Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The
aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of
canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum
canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA
performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin
diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were
tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference
(P < 0.01, Wilcoxons test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected
dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant
difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30
days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearmans test,
rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%)
and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity
is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM
(dermatophyte test medium).
I NTRO D U CTI ON
Canine dermatophytosis is a skin disease caused by
keratinophilic fungi belonging to the genera Microsporum
and Trichophyton. Microsporum canis, M. gypseum and
T. mentagrophytes cause the great majority of infections in clinical cases. Various reports have demonstrated
that M. canis is a common cause of canine dermatophytosis. There is great variation in the proportion in
which these three fungi occur in different parts of the
world, and their incidence and prevalence varies with
climate and natural reservoirs.1 Dermatophytes are
transmitted by contact with infected hair and scale or
fungal elements on animals, in the environment, or on
fomites.2 They are highly contagious and readily transmissible to humans. In dogs, the most consistent clinical sign is one or more circular patches of alopecia.
However, signs and symptoms are highly variable and
depend on hostfungus interaction and, thus, on the
degree of inflammation.2 The differential diagnosis for
dermatophyosis is extensive and this disease should be
considered in any case of alopecic, papular or pustular
lesion.3 The diagnosis can be challenging and fungal
tests are very useful. Woods lamp examination for
fluorescence causes only certain strains of M. canis to
produce a positive yellow-green colour on infected
Correspondence: Luisa Rambozzi, Dipartimento di Produzioni
Animali, Epidemiologia ed Ecologia, Via Leonardo da Vinci 44,
10095 Grugliasco, Turin, Italy. E-mail: luisa.rambozzi@unito.it
102
M AT E R IA L S A N D M E T H O D S
Preparation of the antigen
An isolate of M. canis was cultured on selective
medium agar plates (Mycobios Selective Agar, Biolife
2005 European Society of Veterinary Dermatology
Serum samples
Positive and negative controls were represented by
two pools: five subjects with dermatophytosis due to
M. canis identified by fungal culture and five subjects
with no historical or clinical evidence of dermatophytosis. The serum was separated by centrifugation and
stored at 20 C until assayed. Using a pool reduces
the possibility of technical errors and ensures uniformity in the different phases of the study by creating
reference data for repetitively tested samples. These
sera were also used for the evaluation of the test procedure by intra- and inter-assay precision.
To assess the test performances, blood samples were
collected from 60 subjects: 18 dogs with clinical evidence
of active dermatophytosis caused by M. canis, confirmed
by fungal culture (group A, n = 18); 20 dogs with skin
diseases other than dermatophytosis and 22 dogs with
no cutaneous signs or historical evidence of dermatophytosis (group B, n = 42). In dogs with skin disease,
dermatophytosis was ruled out by negative fungal
cultures from hair and crusts.
These subjects comprised nine dogs with pyoderma,
four with flea allergy, two with sarcoptic mange, one
with demodicosis, one with vasculitis, one with contact
hypersensitivity, and two with food hypersensitivity.
For dogs of group A, the correlation between the
duration of the infection, obtained by anamnesis, and
IgG concentration was evaluated.
In addition, four animals with skin lesions caused by
dermatophytes other than M. canis (three M. gypseum
and one T. mentagrophytes) were tested.
ELISA assays
Assays were performed in polystyrene microtitre plates
(Sigma-Aldrich Corporation, Saint Louis, MO, USA)
coated, for 1 h at 37 C and then overnight at 4 C,
with 100 L per well of 15 g ml1 crude antigen solution in PBS. The last row was left free of antigen as
control. After washing with PBS mixed with Tween 20,
0.05%, pH 7.2 (PBST), the plates were saturated with
5% nonfat dried milk in PBST for 1 h at 37 C. Triplicate
103
Analysis of results
Optical density was defined as the difference between
the adsorbance mean for each triplicated serum tested
and the control wells. Results were then expressed as
OD percentages (OD%) obtained as follows: (OD
sample OD negative control)/(OD positive control OD
negative control). Reproducibility of the test procedure
was determined by intra-assay (CV1) and inter-assay
(CV2) coefficients of variance (CV = standard deviation/
mean) by measuring five replicates each of positive and
negative controls in a single plate and subsequently in
five separate assays. Following the method of Visual
Inspection of Frequency Distributions14,15 the cut-off
value was fixed at the intersection point of the OD%
value distribution curves of the two groups of animals
tested. Comparing these ELISA results against the
gold standard represented by the fungal culture of hair
and scales, the subjects were divided into four categories: true positives (TP) (culture positive, ELISA positive),
true negatives (TN) (culture negative, ELISA negative), false negatives (FN) (culture positive, ELISA
negative) and false positives (FP) (culture negative,
ELISA positive). ELISA sensitivity (Se) and specificity
(Sp) were calculated as follows:
Se = (TP/TP + FN) 100; Sp = (TN/TN + FP) 100.
Statistical evaluation
The results were analysed using Wilcoxon and Spearman correlation tests with R (1-4-1 version) and
EpiInfo 6 software.
R E S U LT S
The absolute OD values of the negative and positive
control sera in a typical experiment were approximately 0.46 (range 0.430.47) and 1.29 (range 1.23
1.3), respectively. The CV1 was 4.1 and 7.6% for the
positive and negative controls, respectively, while the
CV2 was 5.6 and 7.5%. In group A, the mean of the
OD% values corresponded to 112.6 (SD: 52.7); in
group B to 26 (SD: 26). The cut-off value was fixed at
OD% 70 (Fig. 1), giving the test a sensitivity of 83.3%
and a specificity of 95.2% (Table 1). A highly significant
difference (P < 0.001, Wilcoxons test, w = 682) existed
104
A Peano et al.
Positive serology
Negative serology
Total
Group A*
Group A
Group B
Positive culture
Positive culture
Negative culture
Total
5
3
8
10
0
10
2
40
42
17
43
60
D ISCU SSIO N
This study confirmed the production of a humoral
response in canine dermatophytosis, as already
105
106
A Peano et al.
ACKN OWLEDGE ME NT
The authors thank Dr Anna Rita Molinar for valuable
technical help.
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3. Noli C, Scarampella F. Dermatologia del Cane e del
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4. Peano A, Gallo MG, Peyrot R. Epidemiologia, clinica
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diagnosis of Tinea pedis. Archives of Dermatology 1993;
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6. Carroll HF. Evaluation of dermatophyte test medium for
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Veterinary Medical Association 1974; 165: 192 5.
7. Woodfolk JA, Slunt JB, Deuell B et al. Definition of a
Trichophyton protein associated with delayed hypersensitivity in humans. Evidence for immediate (IgE and IgG4)
and delayed hypersensitivity to a single protein. Journal
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107
Rsum Chez le chien, une dermatophytose doit tre suspecte dans tous les cas de lsions alopciques, papuleuses et pustuleuses. Le but de cette tude tait de dvelopper un test ELISA pour le diagnostic de dermatophytose canine. Lantigne utilis tait un extrait de champignon obtenu dun isolat de culture de M. canis provenant
dun chat parasit qui souffrait dune alopcie. Pour dterminer lefficacit du test ELISA, le srum de 18 chiens
dermatophytose M. canis (groupe A, n = 18), de 20 chiens dermatose autre et de 22 chiens sains (group B,
n = 42) ont t tests. Quatre autres chiens ont t tudis (trois prsentant une dermatophytose M. gypseum
et un T. mentagrophytes. Une diffrence significative (P < 0.01, Wilcoxon test, w = 364) a t note pour les taux
dIgG spcifiques des chiens rcemment infests par M. canis (infection < 15 jours) et ceux du groupe contrle
(bien que trois chiens prsentent des titres ngatifs ce stade), et une diffrence hautement significative (P < 0.001,
w = 462) a t note entre les animaux du groupe contrle et ceux prsentant une infestation chronique (> 30
jours). Tous les chiens avaient des titres positifs cette date. Une corrlation hautement significative (P < 0.001,
Spearman test, rho = 0.86) entre la dure de linfection et les concentrations en IgG a t observe. Le test avait
une bonne sensibilit (83.3%) et une spcificit leve (95.2%) mais certains chiens ont prsent des titres positifs
aprs gurison de linfection. La sensibilit est meilleure que celle de lexamen direct des poils et semblable la
culture fongique avec un DTM (Dermatophyte Test Medium).
Resumen En perros con lesiones alopcicas, papulares o pustulares se debe considerar siempre la dermatofitosis. El objetivo de este estudio fue desarrollar un anlisis de ELISA como ayuda en el diagnstico de la dermatofitosis canina. El antgeno utilizado fue un extracto fngico entero obtenido de un cultivo de M. canis en
un medio lquido de pelo infectado de un gato con focos alopcicos. Para evaluar el funcionamiento del ELISA,
se probaron sueros de 18 perros con dermatofitosis causada por M. canis (grupo A, n = 18), 20 perros con
enfermedades cutneas con excepcin de dermatofitosis y 22 perros sanos (grupo B, n = 42). Cuatro animales
ms fueron probados, tres con dermatofitosis causada por M. gypseum y uno por T. mentagrophytes. Exista una
diferencia significativa (P < 0.01, test de Wilcoxon, w = 364) entre los niveles de IgG-especficos de sueros de perros recientemente infectados con M. canis (infeccin < 15 das) y los controles (aunque tres perros tenan ttulos
negativos en esta fase), mientras que se observ una diferencia altamente significativa (P < 0.001, w = 462) entre
los controles y los perros con infeccin de una duracin ms larga (> 30 das). Todos los perros tenan ttulos
positivos en esta fase. Se observ una correlacin altamente significativa (P < 0.001, test de Spearman,
rho = 0.86) entre la duracin de la infeccin y la concentracin de IgG. La prueba tiene la buena sensibilidad
(83.3%) y alta especificidad (95.2%) pero algunos perros conservan ttulos positivos despus de la eliminacin
de la infeccin. La sensibilidad es ms alta que la del examen microscpico directo del pelo y similar a la del cultivo
fngico con DTM (Dermatophyte Test Medium).
Zusammenfassung Bei Hunden sollte bei jedem einzelnen Fall einer alopezischen, papulren und pustulsen
Lsion eine Dermatophytose in Betracht gezogen werden. Das Ziel dieser Studie ist die Entwicklung eines ELISA
(enzyme-linked immunosorbant assay) als Hilfsmittel bei der Diagnostik von caniner Dermatophytose. Das verwandte Antigen war ein Ganzkrperextrakt von einem Isolat von M. canis, welches auf einem Flssigmedium
mit infizierten Haaren einer Katze mit fleckiger Alopezie kultiviert wurde. Um die Leistungsfhigkeit des ELISA
zu beurteilen, wurden die Seren von 18 Hunden mit Dermatophytose durch M. canis (Gruppe A, n = 18), 20
Hunden mit nicht-dermatophytischen Hauterkrankungen und 22 gesunden Hunden (Gruppe B, n = 42) getestet.Vier
weitere Tiere wurden getestet, drei mit Dermatophytose durch M.gypseum und eines durch T. mentagrophytes.
Ein signifikanter Unterschied (P < 0.01, Wilcox-Test, w = 364) bestand zwischen dem Ig-G-spezifischen Gehalt
von Seren von krzlich mit M. canis infizierten Hunden (Infektion < 15 Tage) und Kontrollen (obwohl drei Hunde
in diesem Stadium negative Titer hatten), whrend ein hoch signifikanter Unterschied zwischen den Kontrollen
und den Hunden mit einer Infektion von lngerer Dauer (> 30 Tage) festgestellt wurde. Alle Hunde hatten zu
diesem Zeitpunkt positive Titer. Eine hochgradig signifikante Korrelation (P < 0.001, Spearman-Test, rho =
0.86) wurde zwischen der Dauer der Infektion und der IgG Konzentration festgestellt. Der Test hatte gute
Sensitivitt (83.3%) und hohe Spezifitt (95.2%), aber einige Hunde behielten positive Titer nach Elimination der
Infektion. Die Sensitivitt ist hher als die direkte mikroskopische Haaruntersuchung und ist hnlich der einer
Pilzkultur mit DTM (Dermatophyten-Test-Medium).