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Identification of protozoa by in situ hybridisation

Abstract
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.
Statistik Austria, science classification
106023         Molecular biology
106054         Zoology
403023         Veterinary parasitology
403024         Veterinary pathology
Keywords
in situ Hybridisierung
Paraffin-eingebettete Gewebeproben
Protozoen
Tiere
Lemma
Protozoennachweis/in situ Hybridisierung
Project leader
Weissenböck Herbert
Duration
01.10.08-31.03.11
Type of Research
Basic research
Staff
Dinhopl N., Project team member
Richter B., Project team member
Mostegl M., Project team member
Vetmed Research Units
Institute of Pathology
VetCore
Funded by
FWF - Fonds zur Förderung der wissenschaftlichen Forschung, Sensengasse 1, 1090 Wien, Austria

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39 Publications

Dinhopl, N; Nedorost, N; Mostegl, MM; Weissenbacher-Lang, C; Weissenböck, H (2015): In situ hybridization and sequence analysis reveal an association of Plasmodium spp. with mortalities in wild passerine birds in Austria. Parasitol Res. 2015; 114(4):1455-1462

Binanti, D; Mostegl, MM; Weissenbacher-Lang, C; Nedorost, N; Weissenböck, H (2014): Detection of Pneumocystis infections by in situ hybridization in lung samples of Austrian pigs with interstitial pneumonia. Med Mycol. 2014; 52(2):196-201
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Weissenböck, H; Binanti, D; Mostegl, MM; Nedorost, N; Weissenbacher-Lang, C (2012): Screening for Pneumocystis carinii in lung samples of Austrian pigs using in situ hybridization. 262-262.-22nd International Pig Veterinary Society Congress; JUN 10-13, 2012; Jeju, KOREA.

Mostegl, MM; Richter, B; Nedorost, N; Lang, C; Maderner, A; Dinhopl, N; Weissenböck, H (2012): First evidence of previously undescribed trichomonad species in the intestine of pigs? Vet Parasitol. 2012; 185(2-4):86-90
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Mostegl, MM; Wetscher, A; Richter, B; Nedorost, N; Dinhopl, N; Weissenböck, H (2012): Detection of Tritrichomonas foetus and Pentatrichomonas hominis in intestinal tissue specimens of cats by chromogenic in situ hybridization. Vet Parasitol. 2012; 183(3-4):209-214
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Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Kübber-Heiss, A; Weissenböck, H (2012): Identification of a putatively novel trichomonad species in the intestine of a common quail (Coturnix coturnix). Vet Parasitol. 2012; 183(3-4):369-372
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Mostegl, MM; Wetscher, A; Richter, B; Nedorost, N; Dinhopl, N; Weissenböck, H (2012): Detection of Tritrichomonas foetus and Pentatrichomonas hominis in the Intestine of Cats in Austria. 29th Meeting of the European Society of Veterinary Pathology and the European College of Veterinary Pathologists, Uppsala, Sweden, Sweden, SEP 7-10, 2011. J Comp Pathol (146), 1 76-76.

Schulze, C; Kämmerling, J; Kutzer, P; Richter, B (2012): Cryptosporidium baileyi Infection in Red-breasted Merganser Ducklings. 29th Meeting of the European Society of Veterinary Pathology and the European College of Veterinary Pathologists, Uppsala, Sweden, Sweden, SEP 7-10, 2011. J Comp Pathol (146), 1 70-70.

Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Kübber-Heiss, A; Weissenböck, H (2012): Infection of a Quail (Coturnix coturnix) with a Putative New Intestinal Trichomonad Species. 29th Meeting of the European Society of Veterinary Pathology and the European College of Veterinary Pathologists, Uppsala, Sweden, Sweden, SEP 7-10, 2011. J Comp Pathol (146), 1 76-76.

Mostegl, MM; Richter, B; Dinhopl, N; Weissenböck, H (2011): Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization. J Vet Diagn Invest. 2011; 23(6):1212-1216
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Dinhopl, N; Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H (2011): In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe. Vet Rec. 2011; 169(20):525
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Weissenböck, H; Ondrovics, M; Gurtner, S; Schiessl, P; Mostegl, MM; Richter, B (2011): Development of a chromogenic in situ hybridization for Giardia duodenalis and its application in canine, feline, and porcine intestinal tissues samples. J Vet Diagn Invest. 2011; 23(3):486-491
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Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Kübber-Heiss, A; Weissenböck, H (2011): Infection of a quail (Coturnix coturnix) with a putative new intestinal trichomonad species. 132-132.-29th Meeting of the European Society of Veterinary Pathology; SEP 7-10, 2011; Uppsala, SWEDEN. Cutting Edge Pathology - Abstracts;

Mostegl, MM; Wetscher, A; Richter, B; Nedorost, N; Dinhopl, N; Weissenböck, H (2011): Detection of Tritrichomonas foetus and Pentatrichomonas hominis in the intestine of cats in Austria. 131-131.-29th Meeting of the European Society of Veterinary Pathology; SEP 7-10, 2011; Uppsala, SWEDEN.

Schulze, C; Kämmerling, J; Kutzer, P; Richter, B (2011): Cryptosporidium baileyi-infection in red-brested merganser ducklings. 106-106.-29th Meeting of the European Society of Veterinary Pathology; SEP 7-10, 2011; Uppsala, SWEDEN.

Dinhopl, N; Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H (2011): Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins. Avian Pathol. 2011; 40(3):315-320
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Habenbacher, B; Kunzel, E; Gruber, A; Fragner, K; Weissenboeck, H (2010): Comparative evaluation of immunohistochemistry and in-situ hybridization for the detection of Encephalitozoon cuniculi in rabbit tissues. 28th Meeting of the European Society of Veterinary Pathology, Belgrade, SERBIA, Serbia, SEP 8-11, 2010. J Comp Pathol (143), 4 347-347.

Mostegl, M; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Weissenböck, H (2010): Prevalence and pathogenicity study of intestinal trichomonads in pigs. 28th Meeting of the European Society of Veterinary Pathology, Belgrade, SERBIA, Serbia, SEP 8-11, 2010. J Comp Pathol (143), 4 322-322.

Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Weissenböck, H (2011): Investigations on the prevalence and potential pathogenicity of intestinal trichomonads in pigs using in situ hybridization. Vet Parasitol. 2011; 178(1-2):58-63
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Mostegl, M; Dinhopl, N; Richter, B; Binanti, D; Chvala-Mannsberger, S; Nedorost, N; Maderner, A; Habenbacher, B; Janauschek, V; Ondrovics, M; Wetscher, A; Schiessl, P; Prouzova, P; Gurtner, S; Karakas, Z [and 2 others] (2011): Chromogenic in situ hybridization as a tool for identification of emerging protist species in tissue samples. 155-155.-International Meeting on Emerging Diseases and Surveillance 2011; FEB 4-7, 2011; Vienna, AUSTRIA.

Richter, B; Schulze, C; Kämmerling, J; Mostegl, M; Weissenbock, H (2010): First report of typhlitis/typhlohepatitis caused by Tetratrichomonas gallinarum in three duck species. Avian Pathol. 2010; 39(6):499-503
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Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2010): Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins. 28th Meeting of the European Society of Veterinary Pathology, Belgrade, SERBIA, Serbia, SEP 8-11, 2010. J Comp Pathol (143), 4 350-350.

Habenbacher, B; Künzel, F; Gruber, A; Fragner, K; Weissenböck, H (2010): Detection of Encephalitozoon cuniculi in rabbit tissues by the use of immunohistochemistry and in-situ hybridization. 16-16.-5th. International Meeting of Young Veterinary Pathologists ; JUL 1-3, 2010; Brescia, ITALY.

Mostegl, MM; Richter, B; Dinhopl, N; Weissenböck, H (2010): Influence of prolonged formalin fixation time on the detection of different infectious agents by chromogenic in situ hybridization. 10-10.-5th. International Meeting of Young Veterinary Pathologists ; JUL 1-3, 2010; Brescia, ITALY.

Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Kulda, J; Liebhart, D; Hess, M; Weissenböck, H (2010): Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization. Vet Parasitol. 2010; 171(1-2):1-6
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Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl,N; Kübber-Heiss, A; Weissenböck, H (2010): Entdeckung einer möglichen neuen Trichomonadenart in einer Wachtel (Coturnix coturnix). 21-21.-53. Jahrestagung der Fachgruppe Pathologie in der DVG; MAR 13-14, 2010; Fulda, GERMANY.

Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2010): Aviäre Malaria bei Pinguinen im Zoo: eine retrospektive Studie zum Nachweis von Plasmodium spp. mittels chromogener in-situ Hybridisierung. 13-13.-53. Jahrestagung der Fachgruppe Pathologie in der DVG; MAR 13-14, 2010; Fulda, GERMANY.

Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2010): Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin-embedded tissues from captive penguins. 245-246.-16. DVG-Tagung über Vogelkrankheiten; MAR 4-6, 2010; Munich, GERMANY. (ISBN: 978-3-941703-60-5 )

Mostegl, MM; Richter, B; Nedorost, N; Maderner, A; Dinhopl, N; Kübber-Heiss, A; Weissenböck, H (2010): Case report: A putative new Tritrichomonas sp. found in a quail (Coturnix coturnix). 243-244.-16. DVG-Tagung über Vogelkrankheiten; MAR 4-6, 2010; Munich, GERMANY. (ISBN: 978-3-941703-60-5 )

Mostegl, MM; Richter, B; Dinhopl, N; Weissenböck, H (2009): Design and successful application of an oligonucleotide probe for detection of parasites of the order trichomonadida by chromogenic in-situ hybridization. 27th Meeting of the European Society of Veterinary Pathology, Krakow, POLAND, Poland, SEP 9-12, 2009. J Comp Pathol (141), 4 300.

Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2009): Detection of Leishmania donovani complex using in-situ hybridization. 27th Meeting of the European Society of Veterinary Pathology, Krakow, POLAND, Poland, SEP 9-12, 2009. J Comp Pathol (141), 4 266.

Mostegl, MM; Richter, B; Dinhopl, N; Weissenböck, H (2009): Establishment of a chromogenic in situ hybridization for all members of the order Trichomonadida. 67-67.-43. Jahrestagung der Österreichischen Gesellschaft für Tropenmedizin und Parasitologie; NOV 19-21, 2009; Vienna, AUSTRIA.

Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2009): In-situ hybridization for the detection of Leishmania donovani species complex in paraffin-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe. 63-63.-43. Jahrestagung der Österreichischen Gesellschaft für Tropenmedizin und Parasitologie; NOV 19-21, 2009; Vienna, AUSTRIA.

Richter, B; Mostegl, M; Kübber-Heiss, A; Weissenböck, H (2009): Chromogenic in-situ hybridization for the specific detection of Cryptosporidium spp. in tissue samples from animals. 49-49.-III International Giardia and Cryptosporidium Conference; OCT 11-15, 2009; Orvieto, ITALY.

Mostegl, MM; Richter, B; Dinhopl, N; Weissenböck, H (2009): Design and successful application of an oligonucleotide probe for detection of representatives of the order Trichomonadida by chromogenic in situ hybridization. 19-19.-International Meeting Torino, Giessen, Zürich, Cordoba, Wien, München of Young Veterinary Pathologists; JUL 2-4, 2009; Pombia, ITALY.

Dinhopl, N; Mostegl, MM; Richter, B; Weissenböck, H (2009): Detection of Leishmania donovani species complex using in situ hybridization. 11-11.-International Meeting Torino, Giessen, Zürich, Cordoba, Wien, München of Young Veterinary Pathologists; JUL 2-4, 2009; Pombia, ITALY.

Ondrovics, M (2008): Etablierung und Validierung einer Chromogen in-situ Hybridisierung für Giardia sp. in paraffineingebetteten Gewebeschnitten. Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 29.
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Richter, B; Kübber-Heiss, A; Weissenböck, H (2008): Die Cryptosporidium-Diagnostik bei Reptilien: komplex aber essentiell. 306-307.-54. Jahreskongress der Deutschen Gesellschaft für Kleintiermedizin; Sep 27, 2008; Düsseldorf, GERMANY. (ISBN: 978-3-939902-77-5 )

Richter, B; Schulze, C; Kämmerling, J; Weissenböck, H (2008): Blackhead disease-like typhlohepatitis in two red-breasted mergansers (Mergus serrator) caused by tetratrichomonads. 198--26th Annual Meeting of the European Society of Veterinary Pathology; SEP 17-12, 2008; Dubrovnik, CROATIA.

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