Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis

The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis.     

Evaluation of Paracoccidioides brasiliensis Infection in Dairy Goats

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Brazil and other Latin American countries. The participation of domestic and wild animal species in the ecoepidemiology of paracoccidioidomycosis is not well understood. The objective of this study was to evaluate P. brasiliensis infection in dairy goats. The humoral immune response to the gp43 antigen, the main antigen used for paracoccidioidomycosis serodiagnosis and seroepidemiology, was evaluated in two goats immunized with inactivated P. brasiliensis yeast cells. Both animals produced antibodies against the P. brasiliensis gp43 antigen, detected by ELISA, 2 weeks after immunization. A total of 202 goat serum samples were analyzed by ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens. The seropositivity observed by ELISA was 26.2 % although no reactivity was detected by immunodiffusion. The animals over 18 months of age showed significantly higher positivity (40 %) than animals aged 6–18 months (14.8 %) and 0–6 months (2.6 %). Taking into account that cross-reactivity may occur with other pathogens, the serum samples were also analyzed by ELISA using Histoplasma capsulatum exoantigen as antigen and the positivity observed was 14.3 %. The low correlation (0.267) observed between reactivity to P. brasiliensis gp43 and H. capsulatum exoantigen suggests co-infection rather than cross-reactivity. This is the first report showing serological evidence of P. brasiliensis infection in goats and reinforces that domestic animals are useful epidemiological markers of paracoccidioidomycosis.     

Detection of Antibodies Against Paracoccidioides brasiliensis in Free-Range Domestic Pigs (Sus scrofa)

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.     

Serological Survey of Paracoccidioidomycosis in Cats

The objective of the present study was to evaluate infection of cats by Paracoccidioides brasiliensis. Serum samples of 136 cats from rural (n = 86) and urban areas (n = 50) were analyzed by indirect ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively, and an overall reactivity of 31.6 % was observed by ELISA although no reactivity was detected by immunodiffusion. The positivity observed in animals living in rural areas (48.8 %) with free access to soil was significantly higher (P < 0.0001) than among urban animals (2 %) with limited access to soil, although no significant difference was observed in relation to age or sex. The high rates of positivity observed in cats from rural areas suggest that not diagnosed cases of this mycosis may be occurring in cats living in endemic areas for human paracoccidioidomycosis. This is the first report showing serological evidence of P. brasiliensis infection in cats.     

Occurrence of Antibodies to <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> in Dairy Cattle from Mato Grosso do Sul,Brazil

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: Corumbá, Dourados, Nova Andradina, and São Gabriel d’Oeste. The municipalities of Corumbá (30%) and Nova Andradina (28%) showed higher positivity than Dourados (8%) and São 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.     

Paracoccidioidomycosis in wild monkeys from Paraná State, Brazil

The aim of this study was to evaluate the seroprevalence of Paracoccidioides brasiliensis infection in wild New World monkeys (Cebus sp. and Alouatta caraya). A total of 93 animals (Cebus sp., n = 68 and Alouatta caraya, n = 25) were captured in the Paraná River basin, Paraná State, Brazil and the serum samples were analyzed by ELISA and immunodiffusion using P. brasiliensis gp43 and exoantigen as antigens, respectively. The seropositivity observed by ELISA was 44.1% and 60% for Cebus sp. and A. caraya, respectively, while by immunodiffusion test Cebus sp. showed positivity of 2.9% only. No significant difference was observed in relation to age and sex. This is the first report of paracoccidioidomycosis in wild capuchin monkeys and in wild-black and golden-howler monkeys. The high positivity to P. brasiliensis infection in both species evaluated in our study and the positivity by immunodiffusion test in Cebus sp. suggest that natural disease may be occurring in wild monkeys living in paracoccidioidomycosis endemic areas.     

Serological Evidence of Infection by Paracoccidioides brasiliensis in Dogs with Leishmaniasis

Paracoccidioidomycosis is a systemic mycosis prevalent in Latin American countries, caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. The habitat of these fungi in nature remains undefined, although it is believed that infection occurs by inhalation of infective propagules present in soil. Sentinel animals, such as dogs, can be valuable epidemiological markers of paracoccidioidomycosis. Taking into account that paracoccidioidomycosis and visceral leishmaniasis may occur in the same area, the objective of this study was to evaluate the occurrence of P. brasiliensis infection in dogs positive for Leishmania sp. Serum samples of dogs positive (n = 199) and negative (n = 101) for Leishmania sp. were analyzed by the immunodiffusion test using P. brasiliensis exoantigen, and 22 samples (7.3%) were positive. The serum samples positive in the immunodiffusion test were also analyzed by Western blotting using the P. brasiliensis gp43 recombinant protein, and 86% of the samples were positive. A high positive correlation (r = 0.96) between positivity for Leishmania sp. and P. brasiliensis was observed. These data suggest an association between leishmaniasis and paracoccidioidomycosis in dogs.

     

Serological Evidence of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Infection in Chickens from Paraná and Mato Grosso do Sul States,Brazil

The objective of this study was to detect antibodies against Paracoccidioides brasiliensis in free-range and caged chickens Gallus domesticus. Initially, the humoral immune response of two chickens immunized with P. brasiliensis was evaluated. Both animals showed the production of antibodies to gp43, the major P. brasiliensis antigen. The seroepidemiological survey was conducted in chickens from the Pantanal region in Mato Grosso do Sul State (free-range n = 40) and from northern region of Paraná State (free-range n = 100, caged n = 43). The serum samples were analyzed by indirect ELISA using gp43 as antigen. The positivity observed in free-range chickens from Mato Grosso do Sul (55%) was significantly higher (P = 0.0001) than in free-range chickens from Paraná State (16%). In contrast to the free-range chickens, no positivity was observed in the caged chickens (P = 0.003). This is the first report showing serological evidence of P. brasiliensis infection in chickens. The results suggest that free-range chickens are more frequently infected by P. brasiliensis, probably due to the constant contact with soil than caged chickens and could be useful as epidemiological markers of paracoccidioidomycosis.     

Seroepidemiological Survey of Paracoccidioidomycosis Infection Among Urban and Rural Dogs from Uberaba,Minas Gerais,Brazil

There is some evidence that dogs can be naturally infected by Paracoccidioides brasiliensis in endemic areas of paracoccidioidomycosis. In order to evaluate canine infection with this fungus, a survey with 149 urban and 126 rural dogs was carried out using ELISA and intradermal tests with the gp43 antigen of P. brasiliensis in Uberaba, Minas Gerais state of Brazil. Forty-one out of 149 urban dogs were euthanatized and had their lungs, liver and spleen removed. One slice from each viscera was processed for histopathological examination and the remaining was homogenized and then cultivated on mycobiotic agar at room temperature and Fava-Netto medium at 35°C and observed for 12 weeks. Of urban dogs, 75 (50.3%) were small adult females, 56 (36%) were strays, while 93 (64%) had been donated to the municipal zoonosis control center. Nine (6.2%) had a positive intradermal test without statistical differences regarding gender, race, nutritional status or origin. No colonies with microscopic or morphology appearances resembling P. brasiliensis were isolated, nor granulomatous process or fungal structures were observed from histopathological examination. Eighty (53.6%) of the urban dogs presented seroreactivity, without statistical differences regarding gender, race, nutritional state, origin, or positive intradermal test. Of 126 rural dogs, 102 (80.5%) presented antibodies against gp43 antigen, and this was statistically significant in relation to the reactivity detected in urban dogs (P = 0.0001). Thus, dogs are commonly infected with P. brasiliensis, but they probably present natural resistance to develop paracoccidioidomycosis.     

Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii

Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43

Background

Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43.

Methodology

We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients'' sera from Southwestern and Midwestern Brazil.

Principal Findings

We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species.

Conclusion

Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.     

Sub-inhibitory Concentrations of Antifungals Suppress Hemolysin Activity of Oral Candida albicans and Candida tropicalis Isolates from HIV-Infected Individuals

Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20 %), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.     

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P.␣brasiliensis and more specifically its derived peptide P10, carrying the CD4⁺ T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-γ and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.     

Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

Background  

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations.     

Tinea capitis in the adult: Two case studies

Ascites fluid from hybridoma cell lines producing monoclonal antibodies to an exoantigen of Microsporum canis was assayed for its precipitating properties. Three types of monoclonal antibodies were used for serological analysis of heterologous mycelial cultures. Based on specific antigenic determinants, the fungal strains showed different immunodiffusion reactivity within the species. The monoclonal antibodies also proved to be effective reagents for serotyping heterogeneous species. They may also have potential applications in epidemiology.     

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.     

A rapid and specific method for the immunological identification of mycelial form cultures ofParacoccidioides brasiliensis

We developed two simple methods for extracting specific, cell-free, soluble antigens of the mold form ofParacoccidioides brasiliensis. Detection of these antigens by a microimmunodiffusion test permits the rapid and accurate identification of cultures ofP. brasiliensis. ThirtyP. brasiliensis isolates treated by these techniques produced specific exoantigens detectable by the immunodiffusion test. None of the other 78 fungal pathogens or saprophytes tested produced identical exoantigens. Personnel in any diagnostic laboratory who want a rapid and specific method for identifying or confirming suspected isolates ofP. brasiliensis can use the simple procedures described.     

Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA

Background  

Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples.     

Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros

BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48 kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.