2 MATERIALS AND METHODS
The swab samples of eye, nose and anal from
1,288
clinically diseased cats, tissues from 17 died cats and ascites from 21
cats were
collected
from Harbin, Shenyang, Hohhot, Tangshan, Tianjin, Beijing, Langfang,
Shijiazhuang, Qingdao, Zhengzhou, Luoyang, Hefei, Chongqing, Taizhou,
Guangzhou, Haikou in
China
from 2016 to 2019 (Figure 1).
And
316 blood samples were collected from Tianjin, Beijing, Qingdao,
Zhengzhou. The study was approved by the Animal Care and Ethics
Committee of National Research Center for Veterinary Medicine and
conventional animal welfare regulations and standards were taken into
account.
The primers
for
PCR test of FIPV,
FeLV,
FCV,
FHV-1, FPV,
FIV
were shown in Table 1. The viral nucleotide was extracted using the
viral nucleic acid extraction kit II (Geneaid, Taiwan, China). The
nucleotide samples of FIPV, FeLV, FCV and FIV were used as templates in
the detection of one-step RT-PCR (TransGen Biotech, China), and the
nucleotide samples of FHV-1 and FPV were detected using Premix Taq (Ex
Taq Version 2.0 plus dye) (TakaRa, Japan).
Prior to further testing, the blood samples of cats were centrifuged
(3000 ×g, 15 min) and inactivated (56℃, 30 min). For neutralization
assay,
two-fold diluted sera with the range of 1:2 to 1:4,096 were added in
96-well microtiter plates and preincubated with the virus
(102 TCID50/well) for 1 h before the
addition of F81 cells (2.5×104/well). The plates were
then
incubated at 37℃ in a humidified atmosphere of 5% CO2for 4-5 days. The titer was calculated based on cytopathic effect by the
Reed-Muench method. For haemagglutination inhibition (HI) assay of FPV,
the serum was two-fold diluted from 1:2 to 1:4,096
and
mixed at the equal volume of 0.025 ml with FPV (8 HA unit per sample),
and then incubated at 37℃ for 30
min before the equal volume of 1% suspensions of pig red blood cells
were added. HI titer was defined as the last dilution that shows
completely HI effect after the incubation at 4℃ for 90 min.