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Detection of Feline Immunodeficiency Virus and Feline Leukemia Virus
in tissue samples of domestic cats with in situ hybridization
Anna Szilasi*, Lilla Dénes, Gyula Balka
Department of Pathology, University of Veterinary Medicine Budapest, Hungary *[email protected]
Among the diseases caused by the retrovirus family indomestic cats, Feline Leukemia Virus (FeLV) and FelineImmunodeficiency Virus (FIV) are the most widespreadworldwide. A few years ago the diseases due to FeLVinfection were the reason for most deaths among cats,though recently this seems to be decreased due tovaccination protocols. FIV is a subject of manyresearches nowadays, such as a model for HumanImmunodeficiency Virus (HIV), because they sharenumerous characteristics, other benefit is, that FIV isthe only possible non-primate model. FeLV is a -retrovirus in the Oncornavirus subfamily, while FIV is aretrovirus in the lentivirus group. Both are presentworldwide in domesticated cats. Their nucleic acid isssRNA, which is covered by a helical nucleocapsid, asecond cuboid capsid and an envelop. These virusesare divided into subtypes based on certain sequencesof the genome. In FeLV the most important subtypesare A, B and C, while in FIV there are 6 subtypes (A, B,C, D, E, F), however studies introduce new subtypes,due to the exceptional variability of the genome.
Introduction
The aim of the project was to detect FelineImmunodeficiency Virus (FIV) and Feline LeukemiaVirus (FeLV) in various types of tissues of infecteddomestic cats with a method of RNAscope® in situhybridization.
Objectives
Multiple tissues from cats were harvested using astandard protocol at the Department of Pathology ofUniversity of Veterinary Medicine, Budapest. Tissueswere fixed in 8% neutral buffered formalin for 16–48hours. Fixed samples were dehydrated in a gradedseries of ethanol and xylene, followed by infiltration ofmelted paraffin at 56°C. Sections were cut at athickness of 5 μm and mounted on the SuperFrost®Plus slides. Ready-to-use reagents from RNAscope®2.5HD Reagent Kit-RED (Advanced Cell Diagnostics)were used on our slides and control slides with targetand positive/negative control probes as permanufacturers’ instructions, in a manual assayworkflow. Sections were evaluated with Olympus CX21light microscope, images were acquired using Nikonimaging system. RNA markers were analyzed based onthe average RNA dot number per cell. Tissues weretested with polymerase chain reaction (PCR) previous.
Methods
ResultsFIV and FeLV can be visualized with RNAscope® in situhybridization in various tissue types (spleen, lymphnode, kidney, lung, intestine, bone marrow) and also inneoplastic tissues.
AcknowledgementsThis publication was supported by the 12190-4/2017/FEKUTSTRAT grant of the Hungarian Ministry ofHuman Capacities.
Case No. 1: 8-year-old cat presented with 3 weeks history of hind leg paralysis.FeLV ELISA and PCR tests positive, focal lymphoma in spinal cord (at Th7-8).
Case No. 2: 10-year-old cat presented with ulcerative stomatitis, bloodyvomitus and fever in anamnesis. ELISA and PCR tests were positive to FIV andFeLV.
Images are showing FeLV RNA-containing cells (single red dots or bigger clusters) in smallintestinal propria glands (A), liver (B), bone marrow (C) and mesenteric lymph node (D)(400×magnification).
FeLV RNA-containing cells are dyed red in clusters due to high amount of nucleic acid in tumors.Pictures showing the spinal cord (200× (A) and 400× magnification (B)) and spleen (200× (C) 400×magnification (D)).
In case of FIV, we observed significantly less viral RNA in the cells. Pictures are showing thenegative small intestine (A)(400×), spleen (B)(600×), mesenteric lymph node (C)(400×) andfrontal lobe of brain, white matter (D)(200×).
Case No. 3: 6-year-old cat presented with severe ulcerative-necrotic stomatitisfor months treated with corticosteroids. ELISA and PCR tests were positive toFIV and FeLV.
We can see FeLV RNA-containing cells in ulcerated small intestine and granulation tissue at site ofulceration (A)(40× magnification), spleen (B), mesenteric lymph node (C) and bone marrow(D)(400×magnification).
We found FIV RNA in a smaller amount. Imagesshow small intestinal propria glands (A)(200×magnification), spleen (B), mesenteric lymphnode (C), bone marrow (D)(400×magnification); liver (E) and kidney (F)(400×magnification) on the left.
A DCB
CBA D
DCBA
DCBA
E
DCB
A
A
F
