Sampling of cats
With the owners consents, we sampled 125 cats, that were presented to the Clinic of Small Animals, Vetsuisse-Faculty, Zurich, Switzerland. In order to screen “healthy” cats with respect to PV infections. Furthermore, we included only cats without skin diseases nor any conditions impairing the immune system such as hypersensitivity, auto-immunity, neoplasia, immunomodulatory treatments, FeLV, FIV and FIPV infections. From these 100 healthy cats, 60 were male (44 castrated) and 40 were female (20 spayed). Cat ranged in age from three months to 17 years with a median of seven years. Twenty-two cats were less than 1 year old. The age of 10 cats was unknown. Seventy-six cats were mixed breeds and 24 cats were purebred cats or descendants of two different purebred cats, respectively.
Skin cell samples were taken with a Cytobrush cell sampler (Deltalab; Barcelona, Spain). Two samples were taken from each cat. The first sample was taken from the haired skin around the mouth in the area where the left vibrissae are located. The second sample was taken from the right front paw, interdigitally between P3 and P4. If the described areas were not accessible for any reason (e.g. injury or bandage), the corresponding areas on the contralateral side were used for sampling. Briefly, a Cytobrush was wetted in 0.9 % sterile NaCl solution and rubbed with rotating movement for 30 s on the skin of the described area. The handle of the Cytobrush was then cut off and the brush part placed in a sterile 1.5 ml Eppendorf tube.
Serum samples were taken during routine diagnostics not related to our study or when a new intravenous catheter was placed. Animals with a known or suspected history of immunodeficiency or under treatment with immunosuppressive drugs were not included. If a complete blood count of a candidate was available, it was checked and cats suspected immunodeficiency were excluded.
Two cats with lesions that had been histologically confirmed as BISC served as positive controls. The Cytobrush samples were taken directly from the BISC lesions. One cat was sampled at two lesions on the neck whereas the other cat was sampled at one lesion on the forehead so that in a total of three samples were obtained. Serum samples were taken during routine diagnostics.
As a negative control, Cytobrush and serum samples were taken from 5 specific pathogen-free (SPF) cats . The Cytobrush samples were taken from the same locations as described above. All serum and Cytobrush samples were stored at −20 °C until further analysis.
DNA was extracted from the Cytobrush samples using QIAamp® DNA Mini Kit (Qiagen; Basel, Switzerland) according to the manufacturer’s protocol but with double amount of buffer ATL, proteinase K, buffer AL and ethanol. The extracted DNA was finally dissolved in 100 μl of buffer AE.
Quantitative real-time PCR (qPCR) was performed using the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad; Hercules CA, USA). Reactions contained 10 μl iQ™ SYBR® Green Supermix (Bio-Rad; Hercules CA, USA), 0.6 μl forward primer (10 μM; fdpv2_qpcr_for: 5′-CAG CTC CCA GTC TCC TAA CG-3′), 0.6 μl reverse primer (10 μM; fdpv2_qpcr_rev: 5′-GCT GTG CCA TTA TCT GAG CA-3′), 3.8 μl sterile water and 5 μl template DNA. Negative controls contained no template DNA but additional 5 μl of sterile water. The following amplification conditions were used: 3 min at 95 °C, 41 cycles of 10 s at 95 °C and 30 s at 60 °C and 1 cycle of 1 min at 95 °C and 1 min at 55 °C. Afterwards temperature was raised by 0.5 °C per cycle during 84 cycles of 10 s to create the melt curve.
As a reference gene, feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen. A set of primers (gapdh_qpcr_for: 5′-GTG GAG GGA CTC ATG ACC AC-3′ and gapdh_qpcr_rev: 5′-GTG AGC TTC CCA TTC AGC TC-3′) was designed to amplify cat’s GAPDH. qPCR was performed using the same protocol as described above.
Calibration curves were created with dilution series of plasmid DNA. For FdPV2, the plasmid containing the entire FdPV2 DNA was used. For GAPDH, an amplimer of a PCR reaction (with the primers gapdh_for: 5′-TCA TCA TCT CTG CCC CTT CT-3′ and gapdh_rev: 5′-GTG AGC TTC CCA TTC AGC TC-3′) was cloned, sequenced and then used as template DNA for calibration curve creation.
Antigen production for ELISA
The FdPV2 L1 coding sequence (CDS), lacking the first ten (5′) codons, was amplified by PCR from the cloned whole genome of FdPV2  using Phusion™ High-Fidelity DNA Polymerase (Finnzymes; Espoo, Finland). Flanking BamHI sites at the ends of the amplimer, introduced by the primers (fdpv2_L1_for: 5′-CGA CGG ATC CTT ATA TCT CCC ACC CTC CCC TG-3 and fdpv2_L1_rev: 5′-AAT AGG ATC CTC ATT TGC GGG TGC GTT-3), facilitated the cloning into the BamHI site of the pGEX-6P-1 vector (Pharmacia Biotech; Uppsala, Sweden). Protein expression in E.coli strain BL21(DE3), which express the T7 polymerase upon IPTG induction, was performed as described previously with minor modifications . In brief, bacteria were grown in LB medium containing 100 μg/ml Ampicillin at 25 °C with shaking up to an OD600 of 0.3 when protein expression was induced by adding 0.25 mM isopropyl-β-D-thio-galactoside (IPTG) and incubated over night at 25 °C with shaking. Pelleted bacteria were resuspended in 1/10 of the culture volume of buffer L (40 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA and 2 mM DTT) supplemented with Complete Protease Inhibitor Cocktail (Roche; Mannheim, Germany) and lysed by sonication. ATP (2 mM) and MgCl2 (5 mM) were added and the lysate was incubated for 1 h at room temperature. Urea was slowly added over 5 min to a final concentration of 3.5 M. After incubation of 2 h at room temperature, the mixture was dialysed over night at 4 °C against buffer L using 7 K MWCO Slide-A-Lyzer® Dialysis Cassettes (Thermo Scientific; Rockford IL, USA). After centrifugation the obtained antigen mix was diluted 1:1 with glycerol and stored at −20 °C.
The protein expression procedure was simultaneously performed with three different E.coli strain BL21(DE3) cultures containing different pGEX-6P-1 vector derivatives. The first contained the FdPV2 L1 CDS fused to the GST CDS whereas the second contained a CPV1 L1 CDS fused to the GST CDS . The third culture contained the GST CDS only. All ELISA assays reported in this study were performed with antigen from the same lot of antigen production.
GST capture ELISA
Throughout the protocol, plates were washed three times with PBS buffer supplemented with 0.3 % Tween 20 (PBS-T) between every incubation step. Polysorb 96-well plastic plates (Nunc; Roskilde, Denmark) were prepared for the ELISA. They were coated at 4 °C over night with 50 mM sodium carbonate buffer pH 9.6 containing 0.2 % glutathione casein (kindly provided by Martin Müller DKFZ, Heidelberg, Germany) and then blocked at 37 °C for 1 h with casein buffer (PBS-T containing 0.2 % casein). The GST tagged antigen, diluted 1:10 in casein buffer, was applied to the plates and incubated at 37 °C for 1 h.
Prior to ELISA, the sample sera had been diluted 1:500 in casein buffer, mixed with an equivalent of lysed untransformed E.coli strain BL21 (DE3) and incubated at 4 °C for 30 min to block reactions with contaminating bacterial proteins [26, 27, 30]. The sera, cleared by centrifugation, were applied to the plates and incubated at 37 °C for 1 h. Goat Anti-Feline IgG conjugated to Horseradish Peroxidase (HRP) (Southern Biotech; Birmingham AL, USA) diluted 1:1000 in casein buffer was added as secondary antibody and the plates were incubated again at 37 °C for 1 h. After six final washes with PBS-T, substrate (78 mM CH3COOH, 24 mM CH3COONa, 50 mM NaH2PO4, 2 mM ABTS [Roche; Rotkreuz, Switzerland] with 1.25 mM H2O2 applied shortly before use) was added. Absorbance was measured after 45 min at 405 nm in a Sunrise™ microplate reader (Tecan; Männedorf, Switzerland).
The cat sera were tested in triplicates against the antigen FdPV2 L1-GST and, as a negative control, against CPV1 L1-GST. For a subset of samples the ELISA was repeated. The according samples were then tested in duplicates against CPV1 L1 and against GST alone. In order to normalize the results of the different plates, the same positive and negative control sera were used on every plate. No serum was added in six wells serving as a plate control.
Data analysis and presentation
The Cq-values obtained from qPCR were converted into absolute numbers of copies of FdPV2 and GAPDH in each sample using the equation of the corresponding calibration curve. The Cq values of samples revealing no amplification within forty qPCR-cycles were set to 40 for further calculations. In order to obtain comparable results, in each sample the absolute number of FdPV2 copies was divided by the corresponding absolute number of GAPDH copies.
Serum samples were tested in triplicates in ELISA. To prevent outlier results from influencing the data, the median of the three observed values was used for further analysis. Plate to plate variability was compensated by dividing every value by the mean of the control sera values from the corresponding plate and multiplying the result by the mean of all control sera from all plates.
A cut-off value (COV) was set by the mean of all negative control samples plus two standard deviations. Figures were generated using R (Free Software Foundation; Boston, USA).