Introduction

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Latin American countries. This fungus is thermodimorphic, growing as yeast in the host tissue or when cultured at 37°C and as mycelium when cultured at lower temperatures [1].

Despite the attempts of several researchers to disclose the P. brasiliensis habitat, it remains undefined to date. The soil is considered the probable fungus habitat [2–8], and the infection occurs by the respiratory route [9].

Epidemiological studies have demonstrated that domestic (dogs, cows, horses and sheep) and wild animals (armadillos and monkeys) may be infected by P. brasiliensis [10–19]. The fungus has also been isolated from armadillos in Brazil and Colombia, bats in Colombia, a penguin in Uruguay and a dog in Brazil [20–25]. Recently, the infection by P. brasiliensis was detected by nested-PCR in tissues from road-killed wild animals such as armadillos, a guinea pig, a porcupine, raccoons and grisons [26].

High positivity to P. brasiliensis infection has been described by our group in dogs from Paraná and Mato Grosso do Sul States [11, 12], which are endemic areas for human paracoccidioidomycosis [27, 28].

Taking into account that the animals exposed to soil are at risk of P. brasiliensis infection, the objective of this study was to evaluate the infection by P. brasiliensis in free-range chickens, which are in constant contact with soil and caged chickens that have no contact with the soil.

Materials and Methods

Studied Area

This study was carried out in five municipalities in Paraná State and two municipalities in the Pantanal region of Mato Grosso do Sul State (Fig. 1).

Fig. 1
figure 1

Map showing the municipalities of Aquidauana (a) and Rio Verde (b) in Mato Grosso do Sul State, and Santa Mariana (c), Bandeirantes (d), Itambaracá (e), Andirá (f), Cambará (g) in Paraná State

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The municipalities of Bandeirantes (latitude 23°06′36″ S, longitude 50°27′28″ W, altitude 420 m), Itambaracá (latitude 23°03′45″S, longitude 50°26′15″W, altitude 402 m), Andirá (latitude 23º03′02″S, longitude 50º13′44″W, altitude 479 m) Cambará (latitude 20º02′00″S, longitude 50º06′00″, altitude 450 m), Santa Mariana (latitude 23º11′15″S, longitude 50º33′45″W, altitude 484 m) are located in north Paraná State. The climate is humid subtropical with mean annual temperatures of 22°C and annual rainfall of 1,500 mm to 2,000 mm. The rainy season is from December to February, and oxisol is the predominant soil. The municipalities of Aquidauana (latitude 20° 28′ 15″ S, longitude 55° 47′ 13″ W, altitude 174 m) and Rio Verde (latitude 18° 55′ 4″ S, longitude 54° 50′ 38″ W, altitude 162 m) are located in Pantanal region of Mato Grosso do Sul State, Midwestern Brazil. The climate of both municipalities is humid subtropical with mean annual temperatures of 27°C, annual rainfall of 1,100 mm, and the rainy season is from November and April. Sandy soil is predominant in the Pantanal region.

Animals

The serum samples for the seroepidemiologic study were collected from the northern region of Paraná State and the Pantanal region in Mato Grosso do Sul by convenience sampling, as follows: 100 free-range chickens (88 female and 12 male) and 43 caged chickens (all females) from north Paraná State, 40 free-range chickens (23 female and 17 male) from the Pantanal region of Mato Grosso do Sul State.

P. brasiliensis Antigens

The cellular antigen for chicken immunization was obtained from P. brasiliensis B-339 isolate as previously described [11]. The exoantigen was obtained from culture of P. brasiliensis B-339 as described by Camargo et al. [29], and the gp43 antigen was purified from P. brasiliensis exoantigen by immunoaffinity chromatography as described by Puccia and Travassos [30].

Chicken Immunization

Two chickens, maintained in individual cages with water and commercial feed ad libitum, were inoculated with P. brasiliensis B-339 (1 × 106 inactivated yeast cells) in Incomplete Freund Adjuvant (Sigma, Saint Louis, MO, USA). Each animal received three doses intramuscularly into the breast muscle at two weekly intervals. The antibody production was evaluated in egg yolk samples by indirect ELISA using gp43 as antigen. The yolk antibody extracts were obtained by dilution of yolk 1:1 (vol/vol) in distilled water and heating at 62°C for 15 min in order to separate the yolk lipids from the proteins. The samples were centrifuged at 11,500g for 10 min, and the supernatants were used for the indirect ELISA [31].

Indirect ELISA with gp43

The egg yolk samples from immunized chickens and 183 serum samples from free-range and caged chickens were analyzed by indirect ELISA using gp43 as antigen. Flat bottom microtiter polystyrene plates (Costar Corporation, Corning, NY, USA) were coated at 4°C overnight with 100 μl gp43 (250 ng/well) in carbonate buffer 0.1 M, pH 9.6. After washing with PBS-T (PBS with 0.05% Tween 20), the wells were blocked with 5% skim milk in PBS for 1 h at 25°C. After washing with PBS-T, the egg yolk samples diluted 1:200, or serum samples diluted 1:100 in PBS-1% skim milk (100 μl/well), were incubated at 25°C for 1 h. The plates were washed with PBS-T, and 100 μl/well anti-chicken IgY-peroxidase conjugate (Sigma, St Louis, MO, USA) was added followed by incubation for 1 h at 25°C. After washing with PBS-T, 100 μl substrate-chromogen (H2O2/TMB) was added. The reaction was stopped by the addition of 4 N H2SO4 (50 μl/well). The absorbance at 450 nm was measured in a Microplate Reader (Biotek Instruments Inc., Winooski, VT, USA). All the samples were analyzed twice. The positive and negative controls used in the seroepidemiological study were serum from a chicken immunized with P. brasiliensis and a pool of young caged chickens, respectively. The serum samples with twofold absorbance of the negative control were considered positive.

Data Analysis

The statistical analysis was performed with the program Epi Info 3.5.1, and data were analyzed by the Pearson chi-square and Fisher’s exact test. The values of P < 0.05 were considered statistically significant.

Results

Humoral Immune Response of Caged Chickens Immunized with P. brasiliensis

The two chickens immunized with P. brasiliensis yeast cells produced antibodies to gp43, showing a similar reactivity with a higher response after the third dose of antigen (Fig. 2).

Fig. 2
figure 2

Antibody production against gp43 determined by ELISA in two chickens immunized with three doses (arrows) of inactivated P. brasiliensis yeast cells

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Seroepidemiology of P. brasiliensis Infection in Chickens

The free-range chickens from Mato Grosso do Sul State showed a significantly higher positivity to gp43 in the ELISA test (55%) than free-range chickens from Paraná State (16.0%) (Table 1).

Table 1 Reactivity to P. brasiliensis gp43 antigen evaluated by indirect ELISA in serum samples from free-range chickens from Paraná and Mato Grosso do Sul States
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The caged chickens from Paraná showed a significantly lower positivity to gp43 (0%) than free-range chickens (16%) from the same region (Table 2).

Table 2 Reactivity to P. brasiliensis gp43 antigen evaluated by indirect ELISA in serum samples from free-range and caged chickens from Paraná State
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The positivity to gp43 observed in free-range chickens from both regions showed no significant difference in relation to sex (Table 3).

Table 3 Reactivity to P. brasiliensis gp43 antigen evaluated by indirect ELISA in serum samples from free-range chickens from Paraná and Mato Grosso do Sul States, according to sex
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Discussion

P. brasiliensis infection has been reported in several species of domestic and wild animals initially by means of skin tests and more recently by the ELISA test with purified gp43.

This study evaluated initially the humoral immune response against gp43 in chickens immunized with P. brasiliensis, the main antigen used for immunodiagnosis and seroepidemiology of paracoccidioidomycosis. The high response observed in both animals suggested that gp43 antigen is also immunogenic to chickens, as observed by our group in dogs and cattle [11, 13], and consequently can be used for seroepidemiology of P. brasiliensis infection in chickens.

The higher positivity observed in free-range chickens from the Pantanal region in Mato Grosso do Sul State was probably due to the regional characteristics that could be more favorable for P. brasiliensis development. In a study carried out by our group to evaluate paracoccidioidomycosis infection in dairy cattle from Mato Grosso do Sul, a significantly higher positivity to paracoccidioidomycosis was observed in animals from the Pantanal region [13]. Terçarioli et al. 2007 [7] showed that P. brasiliensis may grow and produce infectious conidia both in sand and clay soils with high humidity, as occurs in the Pantanal region that is usually humid and hot, with periods of flood [32]. Taking into account that the favorable range of altitude for P. brasiliensis is very variable (47–1,300 m above the sea level) [8] and the means of annual temperature and rainfall are similar in both regions studied, it is possible that the high humidity in Pantanal region is a more relevant factor for the production of infective propagules by P. brasiliensis.

The higher positivity observed in free-range chickens when compared to caged chickens reinforce that frequent contact with soil can be considered an important risk factor for P. brasiliensis infection.

As observed in other studies with humans and other animal species, the difference in positivity was not statistically significant in relation to sex, suggesting that this is not a risk factor for paracoccidioidomycosis infection [11, 19].

Free-range chickens, due to their constant contact with soil, are more exposed to infectious diseases and can be used as epidemiological indicators of several pathogens.

Although gp43 had been successfully used in serodiagnosis and seroepidemiology of paracoccidioidomycosis, some cross-reactivity with other pathogen antigens may be occurring due to sharing of common epitopes with other pathogenic fungi as Lacazia loboi (syn. Loboa loboi) [33]. Therefore, more studies are required in order to evaluate whether chickens can develop paracoccidioidomycosis disease. This is the first report showing serological evidence of P. brasiliensis infection in chickens.