Canine paracoccidioidomycosis: a seroepidemiologicstudy

M. A. ONO*,y, A. P. F. R. L. BRACARENSEz, H. S. A. MORAIS§, S. M. TRAPP§, D. R. BELITARDOz& Z. P. CAMARGO*

*Universidade Federal de SaÄo Paulo, Escola Paulista de Medicina, Disciplina de Biologia Celular, Rua Botucatu, SaÄo Paulo, Brazil;yUniversidade Estadual de Londrina, Departamento de CieÃncias PatoloÂgicas, Londrina, Brazil; zDepartamento de MedicinaVeterinaÂria Preventiva, Rua Botucatu, Brazil; §Departamento de Clinicas VeterinaÂrias, Londrina, Brazil

Sera from 305 dogs were analyzed by enzyme-linked immunosorbent assay (ELISA)to determine presence of the antibody anti-gp43, which reacts to a speci�c antigen ofParacoccidioides brasiliensis. The dogs were divided into three groups according totheir origin: urban dogs (animals with little or no contact with rural areas); suburbandogs (from the urban outskirts); and rural dogs. There was a signi�cant differencebetween groups (P <0¢05). Rural dogs reacted positively in 89¢5% of cases, followedby suburban (48¢8%) and urban dogs (14¢8%). There were no differences betweenmale and female dogs. In an attempt to verify the feasibility of skin testing with gp43to determine sensitization against P. brasiliensis in dogs, suburban (n ˆ 61) and rural(n ˆ 21) dogs were tested, showing positivity of 13¢1 and 38¢1%, respectively. Sixdogs that had higher ELISA titers and also showed strong reactions in skin testingwere killed in an attempt to isolate P. brasiliensis. The fungus was not detected byculture or histopathological analysis in these dogs, suggesting that dogs have anatural resistance or that they encounter an inoculum level that is insuf�cient tocause disease. These results indicate that ELISA and skin testing can be useful in theepidemiological study of paracoccidioidomycosis in dogs and that encounter with thefungus in nature is a frequent event.

Keywords dog, ELISA, Paracoccidioides brasiliensis, skin test

Introduction

Paracoccidioidomycosis (PCM) is the most prevalentsystemic mycosis in Latin America, especially in Brazil.Although much progress has been achieved in studies onthe diagnosis and pathogenesis of this mycosis, little isknown about the ecology of the etiological agent,Paracoccidioides brasiliensis. The habitat of this fungushas not yet been determined. It is thought that P.brasiliensis may exist as a saprobe in nature, mainly inthe soil, where it produces propagules that can infecthumans [1]. Since the �rst report of disease by Lutz in

1908 [2] in Sao Paulo, Brazil, only a few isolates ofP. brasiliensis were obtained from nature, in soil [3–5],dog food contaminated with soil [6], bats [7], penguins[8] and armadillos [9,10]. The lack of outbreaks and thelong period between infection and the development ofsymptomatic disease hinder the determination of prob-able infection sources. The dog co-exists in the environ-ment with humans and in theory could be infected byP. brasiliensis in endemic areas, acting as an epidemio-logical marker of PCM, as occurs with blastomycosis [11]and histoplasmosis [12]. Canine behaviors such assnif�ng and digging could facilitate the contact withgeophilic fungi [13,14]. In a seroepidemiological survey,Mos & Fava Netto [15] observed that 75¢2% of mongreldogs had antibodies against P. brasiliensis in thecomplement �xation test using a P. brasiliensis poly-saccharide antigen. However, due its polysaccharide

ã 2001 ISHAM, Medical Mycology, 39, 277±282

Medical Mycology 2001, 39, 277±282 Accepted 27 September 2000

Correspondence: Dr Z. P. Camargo. Universidade Federal deSao Paulo, Escola Paulista de Medicina, Disciplina de BiologiaCelular, 04023-062. Rua Botucatu, 862, 8o andar, Sao Paulo, SP,Brazil. Tel.: ‡55 11 55764523; Fax: ‡55 11 5571 5877; e-mail:zoilo@ecb.epm.br

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nature, this kind of antigen facilitates cross-reactions,and doubts may arise regarding the speci�city of thereaction. Recently, the 43 kDa glycoprotein (gp43), theimmunodominant molecule of P. brasiliensis, has beenused in immunodiagnostic studies of human PCM,showing high speci�city and sensitivity [16]. Gp43 hasalso been used with very good results in skin tests toevaluate delayed-type hypersensitivity (DTH) in PCMpatients [17]. The aim of this work was to evaluate: (i)the presence of speci�c antibodies against gp43, employ-ing an enzyme-linked immunosorbent assay (ELISA)test, in rural, urban and suburban dogs; (ii) theoccurrence of DTH in skin testing, using gp43 asparacoccidioidin; and (iii) the prevalence of actualP. brasiliensis infection as determined by culture andhistopathology of sacri�ced dogs that had shown hightiters of antibodies to gp43 as well as positive DTH.

Materials and methods

Antigens

Cellular antigenP. brasiliensis (B-339) was grown in Sabouraud peptoneglucose broth under agitation (50 rpm) for 7 days at35 oC. The culture was killed by adding thimerosal(0¢2 g l¡1) overnight. It then was �ltered through sterilepaper �lter, and cells were washed with sterile distilledwater.

ExoantigenP. brasiliensis (B-339) was inoculated in 10 tubes ofSabouraud peptone glucose agar and incubated at 35 oCfor 3–4 days. Growth was transferred to a 250 mlErlenmeyer �ask containing 50 ml of neopeptonemedium and incubated under agitation (50 rpm) at35 oC. After 3 days, this pre-inoculum was transferredto a Fernbach �ask containing 500 ml of the samemedium and incubated for 7 days at 35 oC underagitation (50 rpm). The culture was killed by addingthimerosal (0¢2 g l¡1), incubated overnight at 4 oC and�ltered through �lter paper. Crude �ltrate was concen-trated by vacuum at 45 oC, dialyzed for 48 h againstdistilled water and lyophilized [18].

Gp43 antigenGp43 was obtained from exoantigen using an af�nitychromatography column composed of Af�-gel 10 (Bio-Rad, Hercules, CA, USA) bound to anti-gp43 mono-clonal antibody (MAb 17c). Gp43 was eluted with 0¢1 M

citric acid, pH 2¢8, and each milliliter was neutralizedwith 60 m l of 1 M Tris pH 9¢0 [17]. Protein concentrationwas determined by the Bradford method [19].

Dogs

A total of 305 dogs from the northern state of Paranawere divided into three groups according to the locationwhere they were living: urban dogs (54 animals), livingpredominantly in urban areas, in apartments or houses,with little or no contact with rural environments; ruraldogs (38 animals), living exclusively in rural areas, withconstant contact with natural soils and conditions; andsuburban dogs (213 animals), from city outskirts inplaces where streets have no pavement and almost novegetation.

Immunization and skin testing

A suspension of cellular antigen containing 2 £ 106 cellsin 1 ml of sterile saline was mixed in 1 ml of Freundincomplete adjuvant (v/v). One milliliter of this emulsionwas inoculated in clipped areas of the lumbar region ofsix adult male dogs at each of 10 different sites. Theinoculations were repeated every 7 days for 2 weeks(days 7, 14). At the same times, 5 ml of blood wascollected from the cephalic vein to evaluate humoralimmune responses. The blood was allowed to clot andsera were stored at ¡20 oC until the time of analysis.Skin tests with gp43 (10 m g per 100 m l) were performedat days 0, 7, 14 and 21 during the course of immunization,to determine if immunized dogs responded to gp43.

Immunodiffusion test

The test was performed as described previously byCamargo et al. [18], using P. brasiliensis exoantigen asreagent.

ELISA

Polystyrene �at-bottom microtiter plates (CorningCostar Corporation, Corning, NY, USA) were coatedwith 100 m l of gp43 in 0¢1 M carbonate buffer, pH 9¢6(250 ng well¡1), for 2 h at 37 oC and left overnight at4 oC. The plates were washed three times with phos-phate-buffered saline (PBS) with 0¢1% Tween 20 (T),and the wells were blocked with PBS-T 5% skim milk(PBS-T-M) for 4 h at 37 oC. After washing three timeswith PBS-T, the serum samples were diluted 1:100, 1:200,1:400 and 1:800 in PBS-T 0¢25% gelatin (PBS-T-G),0¢1 M galactose and incubated at 37 oC for 1 h. Theplates were washed as above and 100 m l of conjugateanti-dog immunoglobulin (Ig)G-peroxidase (Sigma, StLouis, MO, USA) (diluted 1:1000 in PBS-T-G) wereadded to each well. Plates were then incubated at 37 oCfor 1 h. After washing three times with PBS-T, 100 m l ofsubstrate (5 mg of orthophenylene diamine in 10 ml of0¢1 M citrate buffer, pH 5¢0, plus 5 m l of H2O2) were

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added to each well, and the reaction was stopped byadding 50 m l of 4 N H2SO4. Absorbance was measured ina Multiskan II ELISA reader (Flow Laboratories Inc.,McLean, VA, USA) at 490 nm. Serum from a dogimmunized with P. brasiliensis was used as a positivecontrol. The negative control was a pool of sera fromurban dogs. These sera had shown low absorbance in aprevious test. Sera with twice or more the absorbance ofthe negative control was considered positive.

Skin test

A solution of gp43 in saline (100 m g ml¡1) was sterilizedby �ltration in 0¢2 m m disposable �lters (MilliporeCorporation, Bedford, MA, USA), and stored at¡20 oC until use. Sixty-one suburban dogs and 21 ruraldogs were intradermally inoculated with 100 m l of gp43(10 m g) at a clipped injection site. The diameter ofinduration was measured at 24 and 48 h after injection.Reaction zones extending 5 mm or more were consid-ered positive.

Skin specimens were taken from the sites of antigeninjection with a 5 mm punch after local antisepsis andlocal anesthesia with 2% lidocaine (Astra Quõ micaFarmaceutica Ltd., Barueri, SP., Brazil). The skinfragments were �xed in 10% buffered formalin, andsections were stained with hematoxylin and eosin (HE)by routine methods. The protocol of this gp43 skin testwas approved by the Commission for Medical Ethics ofUniversidade Federal de Sao Paulo, Sao Paulo, SP,Brazil. All animals were maintained in accordance withthe conditions stipulated in European Guidelines(Council Directives on the Protection of Animals forExperimental and other Scienti�c Purposes, 86/609/EEC.Journal Of�ciel des Communautes Europeennes, L358,December 18, 1986).

Isolation of P. brasiliensis from dogs

Six dogs veri�ed as ownerless strays from the suburbangroup, all with ELISA titers ¶1:800 and positive(diameter >8 mm) skin test, were killed by venousinjection of KCl after sedation. The lungs, liver andspleen were removed, and fragments were obtained forhistopathological analysis using HE and Grocott stains.Material was also inoculated in Sabouraud peptoneglucose agar and Mycobiotic agar (Difco, Detroit, MI,USA). The plates were incubated at 37 oC for 40 days.

Statistical analysis

The data were analyzed by Student’s t-test. Thedifference was considered signi�cant when P <0¢05.

Results

Response to immunization and skin tests

The immunized dogs showed humoral responses (byELISA) by day 14 post inoculation, and the maximalresponse was observed at days 21 and 28 (Fig. 1 shows arepresentative result). DTH reaction, with erythema andinduration, was observed at day 21 in animals testedintradermally with puri�ed gp43 (Fig. 2a). Histopatho-logical analysis of skin biopsies showed an intensein�ammatory in�ltrate with mononuclear and polymor-phonuclear cells (Fig. 2b). Negative controls with sterilesaline induced no reaction (data not shown).

Serological analysis

Sera from the three different groups of dogs showeddifferent degrees of reactivity against gp43 antigen whentested by ELISA. The percentages of positive sera weresigni�cantly different among the three groups (Figs 3and 4). Rural dogs showed higher positivity (89¢5%) thansuburban (48¢8%) and urban dogs (14¢8%). No signi�-cant difference was observed in any of the groups amongmale and female dogs (Table 1). Seropositive rural dogs

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Fig. 1 Antibody response against gp43 evaluated by ELISA in adog immunized with P. brasiliensis. Arrows indicate the three dosesof killed cells of P. brasiliensis.

Table 1 Percentage of dogs seropositive to gp43 as evaluated byELISA

% Positivity

Sex Urban(n ˆ 54)

Suburban(n ˆ 213)

Rural(n ˆ 38)

Male 13¢6 51¢5 93¢3Female 15¢6 46¢6 75¢0Total 14¢8* 48¢8* 89¢5*

*, Signi�cant difference by Student’s t-test (P <0¢05).

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frequently showed titers of 1:200, whereas seropositiveurban and suburban dogs often showed titers of 1:100(Fig. 4). The ELISA-positive sera tested in Westernblotting with exoantigen recognized only gp43 antigen(data not shown). None of the 305 sera tested werepositive in immunodiffusion against exoantigen ofP. brasiliensis.

Gp43-skin test

Suburban and rural dogs skin tested with gp43 weresigni�cantly different in their responses showing 13¢1 and

38¢1% positivity, respectively. No signi�cant differenceswere observed among sexes (Table 2).

Attempts to isolate P. brasiliensis from dogs

The attempts to isolate P. brasiliensis from six dogs withpositive skin tests were unsuccessful. No fungal struc-tures were seen in histopathological examination ofspleen, lungs and liver. Culture media showed no growth.

Discussion

Natural PCM in animals is poorly understood. The roleof any animal species as a carrier or reservoir ofP. brasiliensis has not yet been substantiated [1].Infection has been reported in domestic dogs and cats

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Fig. 3 Comparative mean serum reactivity against gp43, evaluatedby ELISA in urban, suburban and rural dogs.

Fig. 2 (A) Site of intradermal skin test with gp43 in a dogimmunized with P. brasiliensis. (B) DTH after skin test showingin�ammatory in�ltrates with mononuclear and polymorphonuclearleukocytes.

Table 2 Intradermal skin test with gp43 in suburban and ruraldogs

% Positivity

Sex Suburban (n ˆ 61) Rural (n ˆ 21)

Male 16¢7 33¢3Female 10¢8 44¢4Total 13¢1* 38¢1*

*, Signi�cant difference by Student’s t-test (P <0¢05).

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as well as in horses, cows and sheep. Skin test surveysshowed that in Brazil, 44¢8% of the cows, 42¢8% of thesheep and 77% of the horses tested were sensitive toparacoccidioidin [20]. In Uruguay, 23% of the horsestested were positive [21]. Further study of the skin-test-positive Brazilian cows and horses revealed that 6¢8%were positive in complement �xation tests [20]. Serolo-gical surveys in mongrel dogs in Sao Paulo showed that75¢1% had a positive complement �xation test, but onlyone had a positive tube precipitin reaction. However,necropsy of the seropositive dogs failed to demonstrateany gross or microscopic tissue lesion, and cultures werenegative [15]. In addition, experimental infection in dogsproduced only a slight in�ammatory reaction, andP. brasiliensis was demonstrated only for a short periodof time [22]. In another apparently contradictory study,however, a dog inoculated with pus from a PCM patientdied three weeks later showing the same symptomsobserved in the human patient [23].

The habits of snif�ng and digging in the ground mayincrease the chance that dogs will contact the fungus’microhabitat and to inhale P. brasiliensis propagules.The constant contact with the fungus could sensitizethe immune system, inducing humoral and cellularresponses. Therefore, dogs could be very sensitiveindicators of the distribution of P. brasiliensis in nature.

The strongly positive results seen in our preliminarystudy in which dogs were immunized with killedP. brasilienis yeast cells were similar to those describedby Thilsted & Shifrine [24], who skin-tested tuberculinand coccidioidin in dogs. A very good correlation (0¢88)between skin test results and lymphocyte blastogenicassay was observed in dogs immunized with tuberculin[25].

The difference of skin test positivity observed betweensuburban (13¢1%) and rural (38¢1%) dogs suggests thatthe later may be more in contact with the fungus. Thehigher levels of speci�c antibodies seen in rural dogscon�rms this.

Dogs immunized with P. brasiliensis cells as well asnaturally seropositive dogs from suburban areas wereunresponsive to a Cryptococcus neoformans skin test,both macroscopically and in microscopic examination(data not shown). These results suggest that skin test togp43-P. brasiliensis antigens is speci�c and effective inthe detection of DTH responses to P. brasiliensis in dogs.

The negativity observed in all 305 dog sera tested byimmunodiffusion indicates that this assay is not useful toevaluate exposure to P. brasiliensis, probably due to alack of sensitivity. An immunodiffusion test also failed todetect Blastomyces dermatitidis antibody in patientsduring an outbreak of blastomycosis [26].

The high ELISA reactivity seen in rural and suburbandogs suggests that these animals readily encounterP. brasiliensis in nature. The rural areas surveyedincluded coffee plantations, a habitat that may connectedwith P. brasiliensis as suggested by two direct soilisolations [4,5]. It was therefore surprising that ourattempts to isolate this fungus from highly seropositivedogs were unsuccessful. The paucity of reports of PCMin dogs may be because dogs possess natural resistance toP. brasiliensis. Alternatively, natural inoculum levelsmay be insuf�cient to cause disease in dogs. It is alsopossible that canine PCM occurs but veterinarians todate have not successfully diagnosed it. Dogs areextremely susceptible to the closely related B. dermati-tidis, showing ratios of dog to human disease of 14:1 inendemic areas [14]. The exposure to airborne dust fromexcavation is an important risk factor for dog infectionby B. dermatitidis [14,26]. On the other hand, dust raisedin PCM areas does not result in reports of canineinfections. A reported PCM increase exclusively involv-ing human cases occurred during the construction of anArgentinean hydroelectric dam, which involved movinglarge quantities of soil [27]. In rural areas, humans andanimals are in constant contact with dust generated byagricultural practices, and this should cause PCM in dogsif they are susceptible.

The ELISA test has a high sensitivity and can detectminimal amounts of antibodies. This favors cross-reactions because fungi may share common epitopes.In human PCM, false-positive reactions may occur withsera from histoplasmosis patients. The antigens involvedin these cross-reactions are mainly those based ongalactose. The cross-reaction may be decreased or evenabolished by the use of galactose in sera to eliminateantibodies against this epitope. In our study, we dilutedthe dogs’ sera in galactose to increase speci�city andreliability of the reaction. Since the study area also is notendemic for histoplasmosis, it appears reasonable toconclude that the reactivity observed was really based oncontact of dogs with P. brasiliensis. Our results indicate

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Fig. 4 Frequency distribution of ELISA titers in sera from urban,suburban and rural dogs.

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that ELISA and skin tests can be useful in epidemiolo-gical studies of PCM in dogs.

Acknowledgements

We thank Nilson J. Carlos, Mari S. Kaminami andWerner Okano for technical assistance, Dr Marina O.Kishima for Grocott analysis and the veterinarians DrAngela M. B. de Campos, Dr Mariza A. Bissolato andDr Claudia T. Kumagai for aid in collection of dog bloodsamples. We also thank CAPES and CNPq to �nancialsupport.

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