Identification of protozoa by in situ hybridization

Protozoal diseases and diseases caused by morphologically similar organisms phylogenetically belonging to fungi or algae are frequent in animals. The present project aims at improving the tools for localizing such organisms in tissues after paraffin embedding, which is a standard technique in histopathology and apart from generating histological slides allows long term storage of samples. Especially certain protozoa belonging to the phyla Parabasala, Metamonada, Amoebozoa and Alveolata as well as some fungal or algal pathogens may be difficult to diagnose in tissue sections using conventional staining techniques. This may be due to indistinct morphology or small size leading to blending in of the organisms with tissue structures. In addition, it is impossible to distinguish pathogenic representatives from morphologically similar symbionts which do not harm the host. Common approaches to specifically identifying protozoa in tissues are immunohistochemistry or PCR amplification. IHC depends on high quality antibodies which are not always available for a number of pathogens of veterinary importance. PCR detects molecular signals but does not allow correlating them with tissue location or lesions. Here we propose the application of chromogenic in situ hybridization as a novel tool for identification of protozoa in tissue sections. As probes we intend to use digioxigenin-labeled oligonucleotides. The advantages of such probes, when compared to other specific detection reagents, such as antibodies, are (1) high flexibility in design - practically any given nucleotide sequence can be probed, (2) quick and affordable synthesis by a number of companies, and (3) that immunohistochemical detection of the digoxigeninin molecules allows generation of a permanent signal in sections which can be re-analysed months and years after the staining procedure. The ISH procedure itself, compared to pure molecular detection methods such as PCR is advantageous for its combination of specific pathogen detection with evaluation of tissue morphology. This is a necessary prerequisite for estimation of pathogenicity of protozoal organisms, which can be unequivocally correlated with lesions they have induced by this technique. In the course of this project we intend to evaluate whether probes designed to hybridize with protozoal ribosomal RNA are able to detect protozoa at different hierarchical levels, e.g. at species, genus, family and sometimes even higher levels. While in many instances it is desirable to identify the species of infectious agents, especially in diagnostic routines there are frequently situations where assignment to a protozoal family or genus has to be made as a first step before further specifying the organism. This goal will be achieved with rigorous test series of various probes on defined reference samples. In the more special and applied part of the project, we will test whether certain protozoa and other morphologically similar agents can be satisfactorily identified in routinely prepared and archived paraffin embedded tissue samples. We will focus on selected protozoa and other organisms of veterinary importance, which in part are remarkable for their zoonotic potential, like trichomonads, Giardia spp., Hexamita spp., Entamoeba spp., Leishmania spp., Plasmodiidae, Pneumocystis spp. and Prototheca spp. Our hypothesis is that infections with some of these organisms have been underdiagnosed because currently and previously performed routine examinations failed to detect these organisms and to correlate certain tissue lesions with the activity of these agents. The long term vision, for which the present project can be seen as a first step is to generate a modular tool-box with numerous vigorously validated probes which allow diagnosing all relevant protozoa present in central Europe. We also think that this concept, as a start focusing on agents of veterinary importance can find broad application in human and experimental protozoology as well.

Protozoal diseases and diseases caused by morphologically similar organisms phylogenetically belonging to fungi or algae are frequent in animals. The present project aims at improving the tools for localizing such organisms in tissues after paraffin embedding, which is a standard technique in histopathology and apart from generating histological slides allows long term storage of samples. Especially certain protozoa belonging to the phyla Parabasala, Metamonada, Amoebozoa and Alveolata as well as some fungal or algal pathogens may be difficult to diagnose in tissue sections using conventional staining techniques. This may be due to indistinct morphology or small size leading to blending in of the organisms with tissue structures. In addition, it is impossible to distinguish pathogenic representatives from morphologically similar symbionts which do not harm the host. Common approaches to specifically identifying protozoa in tissues are immunohistochemistry or PCR amplification. IHC depends on high quality antibodies which are not always available for a number of pathogens of veterinary importance. PCR detects molecular signals but does not allow correlating them with tissue location or lesions. Here we propose the application of chromogenic in situ hybridization as a novel tool for identification of protozoa in tissue sections. As probes we intend to use digioxigenin-labeled oligonucleotides. The advantages of such probes, when compared to other specific detection reagents, such as antibodies, are (1) high flexibility in design - practically any given nucleotide sequence can be probed, (2) quick and affordable synthesis by a number of companies, and (3) that immunohistochemical detection of the digoxigeninin molecules allows generation of a permanent signal in sections which can be re-analysed months and years after the staining procedure. The ISH procedure itself, compared to pure molecular detection methods such as PCR is advantageous for its combination of specific pathogen detection with evaluation of tissue morphology. This is a necessary prerequisite for estimation of pathogenicity of protozoal organisms, which can be unequivocally correlated with lesions they have induced by this technique. In the course of this project we intend to evaluate whether probes designed to hybridize with protozoal ribosomal RNA are able to detect protozoa at different hierarchical levels, e.g. at species, genus, family and sometimes even higher levels. While in many instances it is desirable to identify the species of infectious agents, especially in diagnostic routines there are frequently situations where assignment to a protozoal family or genus has to be made as a first step before further specifying the organism. This goal will be achieved with rigorous test series of various probes on defined reference samples. In the more special and applied part of the project, we will test whether certain protozoa and other morphologically similar agents can be satisfactorily identified in routinely prepared and archived paraffin embedded tissue samples. We will focus on selected protozoa and other organisms of veterinary importance, which in part are remarkable for their zoonotic potential, like trichomonads, Giardia spp., Hexamita spp., Entamoeba spp., Leishmania spp., Plasmodiidae, Pneumocystis spp. and Prototheca spp. Our hypothesis is that infections with some of these organisms have been underdiagnosed because currently and previously performed routine examinations failed to detect these organisms and to correlate certain tissue lesions with the activity of these agents. The long term vision, for which the present project can be seen as a first step is to generate a modular tool-box with numerous vigorously validated probes which allow diagnosing all relevant protozoa present in central Europe. We also think that this concept, as a start focusing on agents of veterinary importance can find broad application in human and experimental protozoology as well.