Mycopathologia (2008) 165:367–371 DOI 10.1007/s11046-008-9095-2
Occurrence of Antibodies to Paracoccidioides brasiliensis in Dairy Cattle from Mato Grosso do Sul, Brazil L. H. Silveira Æ R. C. S. Paes Æ E. V. Medeiros Æ E. N. Itano Æ Z. P. Camargo Æ M. A. Ono
Received: 13 August 2007 / Accepted: 11 February 2008 / Published online: 5 March 2008 Ó Springer Science+Business Media B.V. 2008
Abstract The aim of this study was to evaluate the humoral immune response in cattle immunized with Paracoccidioides brasiliensis and perform a seroepidemiological study of paracoccidioidomycosis in dairy cattle from Mato Grosso do Sul. Two animals (one steer and one heifer) were inoculated with a suspension of P. brasiliensis in Freund incomplete adjuvant. Blood samples were collected periodically to evaluate humoral immune response by immunodiffusion and ELISA, using exoantigen and gp43 as antigens, respectively. The antibody production was detected by immunodiffusion and ELISA, in both animals, 14 days after immunization. The soroepidemiologic study was carried out in 400 cattle of Mato Grosso do Sul from four municipalities: Corumba´, Dourados, Nova Andradina, and Sa˜o Gabriel d’Oeste. L. H. Silveira E. N. Itano M. A. Ono (&) Departamento de Cieˆncias Patolo´gicas, Centro de Cieˆncias Biolo´gicas, Universidade Estadual de Londrina, Campus Universita´rio, Londrina, PR 86051-980, Brazil e-mail:
[email protected] L. H. Silveira E. V. Medeiros Faculdades Luiz Meneghel, Bandeirantes, PR, Brazil R. C. S. Paes Laborato´rio de Sanidade Animal, Ageˆncia Estadual de Defesa Sanita´ria Animal e Vegetal do Mato Grosso do Sul, IAGRO, Campo Grande, MS, Brazil Z. P. Camargo Disciplina de Biologia Celular, Universidade Federal de Sa˜o Paulo, Sao Paulo, SP, Brazil
The municipalities of Corumba´ (30%) and Nova Andradina (28%) showed higher positivity than Dourados (8%) and Sa˜o Gabriel d’Oeste (4%). In this study we concluded that cattle immunized with P. brasiliensis develop humoral immune response for gp43, remaining with high titers of antibodies, and that this animal species could be an epidemiologic marker of paracoccidioidomycosis. Keywords Paracoccidioidomycosis Bovine Epidemiology ELISA
Introduction Paracoccidioides brasiliensis, a thermodimorphic fungus, is the etiological agent of paracoccidioidomycosis, a prevalent systemic mycosis in Brazil and other Latin American countries. The probable habitat of P. brasiliensis is the soil [1, 2], and infection occurs by inhalation of fungal propagules. Male agricultural workers are the most affected by paracoccidioidomycosis. Granulomatous lesions occur in lungs with dissemination to other organs and tissues by hematogenic and/or lymphatic via [3]. Until now, little has been known about the role of other animal species in the fungal ecoepidemiology. P. brasiliensis was isolated from frugivorous bats (Artibeus lituratus) in Colombia [4], a penguin (Pygoscelis adeliae) in Uruguay [5], armadillos
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(Dasypus novemcinctus) in Brazil [6–8] and Colombia [9]. The infection by P. brasiliensis was also evaluated by epidemiological studies using skin or serological tests. Studies with the paracoccidioidin skin test have shown paracoccidioidomycosis in domestic animals such as cattle, equines, sheep [10–12], as well as captive wild animals, such as monkeys and felidaes [13]. Seroepidemiological studies using paracoccidioidin or gp43 as antigens have shown infection by P. brasiliensis in dogs living in paracoccidioidomycosis endemic areas [14, 15], and recently two cases of natural paracoccidioidomycosis in dogs were described in Brazil [16, 17]. Taking into account the lack of data about P. brasiliensis ecoepidemiology, in this study we evaluated the infection by P. brasiliensis in dairy cattle from Mato Grosso do Sul, an endemic area for human paracoccidioidomycosis in Brazil [18]. Materials and Methods Study Area Mato Grosso do Sul State is located in Mid-western Brazil and occupies an area of 358,158 km2. The climate is tropical humid with rainy summers and dry winters. The serum samples were collected in four municipalities: Corumba´ (18°590 5300 S, 57°360 3200 W, altitude 118 m, mean annual temperature of 28°C, and annual rainfall of 1,200 mm), Sa˜o Gabriel d’Oeste (19°230 4400 S, 54°340 0000 W, altitude 658 m, mean annual temperature of 22°C, and annual rainfall of 1,250 mm), Nova Andradina (22°130 5900 S, 53°200 3400 W, altitude 380 m, mean annual temperature of 24°C, and annual rainfall of 1,200 mm), and Dourados (22°130 1800 S, 54°480 1800 W, altitude 430 m, mean annual temperature of 29°C, and annual rainfall of 1,250 mm). The municipalities of Dourados and Sa˜o Gabriel d’Oeste are mainly dedicated to agriculture and have soils with higher fertility (Red Oxisol and Oxisol, respectively) than Nova Andradina and Corumba´ (Sandy Oxisol and Low Humic Gley, respectively) that are important cattle-raising areas. Animals One Hostein steer and one Holstein heifer, 10 months old, maintained on the School Farm belonging to the
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Fundac¸a˜o Faculdades Luiz Meneghel, Bandeirantes, Parana´, were used for immunization with P. brasiliensis. The serum samples for seroepedimiologic study were collected from August to November 2003 from 400 (340 females and 60 males) dairy cattle from municipalities of Dourados (n = 100), Sa˜o Gabriel d’Oeste (n = 100), Corumba´ (n = 100), and Nova Andradina (n = 100). The animals’ ages ranged from 9 months to 10 years. The serum samples were part of a foot and mouth disease survey performed in the State of Mato Grosso do Sul. Cattle Immunization The two animals were subcutaneously inoculated with 1 ml of P. brasiliensis B-339 (1 9 106 inactivated yeast cells) in Incomplete Freund Adjuvant (Sigma, Saint Louis). Each animal received three doses at 7-day intervals. Blood samples were collected from the jugular vein at days 0, 7, 14, 21, 35, 42, 49, 56, 63, 77, and 88 and assayed by ELISA and immunodiffusion test. P. brasiliensis Antigens The cellular antigen for cattle immunization was obtained from P. brasiliensis B-339 culture as described previously [15]. The exoantigen was obtained from culture of P. brasiliensis B-339 as described by Camargo et al. [19], and gp43 antigen was purified from P. brasiliensis exoantigen by immunoaffinity chromatography as described by Puccia and Travassos [20]. ELISA with gp43 The 400 serum samples from dairy cattle were analyzed by ELISA using gp43 as antigen. Flatbottom Microtiter polystyrene plates (Costar Corporation, Corning, NY, USA) were coated at 4°C overnight with 100 ll of gp43 (250-ng well) in carbonate buffer 0.1 mol, pH 9.6. After washing with PBS-T (PBS with 0.05% Tween 20) the wells were blocked with 2% skim milk in PBS for 1 h at 36°C. After washing with PBS-T the serum samples, diluted 1:100 in PBS-1% skim milk (100 ll/well), were incubated at 36°C for 1 h. The plates were washed with PBS-T, and 100 ll/well Protein G-peroxidase conjugate (Biorad, Hercules, CA, USA) was added
Mycopathologia (2008) 165:367–371
Immunodiffusion The serum samples from P. brasiliensis-immunized animals were analyzed by immunodiffusion test as described by Camargo et al. [19] using P. brasiliensis exoantigen as reagent. The serum samples (25 ll) were added at peripheral orifices and the exoantigen (25 ll) at the central orifice.
3.0
Abosrbance (450 nm)
followed by incubation for 1 h at 36°C. After washing with PBS-T 100 ll of substrate-chromogen (H2O2/TMB) was added. The reaction was stopped by the addition of 4N H2SO4 (50 ll/well). The absorbance (450 nm) was analyzed in a Microplate Reader (Biotek Instruments Inc., Winooski, VT, USA). All serum samples were analyzed twice. The serum of cattle immunized with P. brasiliensis and a pool of sera from 6-month-old calves were used as positive and negative controls, respectively. The serum samples with absorbance twice the negative control were considered positive.
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+
The data were analyzed by the chi-square, Fisher, and two-proportion tests. The values of P \ 0.05 were considered statistically significant.
Results Humoral Immune Response of Cattle Immunized with P. brasiliensis The two animals immunized with P. brasiliensis yeast cells produced IgG antibodies to gp43 and maintained response until day 88 (Fig. 1). Similar results were obtained in the immunodiffusion test with exoantigen. Both animals showed positive reactions to exoantigen from days 14 to 88 (Fig. 1).
+
+
+
+
+
+
+
2.0 1.5 1.0 0.5
Steer
Heifer
0 0
7
14
21
35
42
49
56
63
77
88
Days
Fig. 1 Humoral immune response to P. brasiliensis detected by ELISA (gp43) and immunodiffusion (exoantigen) in sera from cattle (one steer and one heifer) immunized with three doses of inactivated P. brasiliensis in Incomplete Freund Adjuvant (arrows). Positive reactions by immunodiffusion (for both animals) are represented by the (+) symbol Table 1 Reactivity to P. brasiliensis gp43 antigen in cattle, evaluated by ELISA, according to age Age (years)
Positive n
Data Analysis
+
2.5
0–2
01
Negative %
n a
1.6
bc
Total %
61
98.4
62
2–4
49
21.5
179
78.5
228
4–6
10
12.0b
73
87.9
83
6–10
10
37.0c
17
62.9
27
Total
70
17.5
330
82.5
400
Percentages in the positive column followed by different letters are statistically different at P \ 0.05
2 years (Table 1). The animals aged 4–6 years showed a lower positivity than those aged 2–4 years although this difference was not statistically significant (Table 1). Animals from the municipalities of Corumba´ and Nova Andradina showed higher positivity frequencies (30% and 28%) than those from Dourados and Sa˜o Gabriel d’Oeste (8% and 4%) (Fig. 2).
Discussion Seroepidemiological Study of Paracoccidioidomycosis in Dairy Cattle The 400 cattle serum samples evaluated by ELISA showed 17.5% positivity for gp43 (Table 1). The cattle over 2 years of age showed significantly higher positivity than animals aged between 0 and
The animals immunized with P. brasiliensis showed humoral immune response against P. brasiliensis gp43 and exoantigen, the main antigens used for paracoccidioidomycosis immunodiagnostic [20] and epidemiology [21]. The response was maintained for several weeks after immunization with P. brasiliensis
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Mycopathologia (2008) 165:367–371
BRAZIL
Mato Grosso do Sul State
30% (a) Corumbá
4% (b) São Gabriel D´Oeste
28% (a) Nova Andradina
Dourados
8% (b)
Fig. 2 Reactivity to gp43 in bovine serum samples from the municipalities of Corumba´, Sa˜o Grabriel d’Oeste, Nova Andradina e Dourados. Percentages followed by different letters are statistically different at P \ 0.05
suggesting that after contact with the fungus antibody levels remain for a long time allowing the detection of infection. Although in naturally infected cattle probably the response could be lower, the high immunogenicity of gp43 observed in this study suggests that this antigen may be useful for epidemiological studies of paracoccidioidomycosis in cattle as observed by our group with dogs [15]. In another study, humoral immune response was also detected in cattle experimentally infected with P. brasiliensis [22]. According to these results both P. brasiliensis antigens could be used to evaluate paracoccidioidomycosis in cattle. The ELISA with gp43 was selected for the seroepidemiological study because it is more sensitive and faster than the immunodiffusion test. In addition, the use of gp43, a purified P. brasiliensis antigen, is advantageous because cross-reactivity is minimized. The two other epidemiological studies of paracoccidioidomycosis in cattle using the skin test with
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paracoccidioidin have shown positivity frequencies of 2.3% in Uruguay [10] and 44.8% in Brazil [12]. These disagreements could be due the fact that we used a serologic test with gp43, a purified antigen, whereas the other studies evaluated a delayed type hypersensitivity to a P. brasiliensis crude polysaccharide antigen. A higher positivity to P. brasiliensis was also observed in dogs aged over 1 year [23] showing that age may be a risk factor for P. brasiliensis infection. The differences in positivity frequencies to P. brasiliensis in the four municipalities may be related to the soil type and quality of the raising areas. The soils of Corumba´ and Nova Andradina are poor in nutrients resulting in low-quality pastures. In this way, the animals need to graze near to the soil surface increasing the probability of infection by P. brasiliensis. In a seroepidemiological study to evaluate infection by P. brasiliensis in dogs from southern Brazil, a higher frequency of infection was observed mainly in animals from rural areas (89.5%), suggesting that dogs, probably due to the habits of sniffing and digging the soil [15], are more exposed to the fungal infection than cattle. The epidemiological studies with different animal species, consequently with different habitats and habits, could be a valuable tool to finally reveal the P. brasiliensis habitat. Acknowledgments The authors thank CNPq, CAPES, and Araucaria Foundation for financial support and Dr. Roberta Lemos Freire for aid with statistical analysis.
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