chromogenic in situ hybridization
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2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Josef Harl ◽  
Tanja Himmel ◽  
Gediminas Valkiūnas ◽  
Mikas Ilgūnas ◽  
Nora Nedorost ◽  
...  

Abstract Background The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.


2021 ◽  
pp. 2894-2905
Author(s):  
Tayseer Anmar Hassan ◽  
Jenan M. Jawad AL-Saffar ◽  
Saad Hasan Mohammed Ali

     Human cytomegalovirus (HCMV) infects a wide range of human cells, resulting in both benign and malignant tumors. In the last few decades, proteins and/or nucleic acids of the virus were found to be often highly expressed in in patients with basal cell hyperplasia and prostatic neoplasia. This research aimed to unravel the rate of HCMV infections among prostatic tissue subsets from Iraqi patients with adenocarcinoma and benign hyperplasia. One hundred, formalin-fixed and paraffin embedded prostatic tissues were obtained from 40 tissue samples collected from different grades of prostate carcinoma; 40 from benign prostatic hyperplasia and 20 from apparently healthy prostatic tissues. These tissue specimens were collected from the archives of different public and private histopathological laboratories in Baghdad. Detection of HCMV-DNA was achieved by a highly sensitive version of chromogenic in situ hybridization technique. The signals of chromogenic in situ hybridization reactions for HCMV-DNA detection in prostatic adenocarcinoma tissues were found in 65% (26 out of 40) of the tissues, whereas in BPH (Benign Prostatic Hyperplasia), HCMV-DNA was detected in 57.5% (23 out of 40) of the tissues, and in the healthy control group in 25% (5 out of 20) of the tissues. The highest percentage of positive- HCMV- DNA-CISH reactions (57.5%) was found in prostatic adenocarcinomatous tissues that showed poor differentiation. Our results could show that HCMV might contribute to the development of the studied subsets of prostatic adenocarcinoma and benign prostatic hyperplasia.


Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 600
Author(s):  
Yongshuai Peng ◽  
Chenyang Lu ◽  
Yaqun Yan ◽  
Jinxing Song ◽  
Zhiyang Pei ◽  
...  

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295–518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charles Caldwell ◽  
James B. Rottman ◽  
Will Paces ◽  
Elizabeth Bueche ◽  
Sofia Reitsma ◽  
...  

AbstractDickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.


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