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Abstract
BackgroundMicrosporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques.ResultsSeven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target.ConclusionsThe IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.
Highlights
Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens
It is important to highlight the detection of Discussion The present study reports the production, characterization and reactivity of seven Monoclonal antibody (MAb) directed against E. intestinalis, the second most common microsporidia infecting immunodeficient patients
Immunofluorescence assays using polyclonal serum produced in rabbits against E. hellem, E. cuniculi, E. intestinalis and E. bieneusi have shown the existence of cross-reactions among the different species of Encephalitozoon and with E. bieneusi, indicating the presence of common antigens between genus and species [38]
Summary
Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. Considered as human opportunistic pathogens, microsporidiosis is traditionally associated with immunosuppressed patients, such as those that are HIV-positive [3] Their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult, the development of reliable differential staining techniques, such as Weber’s chromotrope-based stain and its modifications [6, 7], has improved the diagnosis of microsporidia These techniques are time consuming and intensive training is necessary to use them reliably. The presence of inhibitors of the polymerase enzyme in clinical samples (mainly stools), discourage their use [9,10,11,12]
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