2.1. Animals and Tissue Processing

This study was undertaken on three groups of cats. Group 1.1 comprised 16 cats with FIP, further subdivided into natural infection (12 pet cats; age: 5 months to 2 years; Group 1.1a) and experimental infection (four female specific pathogen free (SPF) cats; age: 14–16 weeks; Group 1.1b), see Table 1. All 1.1a cats were submitted for diagnostic post mortem examination with full owner consent. The 1.1b cats had been euthanased with FIP after experimental intra-peritoneal infection with the serotype I FCoV strain FIPV-UCD at the University of Utrecht, The Netherlands. Approval for this experiment was obtained from the Ethical Committee of Utrecht University (approval number: 0502.0802). All Group 1.1b cats showed clinical signs of FIP which necessitated euthanasia of two cats at 3.5 and 4 weeks post infection (p.i.) whilst the remaining two were euthanased at the end of the experiment (11 weeks p.i.).

Signalment and lesion distribution of cats in Group 1 (naturally and experimentally infected cats with feline infectious peritonitis (FIP) used for the liver study).

m: months; y: years; ME: male entire; MN: male neutered; FE: female entire; CNS: brain and spinal cord; N: no effusions; Y: effusions present; blank: data not available; NR: not recorded.

The diagnosis of FIP was confirmed in all cases by gross, histological, and immunohistological examination [3]. Six of the 16 cats with FIP had effusions, including three of the four experimental cases (data was unavailable for three animals).

Group 1.2 consisted of 14 clinically healthy, male SPF cats that had been per-orally infected at an age of 8.5 to 27 weeks with previously isolated serotype I FCoV field strains of enteric pathotype (FCoVZu1, 2, 3, and 5 -feline enteric coronavirus; FECV) and had been euthanased between 2 and 12 weeks p.i. [34]. This experiment was performed under the Swiss regional legislation (project license number TVB 66/2000). All cats had tested positive for FCoV shedding and those euthanased more than 2 weeks p.i. seroconverted. All were confirmed to be systemically FCoV infected by the presence of a FCoV viraemia [34]. These cats were used as a comparison group to provide relatively uniform baseline cytokine level as pet cats without FIP would be subject to wide variations in terms of pathogen exposure, FCoV infection status, and concurrent disease. This also allowed evaluation of the effect of FIP on the animal rather than FCoV infection per se.

All group 1.1 and 1.2 animals were necropsied within 1 h of death. Liver samples from grossly normal regions (i.e., without FIP lesions) were collected and immediately frozen at −80 °C for RNA extraction, whilst normal and lesion samples were fixed in 10% buffered formalin for 24–48 h and routinely paraffin wax embedded for histological and immunohistological examination.

The third group (Group 1.3) comprised six healthy untreated SPF cats, aged 36–38 months, that had been euthanased at the University of Glasgow, UK as part of a study performed under UK Home Office Project Licence PPL 60/3735. From these cats, formalin fixed, paraffin embedded liver samples were kindly provided by Prof M Hosie.

This study was undertaken on three additional groups of cats. Group 2.1 comprised 18 pet cats (age: 2 months to 3 years; mean age: 14 months) that had died or were euthanased with FIP; the diagnosis was confirmed as described above. See Table 2.

Signalment and lesion distribution of cats in Group 2.

* in the case of FIP; ^† in all non FIP cats; m: months; y: years; ME: male entire; MN: male neutered; FE: female entire; mes: mesenteric; med: mediastinal; ln: lymph node.

Group 2.2 comprised 10 cats that had been euthanased due to non-inflammatory diseases not expected to have any systemic impact (24 months to 19 years; mean age: 9 years) which were further grouped by age (Group 2.2a (n = 4), (two each); Group 2.2b (n = 6), aged 9–19 years, mean age: 13.4 years) to acknowledge the fact that age has an effect on constitutive cytokine expression in the myocardium [35]. See Table 2.

Group 2.3 comprised three cats with systemic inflammatory diseases other than FIP. See Table 2.

All cats had been euthanased and submitted for diagnostic post mortem examination with full owner consent. They were necropsied within 1 h of death. Pleuritis involving the outer pericardium was observed in one of the FIP cats, however, neither this cat nor any of the others exhibited any gross changes in the heart. 14 of the 18 cats with FIP had effusions (data was unavailable for one animal). Hearts were removed and samples collected from both atria, both ventricular free walls, and the interventricular septum into RNAlater^TM Stabilization Solution (Thermo Fisher Scientific, Ilkirch Cedex, France) and stored at −80 °C until RNA extraction. The remaining heart was fixed in 10% buffered formalin for 24–48 h, trimmed, routinely paraffin wax embedded and subjected to a histological examination. This did not reveal pathological changes in the myocardium of any of the cats.