scholarly journals Diagnosis of Polyomavirus-Induced Hepatic Necrosis in Psittacine Birds Using DNA Probes

1994 ◽  
Vol 6 (3) ◽  
pp. 308-314 ◽  
Author(s):  
AnaPatricia Garcia ◽  
Kenneth S. Latimer ◽  
Frank D. Niagro ◽  
Branson W. Ritchie ◽  
Raymond P. Campagnoli
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Dna Amplification ◽  
Hepatic Necrosis ◽  
Dna Probes ◽  
Intranuclear Inclusions ◽  
Hepatocellular Necrosis ◽  
Hematoxylin And Eosin ◽  
Avian Polyomavirus

Liver sections from 32 psittacine birds with multifocal to coalescing hepatocellular necrosis were examined to determine the cause of disease. Avian polyomavirus (APV) infection (19 of 32 birds), bacterial hepatitis (5 of 32 birds), and chlamydiosis (3 of 32 birds) were major causes of hepatic disease. The presence of APV inclusions or nucleic acid was demonstrated using hematoxylin and eosin (HE) staining, DNA in situ hybridization, and DNA amplification with Southern blotting. Amphophilic intranuclear inclusions, suggestive of APV infection, were observed in HE-stained liver sections from 5 of 32 birds. Hepatocellular karyomegaly was present in liver tissues from 10 birds (5 birds with typical APV inclusions and 5 birds without discernable inclusions). DNA in situ hybridization recognized intranuclear APV nucleic acid in liver sections of 18 of 32 birds. DNA amplification with Southern or dot blots also identified APV nucleic acid in processed, paraffin-embedded livers of 18 of 32 birds. This study demonstrates that acute APV infection is a frequent cause of multifocal to coalescing hepatocellular necrosis in psittacine birds. Furthermore, APV infection is best diagnosed using DNA probes, especially when typical intranuclear inclusions are not observed microscopically.

scholarly journals Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

2006 ◽  
Vol 72 (8) ◽  
pp. 5311-5317 ◽  
Author(s):  
Kengo Kubota ◽  
Akiyoshi Ohashi ◽  
Hiroyuki Imachi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Dna Probes ◽  
Locked Nucleic Acid ◽  
Factors Affecting ◽  
Hybridization Efficiency ◽  
Probe Hybridization ◽  
Major Factors

ABSTRACT Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

scholarly journals Polyomavirus Encephalopathy in a Ducorps Cockatoo (Cacatua Ducorpsii) with Psittacine Beak and Feather Disease

1996 ◽  
Vol 8 (3) ◽  
pp. 291-295 ◽  
Author(s):  
Kenneth S. Latimer ◽  
Frank D. Niagro ◽  
W. L. Steffens ◽  
Branson W. Ritchie ◽  
Raymond P. Campagnoli
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Neuronal Degeneration ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Histologic Evaluation ◽  
Tissue Specimens ◽  
Avian Polyomavirus ◽  
Formalin Fixed

Necropsy tissues were examined from an adult wild-caught Ducorps cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.

scholarly journals New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1819
Author(s):  
Tatyana Karamysheva ◽  
Svetlana Romanenko ◽  
Alexey Makunin ◽  
Marija Rajičić ◽  
Alexey Bogdanov ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sex Chromosomes ◽  
Interphase Nucleus ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Dna Probes ◽  
B Chromosomes ◽  
Apodemus Flavicollis ◽  
Yellow Necked Mouse

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

scholarly journals Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

2009 ◽  
Vol 15 (12) ◽  
pp. 4009-4016 ◽  
Author(s):  
Nobutake Yamamichi ◽  
Ryoichi Shimomura ◽  
Ken-ichi Inada ◽  
Kouhei Sakurai ◽  
Takeshi Haraguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Hybridization Analysis ◽  
Locked Nucleic Acid ◽  
Cancer Development

Identification of the origin of centromeres in whole-arm translocations using fluorescent in situ hybridization with α-satellite DNA probes

1991 ◽  
Vol 40 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Avirachan T. Tharapel ◽  
Mazin B. Qumsiyeh ◽  
Paula R. Martens ◽  
Sugandhi A. Tharapel ◽  
James D. Dalton ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Satellite Dna ◽  
Fluorescent In Situ Hybridization ◽  
Dna Probes

Canine Hepatitis Associated with Intrahepatic Bacteria in Three Dogs

2018 ◽  
Vol 54 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Joon Im ◽  
Derek P. Burney ◽  
Sean P. McDonough ◽  
Brigid Nicholson ◽  
Adam Eatroff ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Case Report ◽  
Fluorescence In Situ Hybridization ◽  
Antimicrobial Therapy ◽  
Granulomatous Inflammation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Hematoxylin And Eosin ◽  
Formalin Fixed

ABSTRACT This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.

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

2011 ◽  
Vol 23 (6) ◽  
pp. 1212-1216 ◽  
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.

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