Detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction

Abstract

Feline coronavirus infections in cell cultures and in fresh and fixed feline tissues were detected using a polymerase chain reaction (PCR) test. Cell cultures were inoculated with feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV)_or sham inoculum. The tissue samples of liver, kidney and spleen were taken from specific-pathogen-free (SPF) cats that were inoculated intranasally with 10 3 TCID 50 of FIPV 79-1146 ( n = 10), FIPV UCD1 ( n = 3) or sham inoculum ( n = 3), from clinical cats ( n = 43), and from formalin-fixed archived feline tissues ( n = 49), respectively. Additional tissue samples were taken from the FIPV-inoculated cats ( n = 6) and were kept at 4°C, room temperatures (20–24°C) and 37°C respectively for 0, 6, 12, 24, 48, 72, and 96 hours before frozen (−70°C) for PCR to evaluate the effects of the ambient temperatures and post-mortem intervals on the test. The samples were also fixed in 10% neutrally buffered formalin, 95% ethanol, and Bouin's solution respectively to evaluate the effects of the fixatives on the test. Positive PCR results were obtained from the cell cultures that were inoculated with FIPV and FECV and from the FIPV-inoculated cats (13/13). Negative PCR results were obtained from the sham-inoculated cell cultures and cats (3/3). Of the 92 clinical cats, 7 of the 8 FIP-suspected cats (87.5%) and 51 of the 84 non-FIP-suspected cats (60.7%) were shown to be virus-positive in at least one of the tissue samples. There was no significant difference in the PCR results between the fresh and the formalin-fixed tissues of the clinical cats ( P > 0.05). Of the FIPV inoculated cats, the virus was detectable equally well in fresh and formalin-, Bouin's solution- or ethanol-fixedtissues. However, the amounts of total RNA extracted from the fixed tissues were significantly less than those from fresh tissues ( P < 0.01). In tissues that were kept at 4°C, the virus was detectable up to 96 h; at room temperatures, up to 48 h; and at 37°C, up to 24 h, respectively.