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Clinical and Vaccine Immunology, January 2008, p. 164-167, Vol. 15, No. 1
1071-412X/08/$08.00+0     doi:10.1128/CVI.00201-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of Lacazia loboi Antigens{triangledown}

Leonel Mendoza,1* Andréa F. F. Belone,2 Raquel Vilela,1 Manuela Rehtanz,3 Gregory D. Bossart,4 John S. Reif,5 Patricia A. Fair,6 Wendy N. Durden,7 Judy St. Leger,8 Luiz R. Travassos,9 and Patricia S. Rosa2

Biomedical Laboratory Diagnostics Program, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1031,1 Instituto Lauro de Souza Lima, Bauru, São Paulo, Brazil,2 New York University, Department of Basic Sciences, Infectious Diseases, New York, New York 10010,3 Harbor Branch Oceanographic Institution, Center for Coastal Research, Marine Mammal Research and Conservation, Fort Pierce, Florida 34946,4 Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523,5 National Oceanic and Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina 29412,6 Hubbs-SeaWorld Research Institute, Orlando, Florida 32821-8043,7 Department of Pathology, SeaWorld, San Diego, California 92109,8 Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil9

Received 14 May 2007/ Returned for modification 5 August 2007/ Accepted 16 October 2007


   ABSTRACT
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Antibodies in the sera of patients with lacaziosis recognized an ~193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.


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Lacazia loboi is an uncultivated fungal pathogen predominantly restricted to Latin America. Lacaziosis has been recorded among humans dwelling in this geographical area, tourists visiting areas of endemicity (4, 10), and workers handling infected dolphins (19, 24). Lacaziosis has also been diagnosed in dolphins inhabiting the coastal areas of the United States (9, 12, 19), Brazil (16), and France (25). Using molecular methods, the phylogenetic features of L. loboi were only recently deciphered (13, 18, 31). These studies placed L. loboi as a sister group to Paracoccidioides brasiliensis and, in turn, linked to the other dimorphic members of the Onygenales in the family Ajellomycetaceae.

The clinical features of Lacazia loboi and P. brasiliensis infections are different, but both fungi develop yeast cells, which are difficult to differentiate microscopically, in the host's infected tissues (7). This resemblance was used by some investigators to classify this pathogen in the genus Paracoccidioides (1, 6, 8). Thus, due to L. loboi's inability to grow in culture, immunological studies have traditionally been carried out with antigens extracted from mycelial cultures of P. brasiliensis (15, 17, 21, 23-27, 30). The objective of our study was to characterize the immunogenic proteins extracted from L. loboi yeast-like cells in Western blotting analyses, using sera from humans and dolphins with lacaziosis and sera from experimentally infected mice.

Sera from four human patients with lacaziosis from Acre State, Brazil, and from one Brazilian patient with proven paracoccidioidomycosis and four sera from experimentally L. loboi-infected mice were collected and stored at –80°C until use (2). Sera from three bottlenose dolphins (Tursiops truncatus) infected with L. loboi were evaluated: one wild dolphin infected with L. loboi was captured in the Indian River Lagoon, FL, one was a stranded Indian River Lagoon bottlenose dolphin, and one was a captive bottlenose dolphin from SeaWorld in San Diego, CA. In addition, two sera from apparently healthy humans, two sera from healthy dolphins, and two sera from healthy laboratory mice were used as negative controls.

Lacazia loboi yeast-like cell extracts were obtained from the footpads of a previously infected BALB/c mouse 6 months after inoculation, as described by Belone et al. (2). Briefly, L. loboi yeast-like cells were then centrifuged for 10 min at 3,000 rpm, and the supernatant was discarded. Cells were washed twice with saline, and the pellet was suspended in 1 ml of saline. Fungal cells were macerated to a powder with liquid nitrogen inside a mortar and used in the experiments. The gp43 protein of P. brasiliensis was extracted and purified per the method of Taborda et al. (28). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were done per the methods of Laemmli (14) and Towbin et al. (29).

The diagnosis of lacaziosis in the investigated humans and dolphins was confirmed by clinical and histopathological methods (Fig. 1). The stained gels of L. loboi protein extracts showed at least 10 bands, ranging from ~193 kDa to 9.0 kDa (Fig. 2). The L. loboi protein extract lacked a visible protein band at 43 kDa. The purified gp43 protein of P. brasiliensis showed a strong band at the expected molecular mass and did not contain other proteins (Fig. 2). Normal mouse skin tissue samples processed as described above did not show visible bands (data not shown).


Figure 1
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  		FIG. 1. Human parakeloidal (A) and dolphin verrucous (B) lesions from two of the hosts with lacaziosis used in this study. The histopathology of these lesions is shown in panels C (human) (silver stain; magnification, x100) and D (dolphin) (silver stain; magnification, x40).

 

Figure 2
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  		FIG. 2. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel showing purified Paracoccidioides brasiliensis gp43 (lane 1) and the profile of an Lacazia loboi protein extract (lane 2). The figure shows the ~193-kDa immunodominant antigen (arrow) as well as other proteins in the L. loboi protein extract. The asterisks indicate common proteins detected by the sera tested in this study. Lane M, molecular mass marker.

 
The immunoglobulin G (IgG) antibodies in the sera of all tested hosts with lacaziosis detected the gp43 protein of P. brasiliensis fairly well (Fig. 3). The tested dolphin, human, and experimentally infected mouse sera detected a major, immunodominant, ~193-kDa antigen in the L. loboi protein extract (Fig. 3) and also reacted weakly with other immunogenic antigens (Fig. 3). The experimentally infected mouse antibodies mildly detected five other immunogens, including an ~48-kDa protein not detected by the other sera (Fig. 3, panel 2, lane b, arrowhead). The IgG antibodies of the dolphins detected only two high-molecular-mass antigens in the L. loboi protein extract, including the immunodominant, ~193-kDa antigen (Fig. 3, panel 3, lane b). The antibodies in the serum from a patient with paracoccidioidomycosis weakly detected at least 13 other antigenic components in the L. loboi protein extract, ranging from ~193 kDa to 9.0 kDa. Some of these antigens were not detected by the antibodies in the sera from humans and dolphins with lacaziosis or the antibodies in the sera from experimentally infected mice (Fig. 3, panel 4, lane b). Only the antibodies in the serum from the patient with paracoccidioidomycosis weakly detected a molecular antigen of 43 kDa in the L. loboi protein extract (Fig. 3, panel 4, lane b). The healthy control human, dolphin, and mouse sera did not react with the antigens used in this study (data not shown). One of the two control dolphin sera possessed antibodies against the 43-kDa purified protein of P. brasiliensis and the major antigens of L. loboi normally detected by the dolphins with lacaziosis. Putative residual normal mouse skin cells were not detected by the tested sera (data not shown).


Figure 3
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  		FIG. 3. Western blot analyses of transferred proteins on nylon membranes. The antibodies in the serum from a human with lacaziosis weakly detected Paracoccidioides brasiliensis gp43 (lane 1a). This serum strongly detected an ~193-kDa immunodominant antigen (arrow) as well as six other weak bands (asterisks) (lane 1b). The IgG in the serum from the experimentally infected mouse weakly detected P. brasiliensis gp43 (lane 2a). The mouse antibodies strongly detected the ~193-kDa immunodominant antigen (arrow) as well as seven other proteins (asterisks) and an ~48-kDa protein not detected by the other sera (lane 2b, arrowhead). The antibodies in the dolphin with lacaziosis slightly detected P. brasiliensis gp43 (lane 3a). The antibodies strongly recognized the ~193-kDa immunodominant antigen (arrow) and only one of the minor antigens previously detected in the L. loboi extract (lane 3b). The antibodies in the serum from the patient with paracoccidioidomycosis strongly detected gp43 (lane 4a). The antibodies of this patient also detected the immunodominant ~193-kDa antigen (arrow) in the L. loboi extract as well as other antigens (asterisks), including a 43-kDa antigen (arrowhead) (lane 4b). The molecular mass marker (lanes M) appears in each of the panels.

 
With the exception of three previous studies (1, 23, 30), in the past 75 years L. loboi immunological research has been carried out using antigens of P. brasiliensis (6, 8, 15, 17, 21, 24, 26, 27, 32). Our study showed that patients with lacaziosis possessed antibodies not only against the gp43 antigen of P. brasiliensis but mainly against an ~193-kDa immunodominant L. loboi antigen. The possibility that the ~193-kDa antigen comprises several compressed L. loboi immunogens is currently under investigation in our laboratory. The IgGs in the sera from patients with lacaziosis failed to detect the gp43-like antigen in the protein extract of L. loboi. However, the serum from the patient with paracoccidioidomycosis did detect an antigen with a similar molecular mass. The cross-reactive antigens reported in this study, including purified gp43 and those reported by other investigators (1, 6, 15, 21, 24, 26, 30), were not unexpected, since recent molecular studies have shown that both L. loboi and P. brasiliensis share the same ancestor (13, 18, 31).

The many reports of lacaziosis in dolphins have puzzled investigators for decades (3, 5, 9, 12, 19, 22, 25, 32). However, studies to elucidate its etiology, epidemiology, and immunology have been equally challenging (5, 22, 25). Our study showed that dolphins develop IgG antibodies against similar prominent antigens to those recognized by humans with lacaziosis and by experimentally infected mice. Interestingly, one of the negative control dolphins detected the gp43 antigen of P. brasiliensis and the other major antigens of L. loboi identified by dolphins with lacaziosis. This unexpected result suggests a previous exposure to L. loboi without disease, as it is frequent among the other dimorphic members of the Onygenales (15, 17, 20). Thus, Western blotting analyses could be a useful tool for epidemiological studies of L. loboi. Although some investigators have reported morphological differences between the yeast-like cells of L. loboi in both species (11), our study is the first to suggest that dolphins are infected with similar L. loboi strains to those infecting humans.

In contrast to previous serological reports (1, 23, 30), this study suggests that during infection, L. loboi presents immunogens to the immune system which are different from that in P. brasiliensis infection. The molecular characterization of these antigens, especially the ~193-kDa immunodominant antigen, could generate valuable information to better understand the immunology and serology of L. loboi infection and possibly aid in the development of new therapies for infections caused by this resilient fungal pathogen.


   ACKNOWLEDGMENTS
 
Lesions from the free-ranging dolphin were collected under National Marine Fisheries Service scientific research permit no. 998-1678, issued to Gregory D. Bossart as part of the Bottlenose Dolphin Health and Risk Assessment (HERA) Project conducted in the Indian River Lagoon, FL, and the estuarine waters of Charleston, SC. Samples from the stranded dolphin were collected under a National Marine Fisheries Service letter of agreement, and this collection was supported in part under award NA06NMF4390138 from the National Oceanic and Atmospheric Administration, U.S. Department of Commerce. This research was supported in part by the Biomedical Laboratory Diagnosis Program, Michigan State University, MI.


   FOOTNOTES
 
* Corresponding author. Mailing address: Biomedical Laboratory Diagnostics Program, Michigan State University, 322 North Kedzie Hall, East Lansing, MI 48824-1031. Phone: (517) 353-7800. Fax: (517) 432-2006. E-mail: mendoza9{at}msu.edu Back

{triangledown} Published ahead of print on 24 October 2007. Back


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Clinical and Vaccine Immunology, January 2008, p. 164-167, Vol. 15, No. 1
1071-412X/08/$08.00+0     doi:10.1128/CVI.00201-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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