Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

Abstract Background Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos) and to determine parasite burden and associated histological effects.Methods For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite load was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections.Results 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and P. vaughani SYAT05 showing the highest prevalences. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high parasite loads in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses.Conclusion This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Download Full-text

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

10.21203/rs.2.19034/v2 ◽

2020 ◽

Author(s):

Tanja Himmel ◽

Josef Harl ◽

Simone Pfanner ◽

Nora Nedorost ◽

Norbert Nowotny ◽

...

Keyword(s):

In Situ Hybridization ◽

Bird Species ◽

Parasite Burden ◽

Passerine Birds ◽

Haemosporidian Parasite ◽

Pathogenic Potential ◽

Tissue Samples ◽

Inflammatory Reactions ◽

Turdus Merula

Abstract Background Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. Methods For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite load was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. Results By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. Conclusion This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Download Full-text

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

Malaria Journal ◽

10.1186/s12936-020-3147-6 ◽

2020 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Tanja Himmel ◽

Josef Harl ◽

Simone Pfanner ◽

Nora Nedorost ◽

Norbert Nowotny ◽

...

Keyword(s):

In Situ Hybridization ◽

Parasite Burden ◽

Hybridization Study ◽

Turdus Merula

Download Full-text

A PILOT STUDY OF HER-2/NEU GENE AMPLIFICATION ASSESSED BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN GASTRIC CANCER

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2014.3.2 ◽

2014 ◽

pp. 15-20

Author(s):

Van Huy Tran ◽

Thi Minh Thi Ha ◽

Trung Nghia Van ◽

Viet Nhan Nguyen ◽

Phan Tuong Quynh Le ◽

...

Keyword(s):

Gastric Cancer ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Cancer Patients ◽

Gene Amplification ◽

Cancer Tissue ◽

Tissue Samples ◽

Her 2 ◽

Gastric Cancer Patients

Background: HER-2/neu is a predictive biomarker for treatment of gastric cancer using trastuzumab in combination with chemotherapy. This study aimed to evaluate the status of HER-2/neu gene amplification using fluorescence in situ hybridization (FISH) in gastric cancer. Patients and methods: thirty six gastric cancer patients were assessed HER-2/neu gene amplification by FISH using PathVysionTM HER-2 DNA Probe kit (including HER-2/neu probe and CEP-17 probe) with biopsy and surgical specimens. Results: The HER-2/neu gene amplification was observed in three cases (8.3%), the HER-2/neu gene amplification rate in Lauren’s intestinal-type and diffuse-type were 11.8% and 5.2%, respectively. Conclusion: We applied successfully FISH technique with gastric cancer tissue samples. This technique could be performed as routine test in gastric cancer in order to select patients that benefit from trastuzumab in combination with chemotherapy.

Download Full-text

Detection and Confirmation of Reptilian Adenovirus Infection by in Situ Hybridization

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870101300418 ◽

2001 ◽

Vol 13 (4) ◽

pp. 365-368 ◽

Cited By ~ 20

Author(s):

Laura E. Leigh Perkins ◽

Raymond P. Campagnoli ◽

Barry G. Harmon ◽

Christopher R. Gregory ◽

W. L. Steffens ◽

...

Keyword(s):

Electron Microscopy ◽

In Situ Hybridization ◽

Adenovirus Infection ◽

Pathogenic Potential ◽

Suitable Alternative ◽

Viral Particles ◽

Viral Inclusions ◽

Diagnostic Setting ◽

Formalin Fixed

Adenovirus infections are documented in at least 12 different species of reptiles. In contrast to their mammalian and avian counterparts reptilian adenoviruses are not well characterized as to their pathogenic potential and their ability to cause primary disease. In the diagnostic setting, fresh tissues are often not available for virus isolation, and the confirmation of reptilian adenovirus infections is dependent largely upon electron microscopy for the identification of intranuclear viral inclusions associated with histopathologic changes. The diagnosis of adenovirus infection in 2 different species of snake was confirmed by the application of DNA in situ hybridization. Using an aviadenovirus specific oligoprobe, adenoviral DNA was observed in the nuclei of hepatocytes, Kupffer cells, endothelial cells, and enterocytes. Electron microscopy of the liver confirmed the presence of intranuclear viral particles morphologically consistent with an adenovirus. DNA in situ hybridization on formalin-fixed tissues can serve as a suitable alternative to electron microscopy in the diagnosis of reptilian adenovirus infections. Both affected snakes had other concurrent diseases, suggesting that the adenovirus may not have been the primary pathogen.

Download Full-text

Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711425584 ◽

2011 ◽

Vol 23 (6) ◽

pp. 1212-1216 ◽

Cited By ~ 10

Author(s):

Meike M. Mostegl ◽

Barbara Richter ◽

Nora Dinhopl ◽

Herbert Weissenböck

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Signal Intensity ◽

Formalin Fixation ◽

Proteinase K ◽

Cross Linking ◽

Fixation Time ◽

Tissue Samples ◽

Chromogenic In Situ Hybridization

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

Download Full-text

HER-2/neu Analysis in Archival Tissue Samples of Human Breast Cancer: Comparison of Immunohistochemistry and Fluorescence In Situ Hybridization

Journal of Clinical Oncology ◽

10.1200/jco.2001.19.2.354 ◽

2001 ◽

Vol 19 (2) ◽

pp. 354-363 ◽

Cited By ~ 208

Author(s):

Annette Lebeau ◽

Daniela Deimling ◽

Christine Kaltz ◽

Andrea Sendelhofert ◽

Anette Iff ◽

...

Keyword(s):

Breast Cancer ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Gene Amplification ◽

Cancer Tissue ◽

Breast Cancers ◽

Paraffin Sections ◽

Tissue Samples ◽

Her 2

PURPOSE: The objective of our study was to compare the methods used in the literature to analyze HER-2/neu status on archival breast cancer tissue. Therefore, a series of antibodies was evaluated to assess their immunohistochemical (IHC) sensitivity in correlation to gene amplification determined by fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: HER-2/neu overexpression was studied on paraffin sections of 85 invasive breast cancers using a panel of five monoclonal (9G6, 3B5, CB11, TAB250, GSF-HER2) and two polyclonal antibodies (A8010, A0485) in addition to the HercepTest (DAKO, Glostrup, Denmark). HER-2/neu gene amplification was determined by FISH using a dual-color probe (PathVysion; Vysis, Stuttgart-Fasanenhof, Germany). RESULTS: HER-2/neu overexpression was demonstrated in 26% (9G6, TAB250, GSF-HER2), 27% (3B5, CB11), 33% (A8010) and 42% (A0485, HercepTest) of the tumors. FISH on paraffin sections identified gene amplification in 28% of the tumors. Strongly positive IHC results (3+) were always associated with gene amplification. Among the 16 tumors presented with weakly positive IHC results (2+) using the HercepTest, 12 (75%) lacked gene amplification. CONCLUSION: The comparison of IHC and FISH demonstrated an excellent correlation of high-level HER-2/neu overexpression (3+) with gene amplification; ie, FISH does not provide further information in these tumors. However, weakly positive IHC results (2+) obtained with the HercepTest share only a minor association with gene amplification.

Download Full-text

Specific Detection of Lawsonia intracellularis in Porcine Proliferative Enteropathy Inferred from Fluorescent rRNA In Situ Hybridization

Veterinary Pathology ◽

10.1177/030098589803500212 ◽

1998 ◽

Vol 35 (2) ◽

pp. 153-156 ◽

Cited By ~ 24

Author(s):

M. Boye ◽

T. K. Jensen ◽

K. Møller ◽

T. D. Leser ◽

S. E. Jorsal

Keyword(s):

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Lawsonia Intracellularis ◽

Specific Detection ◽

Fixed Tissue ◽

Tissue Samples ◽

Formalin Fixed Tissue ◽

Proliferative Enteropathy ◽

Formalin Fixed

Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy. A specific oligonucleotide probe was designed and the specificity of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L. intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy. We used 10 tissue samples from pigs without proliferative mucosal changes as negative controls. The results showed that the oligonucleotide probe is specific for L. intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L. intracellularis in formalin-fixed tissue.

Download Full-text