature with
50 L of 1 sulfuric acid, and the absorbance at 450 nm
was measured immediately using a spectrophotometer
(Multiskan RC, Thermolabsystems, Helsinki, Finland).
On each plate, positive and negative reference antisera
were also included in duplicate. The positive reference
antiserum was collected by Mignon from a guinea pig
immunized with the crude exo-antigen.17 The negative
reference antiserum was a pool of the sera obtained on
day 56 PI from the guinea pigs in group 4.
For each serum tested, the optical density (OD) was
defined as the mean difference in absorbance between
the antigen-sensitized wells and the controls. Results
were expressed as OD percentages obtained as follows:
OD percentage = (ODx ODn)/(ODp ODn) where
ODx is the OD obtained with the tested serum, ODp
and ODn are the OD obtained on the same plate
with the positive and the negative reference antisera,
respectively.
Lymphocyte blastogenesis test
CMI to r-SUB3 and exo-antigen were evaluated in vitro
using the lymphocyte blastogenesis test (LBT). For this
experiment, animals from groups 1, 2 and 3 were randomly divided into two subgroups of three guinea pigs
each. Two millilitres of heparinized blood were sampled from each guinea pig and diluted 1:2 in PBS. Samples from guinea pigs belonging to the same subgroup
were then mixed, pipetted over 9 mL of Ficoll-Paque
solution (Amersham, Uppsala, Sweden) in 50-mL conical tubes and centrifuged for 30 min at 400 g. The
peripheral blood mononuclear cell (PBMC) rings were
harvested and washed twice in PBS for 10 min at 400 g.
PBMC were then diluted at a final concentration of
1 106 mL1 in RPMI-1640 medium (Bio Media, Boussens, Belgium) containing 2% horse serum (Gibco Brl,
Paisley, UK), 1% nonessential amino acids, 1% sodium
pyruvate, 10 IU mL1 penicillin, 0.1 mg mL1 streptomycin and 50 2-mercaptoethanol. Aliquots of the
cell suspension (250 L) were incubated in 96-well sterile
tissue-culture plates (Corning, New York, USA) containing in quadruplicates either PBS (negative control
wells), 1 g concanavalin A (positive control wells),
500 ng heat-inactivated r-SUB3 or exo-antigen (test
wells). Plates were cultured for 3 days at 37 C in an
atmosphere containing 5% CO2 and pulsed with 1 Ci
of [3H] thymidine (Amersham) for 24 h. Cells were
harvested onto glass fibre filters (Skatron, Sterling, VA,
USA) using a Skatron cell harvester. The dried filter
discs were transferred to vials containing 4 mL of scintillation fluid (Ecoscint A, National Diagnostics, Hessle
Hull, UK), and incorporation of [3H] thymidine was
quantified for 3 min with a Beckman LS 5000 CE
scintillation counter (Beckman Instruments, Fullerton,

307

CA, USA). The results of the LBTs were expressed as

a stimulation index (SI), calculated as the ratio of the
mean count per minute (cpm) in the test wells divided
by the mean cpm in the negative control wells.
Statistics
The MannWhitney U-test was used for the statistical
comparison of both mean global scores and ELISA
results between the vaccinated and the nonvaccinated
groups. This test was performed using the software . A P-value < 0.01 was considered significant.
The necessity of pooling the PBMC samples did not
allow statistical tests to be performed on LBT results.

RESULTS
Antibody responses
Vaccinated animals from groups 1 and 2 developed
very high and significant antibody responses towards
both r-SUB3 and exo-antigen (Figs 1 and 2). Maximal

Figure 1. Kinetics of the median antibody response (OD percentage)

to r-SUB3 tested in ELISA in vaccinated and control guinea pigs
after vaccination and subsequent M. canis challenge exposure. Vertical
bars = values ranging from 25th to 75th percentile. *Statistically
different (P < 0.01) from values of control guinea pigs.

Figure 2. Kinetics of the median antibody response (OD percentage)

to the exo-antigen tested in ELISA in vaccinated and control guinea
pigs after vaccination and subsequent M. canis challenge exposure.
Vertical bars = values ranging from 25th to 75th percentile. *Statistically different (P < 0.01) from values of control guinea pigs.

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308

F. F. Descamps et al.

responses were observed on day 0, i.e. 6 weeks after the

last immunization. The antibody titres remained high
until the end of the experiment and were significantly
higher than those observed in the group of control
nonvaccinated animals (group 3) during all the infection period except for exo-antigen antibody titres in
group 2 on day 28 and 56 PI, and in group 1 on day 56
PI (Figs 1 and 2). Antibody titres recorded in the control noninfected animals (group 4) remained at a basal
level along the experiment (data not shown).
CMI
Lymphocyte blastogenesis tests were performed at
biweekly intervals to provide an indication of the antirSUB3 and antiexo-antigen CMI. Considerable variation
was observed for some SI results obtained in a given
group depending of the subgroup. However, the mean
SI obtained with r-SUB3- and exo-antigen-stimulated
lymphocytes increased notably in both group 1 and
group 2 after the first immunization, i.e. on day 56
(Figs 3 and 4). At that time, mean SI with r-SUB3 were
10.3 and 20.02 in groups 1 and 2, respectively, whereas

Figure 3. Kinetics of CMI to r-SUB3 evaluated as the mean

stimulation index (+ SE) of lymphocyte proliferation in vaccinated
and control guinea pigs after vaccination and subsequent M. canis
challenge exposure.

Figure 4. Kinetics of CMI to exo-antigen evaluated as mean

stimulation index (+ SE) of lymphocyte proliferation in vaccinated
and control guinea pigs after vaccination and subsequent M. canis
challenge exposure.

mean SI with exo-antigen were 15.6 and 37.8 in

groups 1 and 2, respectively. The SI decreased dramatically six weeks after the last immunization, which
corresponded with the infection challenge day, but nevertheless remained higher than in the control group 3.
Stimulation indexes increased again following challenge
exposure, albeit irregularly, in vaccinated guinea pigs
as well as in controls (Figs 3 and 4). Stimulation indexes
recorded in noninfected animals from group 4 remained at a basal level (< 1.5) throughout the experiment
(data not shown).
Clinical and mycological follow-up
Noninfected animals from group 4 did not develop any
infection. All guinea pigs from challenged groups developed clinical signs consistent with dermatophytosis.
However, considerable variation in lesion intensity was
observed among animals in a given group. Typical dermatophytic signs were already observed on day 7 PI
and consisted in mild erythema and scaling of the skin
at the application site. All these animals were found to
be culture positive after the first sampling (day 14 PI)
and Woods lamp examination positive on day 21 PI or
sooner, except for one animal from group 1 and one
from group 3 which remained Wood negative. Mean
global scores, accounting for both clinical and mycological evaluations, are illustrated for each group in
Figs 5 and 6. In all the challenged groups, the maximum level of infection was observed between day 14
and day 28 PI, the lesions then cured progressively. The
lower mean score observed in the group 1 was attributed to the less important erythema and alopecia than
in control group. However, no significant difference
was observed at any time of the infection between the
vaccinated and nonvaccinated groups.

DISCUSSION
Vaccination of guinea pigs either with r-SUB3 or
M. canis crude exo-antigen was not protective against

Figure 5. Kinetics of the mean global score ( SD) reflecting the

severity of dermatophytic lesions in r-SUB3-vaccinated and control
guinea pigs after M. canis challenge exposure.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 305 312

Experimental vaccine to Microsporum canis

Figure 6. Kinetics of the mean global score ( SD) reflecting the

severity of dermatophytic lesions in exo-antigen vaccinated and
control guinea pigs after M. canis challenge exposure.

experimental infection and did not allow the vaccinated

animals to recover more quickly than the control ones.
Vaccinated animals developed strong and specific
antibody responses towards both r-SUB3 and exo-antigen.
These antibody cross-reactions were not surprising as
SUB3 is one of the major proteins of the exo-antigen.15
Challenge in nonvaccinated animals induced an important antibody response to exo-antigen, but no anti-SUB3
Igs, which is in accordance with previous published
experiments performed in experimentally infected guinea
pigs.17
Cell-mediated immune response to M. canis antigens was also induced by the immunization procedure.
Stimulation indexes ranging from 10 to 37 were recorded after the first immunization; a SI > 2.53 is usually
interpreted as positive.25 For the reason given above,
animals vaccinated with r-SUB3 developed a CMI
response to the exo-antigen and vice versa. The challenge exposure induced CMI to both r-SUB3 and exoantigen in control nonvaccinated guinea pigs which is
in accordance with previously reported observations.17
Several reasons may account for the failure of vaccination to induce prophylactic immunity against the
experimental challenge exposure. First, it is possible that
the challenge exposure reported here was too vigorous
and largely overcame the immune response induced by
vaccination. Indeed, even a strong immunity associated
with recovery from a previous infection can sometimes
be overcome by a challenge exposure of sufficient intensity.26,27 It is possible that a less intense challenge exposure (e.g. contact with an infected animal) would have
failed to produce infection in vaccinated guinea pigs as
suggested by DeBoer et al. who evaluated M. canis vaccines in cats.14 Less severe challenge, such as by infected
animal contact, was used in a guinea pig model by Pier
et al. who performed a vaccination trial using a broad
spectrum killed vaccine.28 All the vaccinated animals
were protected against the challenge which induced
infection in 7 of 10 control unvaccinated guinea pigs.
However, protocols using cutaneous fungal direct infection as challenge exposure, as used in this study, are

309

commonly reported in dermatophyte vaccine studies,

and several vaccines were shown to protect against
such a challenge in different animal species, including
the guinea pig.29 Moreover, in our study, a control
unvaccinated guinea pig remained Woods lamp examination negative after challenge. This would suggest
that the challenge procedure used here was not too
severe.
It is also possible that the nature of the vaccine antigens, and subsequently the nature of the immune response,
were inappropriate to prevent infection. r-SUB3 and
exo-antigen are both secreted fungal antigens. When
using such vaccine antigens, the most probable mechanism allowing either the prevention or the more rapid
clearance of dermatophyte infection is the stimulation
of CMI allowing in turn the recruitment of polymorphonuclear neutrophils and macrophages on the inoculation site. Indeed, these cells are considered as the
final effectors of an efficient immune response during
many fungal infections, including dermatophytoses.30
A first immunization induced CMI directed to both
r-SUB3 and exo-antigen, but, for unknown reasons,
these responses had largely decreased on the infection
challenge day. Under these conditions, these potentially
useful responses might be inefficient. Moreover, although
systemic CMI was recorded, further investigations
should be required to assess which cellular types are
attracted at inoculation sites, and which cytokine profile is elicited, following vaccination. In contrast to the
CMI, the role of the antibody response raised towards
those secreted antigens is much more hypothetical, as
is the role of the antidermatophyte antibody response
in general.31 Nevertheless, considering the potentially
important role of secreted keratinases in M. canis infection, SUB3-specific antibodies could be implied in the
clearance of the fungus by inactivating its proteolytic
activity, as previously suggested for other dermatophyte
keratinases.15,16,32,33
Apart from the nature of the antigens, the route of
administration may have substantial importance on
the type or degree of immunity conferred. Intramuscular, subcutaneous and intradermal routes were reported
and all were shown to induce some degree of immunity.3437 The intradermal route is empirically considered more appropriate by some authors. 12,38 They argue
that this route might represent an exposure to fungal
antigens mimicking that produced during a natural
infection. Such a route would apparently enhance antigen processing.39 In a study in cattle, subcutaneous
vaccine administration was more protective than
intradermal one against a Trichophyton verrucosum
challenge.40 This prompted us to select this route of
administration. Moreover, the most successful antidermatophyte vaccination (i.e. vaccination of cattle against
T. verrucosum with LTF 130 strain) is performed
subcutaneously.41
In order to promote a strong CMI, the vaccination
protocol included the use of Freunds complete adjuvant in association with relatively low doses of antigens. Indeed, the Freunds complete adjuvant is known

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F. F. Descamps et al.

to stimulate the CMI, while administration of high

antigen quantities seems to favour antibody formation
rather than CMI.42,43 The use of another adjuvants as
well as different antigen dosages could be useful. For
example, the use of interleukins, as IL-12, a specific
adjuvant able to promote the development of CMI, is
currently assessed.44
Finally, the guinea pig is not a natural host for M. canis
and consequently r-SUB3 and exo-antigen could represent protective immunogens in cat, the natural host
and the main reservoir of M. canis. This would require
further investigation. Experimental vaccination trials
in cats against M. canis infection were ineffective.1214
Several commercial vaccines against feline dermatophytosis are available but their prophylactic efficacy
has not been reported. In contrast, vaccination against
ringworm in other species (cattle, horses, fur-bearing
animals) has been spectacularly successful in many
countries.45 47 Their use has reduced the incidence of
the disease in these animals considerably and indirectly
contributed to the reduction of human infections.48 However, these attenuated vaccines frequently induce small
lesions at the injection site and subsequent dissemination of arthrospores into the environment. In a farm or
a ranch situation, this may be of little consequence as
long as all animals are included in the vaccination programme. However, for cats which are generally housed
indoors and have frequent and close contact with their
owners, a live-fungus vaccine, able of producing active
lesions is highly undesirable because of the zoonotic
hazard.12
Further studies should then be directed to the identification, characterization, recombinant expression and
immunological evaluation of another potential M. canis
antigens. In this context, other proteins from exo-antigen
should be evaluated as this crude complex antigen induces
a very high CMI in experimentally infected guinea pigs.

ACKNOWLEDGEMENTS
We thank Jacques Detry and Franoise Marchal for
their excellent technical assistance. This work was supported by grant no. 3.4534.01 from Fonds de la Recherche Scientifique Mdicale (FRSM). Frdric Descamps
and Sandy Vermout are recipients of a studentship of
F.R.I.A. (Fonds pour la Formation la Recherche
dans lIndustrie et dans lAgriculture, rue dEgmont 5,
1000 Bruxelles).

REFERENCES
1. Scott, D.W., Miller, W.H., Griffin, C.E. Fungal skin diseases. Muller and Kirks Small Animal Dermatology.
W.B. Saunders Co., Philadelphia, 2001: 339 61.
2. Moriello, K.A. Management of dermatophyte infections
in catteries and multiple-cat households. Veterinary Clinics of North America: Small Animal Practice 1990; 20:
1457 74.

3. Moriello, K.A., DeBoer, D.J. Feline dermatophytosis.

Recent advances and recommendations for therapy. Veterinary Clinics of North America: Small Animal Practice
1995; 25: 90121.
4. Hnilica, K.A., Medleau, L. Evaluation of topically
applied enilconazole for the treatment of dermatophytosis in a Persian cattery. Veterinary Dermatology 2002; 13:
238.
5. Moriello, K.A., DeBoer, D.J. Efficacy of griseofulvin
and itraconazole in the treatment of experimentally induced
dermatophytosis in cats. Journal of the American Veterinary
Medical Association 1995; 207: 43944.
6. Ben-Ziony, Y., Arzi, B. Use of lufenuron for treating fungal infections of dogs and cats. Journal of the American
Veterinary Medical Association 2000; 217: 151013.
7. Guillot, J., Malandain, E., Jankowski, F. et al. Evaluation of the efficacy of oral lufenuron combined with
topical enilconazole for the management of dermatophytosis in catteries. Veterinary Record 2002; 150: 718
24.
8. Sparkes, A.H., Werrett, G., Stokes, C.R. et al. Microsporum canis. Inapparent carriage by the cats and the viability of arthrospores. Journal of Small Animal Practice
1994; 35: 397401.
9. Lunder, M., Lunder, M. Is Microsporum canis infection
about to become a serious dermatological problem?
Dermatology 1992; 184: 879.
10. Mignon, B., Losson, B. Prevalence and characterization
of Microsporum canis carriage in cats. Journal of Medical
and Veterinary Mycology 1997; 35: 24956.
11. Symoens, F., Fauvel, E., Nolard, N. Evolution de la contamination de lair et des surfaces par Microsporum canis
dans une habitation. Bulletin de la Socit Franaise de
Mycologie Mdicale 1989; 18: 2938.
12. DeBoer, D.J., Moriello, K.A. The immune response to
Microsporum canis induced by a fungal cell wall vaccine.
Veterinary Dermatology 1994; 5: 4755.
13. DeBoer, D.J., Moriello, K.A. Investigations of a killed
dermatophyte cell wall vaccine against infection with Microsporum canis in cats. Research in Veterinary Sciences 1995;
59: 11013.
14. DeBoer, D.J., Moriello, K.A., Blum, J.L. et al. Safety
and immunologic effects after inoculation of inactivated
and combined liveinactivated dermatophytosis vaccines
in cats. American Journal of Veterinary Research 2002;
63: 15327.
15. Mignon, B., Swinnen, M., Bouchara, J.P., et al. Purification and characterization of a 31.5 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and
evidence of its secretion in naturally infected cats. Medical Mycology 1998; 36: 395404.
16. Descamps, F., Brouta, F., Monod, M. et al. Isolation of
a Microsporum canis gene family encoding three subtilisinlike proteases expressed in vivo. Journal of Investigative
Dermatology 2002; 119: 8305.
17. Mignon, B., Leclipteux, T., Focant, C. et al. Humoral
and cellular immune response to a crude exo-antigen and
purified keratinase of Microsporum canis in experimentally infected guinea pigs. Medical Mycology 1999; 37:
1239.
18. Calderon, R.A., Hay, R.J. Cell-mediated immunity in experimental murine dermatophytosis II. Adoptive transfer of
immunity to dermatophyte infection by lymphoid cells
from donors with acute or chronic infections. Immunology
1984; 53: 46572.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 305 312

Experimental vaccine to Microsporum canis

19. Sohnle, P.G. Dermatophytoses. In: Cox, R.A., ed. Immunology of Fungal Diseases. CRC Press, Boca Raton, FL,
1989: 1 27.
20. Descamps, F., Brouta, F., Vermout, S. et al. Recombinant
expression and antigenic properties of a 31.5 kDa keratinolytic subtilisin-like serine protease from Microsporum
canis. FEMS Immunology and Medical Microbiology (in
press).
21. Brouta, F., Descamps, F., Fett, T. et al. Purification and
characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis. Medical Mycology 2001;
39: 269 75.
22. Mignon, B.R., Coignoul, F., Leclipteux, T. et al. Histopathological pattern and humoral immune response to
a crude exo-antigen and purified keratinase of Microsporum canis in symptomatic and asymptomatic infected
cats. Medical Mycology 1999; 37: 1 9.
23. Bradford, M.M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of proteindye binding. Analytical Biochemistry
1976; 72: 248 54.
24. Van Cutsem, J. Animal models for dermatomycotic
infections. In: McGinnis, M.R., Borgers, M., eds. Current Topics in Medical Mycology, Vol. 3. Springer, New
York, 1989: 1 35.
25. Kristensen, F., Kristensen, B., Lazary, S. The lymphocyte
stimulation test in veterinary immunology. Veterinary
Immunology and Immunopathology 1982; 3: 20377.
26. Grappel, S.F., Bishop, C.T., Blank, F. Immunology of
dermatophytes and dermatophytosis. Bacteriological
Reviews 1974; 38: 840 8.
27. Jones, H.E., Reinhardt, J.H., Rinaldi, M.G. Acquired
immunity to dermatophytes. Archives of Dermatology
1974; 109: 840 8.
28. Pier, A.C., Hodges, A.B., Lauze, J.M. et al. Experimental immunity to Microsporum canis and cross reactions
with other dermatophytes of veterinary importance.
Journal of Medical and Veterinary Mycology 1995; 33:
93 7.
29. Wawrzkiewicz, K., Ziolkowska, G. Specific immunoprophylaxis in Microsporum canis infection in guinea pigs.
Journal de Mycologie Mdicale 1996; 6: 56 62.
30. Calderon, R.A., Shennan, G.I. Susceptibility of Trichophyton quinckeanum and Trichophyton rubrum to products of oxidative metabolism. Immunology 1987; 61:
283 8.
31. Sparkes, A.H., Stokes, C.R., Gruffydd-Jones, T.J. Experimental Microsporum canis infection in cats: correlation
between immunological and clinical observations. Journal of Medical and Veterinary Mycology 1995; 33: 177
84.
32. Brouta, F., Descamps, F., Vermout, S. et al. Secreted
metalloproteases of Microsporum canis. Infection and
Immunity 2002; 70: 5676 83.
33. Grappel, S.F., Blank, F. Role of keratinases in dermatophytosis. I. Immune responses of guinea pigs infected with

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

311

Trichophyton mentagrophytes and guinea pigs immunized

with keratinases. Dermatologica 1972; 145: 24555.
Mosher, C.L., Langendoen, K., Stoddard, P. Treatment
of ringworm (Microsporum canis) with inactivated fungal vaccine. Veterinary Medicine/Small Animal Clinician
1977; 72: 13435.
Hussin, Z., Smith, J.M.B. Vaccination procedures and
the infectivity of dermatophyte lesions. Mycopathologia
1983; 81: 716.
Wharton, M.L., Reiss, F., Wharton, D.R.A. Active
immunization against Trichophyton purpureum infection
in rabbits. Journal of Investigative Dermatology 1950; 14:
291303.
Reiss, F., Leonard, L. Failure of active immunization
against Trichophyton gypseum infection in guinea pigs.
Journal of Investigative Dermatology 1956; 26: 44952.
Elad, D., Segal, E. Immunogenicity in guinea pigs of a
crude ribosomal fraction from Microsporum canis.
Vaccine 1994; 12: 1348.
Dreesen, D.W., Summer, J.W., Brown, J. et al. Intradermal use of human diploid cell vaccine for pre-exposure
rabies immunization. Journal of the American Veterinary
Medical Association 1982; 181: 151923.
Rybnikar, A., Vrzal, V., Chumela, J. Protective efficacy
of vaccine against bovine trichophytosis after intramuscular, subcutaneous, and intradermal administration.
Acta Veterinaria Brno 1997; 66: 5760.
Mignon, B., Losson, B. Vaccination against ringworm
in cattle. In: Pastoret, P.-P., Blancou, J., Vannier, P.,
Verschueren, C., eds. Veterinary Vaccinology. Elsevier
Science BV, Amsterdam, 1997: 4901.
Grun, J.L., Maurer, P.H. Different T-helper cell subsets
elicited in mice utilizing two different adjuvant vehicles:
the role of endogenous interleukin 1 in proliferative responses.
Cellular Immunology 1989; 121: 13445.
Bretscher, P.A. A strategy to improve the efficacy of vaccination against tuberculosis and leprosy. Immunology
Today 1992; 13: 3425.
Zuckermann, F.A., Martin, S., Husmann, R.J. et al. Use
of interleukin 12 to enhance the cellular immune response
of swine to an inactivated herpesvirus vaccine. Advances
in Veterinary Medicine 1999; 41: 44761.
Gudding, R., Naess, B. Vaccination of cattle against
ringworm caused by Trichophyton verrucosum. American
Journal of Veterinary Research 1986; 47: 241517.
Pier, A.C., Zancanella, P.J. Immunization of horses
against dermatophytosis caused by Trichophyton equinum.
Equine Practice 1993; 15: 237.
MacEnzie, D.W.R., Loeffler, W. et al. Guidelines for the
Diagnosis, Prevention and Control of Dermatophytoses in
Man and Animals World Health Organization, Geneva,
1986: 5568.
Pier, A.C., Smith, J.M.B., Alexiou, H. et al. Animal ringworm its aetiology, public health significance and control. Journal of Medical and Veterinary Mycology 1994;
32 (Suppl. 1): 13350.

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312

F. F. Descamps et al.
Rsum Une kratinase recombinante de poids molculaire 31.5 kDa et un exo-antigne de Microsporum canis
ont t tests comme vaccins chez des Cobayes prsentant une infection exprimentale. Les animaux ont t
vaccins trois fois deux semaines d'intervalle, soit avec la kratinase, soit avec l'exo-antigne soit avec l'adjuvant.
Une provocation a t ralise l'aveugle. Les rponses immunologiques humorales et cellulaires spcifiques de
M. canis ont t values tous les 14 jours et les lsions cliniques et la prsence de champignons dans la peau ont
t tests toutes les semaines. La vaccination a provoqu des rponses anticorps trs leves et significatives vis
vis des deux antignes (P<0.01). Des rponses cellulaires leves ont galement t notes aprs la vaccination.
Aprs provocation, les scores cliniques refltant la svrit des lsions fongiques n'taient pas significativement
diffrentes entre les groupes vaccins et le groupe contrle. Ces rsultats suggrent que, chez le Cobaye, l'induction
de rponses immunologiques spcifiques contre les antignes de M. canis utiliss dans cet essai ne sont pas
protecteurs vis vis d'une infection provoque exprimentalement.
Resumen Una queratinasa recombinante de 31 kDa de Microsporum canis y un exo-antgeno crudo de M. canis
fueron probados como vacunas en un modelo experimental en cobayas. Los animales fueron vacunados
subcutneamente tres veces, en intervalos de dos semanas, con la queratinasa, el exo-antgeno o el coadyuvante
solamente. La administracin fue realizada ciegamente. Las respuestas inmunes humoral y celular especfica a
los antgenos de M.canis fueron evaluadas cada 14 das mientras que la evaluacin ciega del desarrollo clnico
de la lesin y la persistencia fngica en la piel fueron monitorizadas semanalmente. La respuesta de anticuerpos
inducida por la vacunacin, hacia ambos antgenos, fue muy alta y significativa (P<0.01). Mediante la vacunacin
tambin fue inducida una elevada respuesta inmune celular hacia ambos antgenos. Despus de la vacunacin,
sin embargo, los ndices que reflejaban la severidad de las lesiones dermatofticas no se diferenciaron perceptiblemente entre los grupos de animales vacunados y los controles. Estos resultados sugieren que, en el cobaya, la
induccin de una respuesta inmunoespecfica contra los antgenos secretados por M. canis usados en este estudio
no son protectores contra la exposicin.
Zusammenfassung Eine rekombinante Microsporum canis 31.5 kDa Keratinase und ein unbehandeltes M. canis
Exo-Antigen wurden als Vakzine in einem experimentellen Infektionsmodell beim Meerschweinchen getestet.
Die Tiere wurden entweder mit der Keratinase, dem Exo-Antigen oder dem Adjuvans allein dreimal im
zweiwchigen Intervall subkutan geimpft. Die Challenge-Infektion wurde blind durchgefhrt. Sowohl die
humorale als auch die zellulre, spezifische Immunantwort auf M. canis-Antigene wurden alle vierzehn Tage
bewertet, die Blindbewertung der klinischen Lsionen und Erregerpersistenz wurde wchentlich durchgefhrt.
Die Impfung induzierte sehr hohe und signifikante (P<0.01) Antikrperantworten auf beide Antigene. Auch eine
hohe zellmediierte Immunantwort auf beide Antigene wurde durch die Impfung hervorgerufen. Nach Challenge
jedoch unterschieden sich die die Schwere der dermatophytischen Lsionen widerspiegelnden Scores der behandelten Tiere zu keinem Zeitpunkt signifkant von denen der Kontrollgruppe. Diese Ergebnisse deuten darauf hin,
dass bei Meerschweinchen die Induktion spezifischer Immunantworten gegen die in dieser Studie verwandten,
von M. canis sezernierten Antigene nicht gegen eine Challenge-Infektion schtzt.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 305 312