Multiplex fluorescent immunocytochemistry for the diagnosis of feline infectious peritonitis: Determining optimal storage conditions

Abstract

BackgroundFeline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF‐ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples.ObjectiveWe aimed to develop and optimize an MF‐ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay.MethodsA feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF‐ICC were made at 0, 1, 2, 3, and 5 days post‐collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF‐ICC for the presence of mononuclear cells with co‐expression of vimentin and coronaviral antigen.ResultsMF‐ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post‐harvest, 22℃ through 48 hours post‐harvest, and 37℃ through 24 hours post‐harvest. The MF‐ICC assay was successfully performed on fixed slides through the 12‐week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP.ConclusionsThe MF‐ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.

2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines

“…130 ICC with dual staining for macrophages as well as FCoV antigen might potentially increase specificity. 161 FCoV immunostaining using ICC was successful in detecting FCoV antigen in the CSF of a cat with neurological FIP. 162 However, one larger study evaluating CSF ICC in cats with and without FIP (with or without neurological signs) found positive results in 17/20 cats with FIP but also in 3/18 cats without FIP (one cat each with mediastinal lymphoma, meningoencephalitis, and hypertensive angiopathy with brain hemorrhage), indicating low specificity.…”

“…130 ICC with dual staining for macrophages as well as FCoV antigen might potentially increase specificity. 161…”

Feline Infectious Peritonitis: European Advisory Board on Cat Diseases Guidelines

“…In the former study, eight (three cats with heart failure and five cats with neoplasia) of twenty-nine non-FIP effusions were found to be positive by ICC [ 297 ], whilst in the latter study, two of seven non-FIP effusions (one of the two cats had heart failure, the other cholangiocarcinoma) were found to be positive by IF [ 299 ]. Whilst a multiplex fluorescent ICC assay utilising dual antibodies (vimentin and FCoV) has been developed [ 302 ], specificity and sensitivity data using this technique are yet to be determined. It is therefore important for clinicians to be aware of variations in immunostaining techniques and to be familiar with the specificity of the methodology employed by their local laboratory, as well as confirmation of the inclusion of negative controls in testing when interpreting positive results.…”

Clinical and Pathological Studies on Feline Infectious Peritonitis in Egypt

“…Feline infectious peritonitis (FIP) is a deadly immune mediated viral disease that affects wild and domestic cats. It arises because of the mutation of the avirulent feline enteric coronavirus FeCoV within a small percentage of infected cats (Michimae et al 2010;Kipar and Meli 2014;Tekes and Thiel 2016;Howell et al 2020). Both FeCoV and FIP are belonged to the same virus species feline coronavirus FeCoV but they are totally different distinct pathotypes (Howell et al 2020).…”

“…It arises because of the mutation of the avirulent feline enteric coronavirus FeCoV within a small percentage of infected cats (Michimae et al 2010;Kipar and Meli 2014;Tekes and Thiel 2016;Howell et al 2020). Both FeCoV and FIP are belonged to the same virus species feline coronavirus FeCoV but they are totally different distinct pathotypes (Howell et al 2020). Infection with FeCoV is very common and about 40-50% of the domestic cat population has been infected with this type, this figure could increase to reach 90-100% in multi-cat household (Meli et al 2004;Vogel et al 2010) .…”