This study was approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol No. 2031014) and the Institutional Biosafety Committee (IBC) (Protocol No. 1931036) of Chulalongkorn University, Thailand.

The PCR-positive CanineCV sample was obtained from a prior study (11). Briefly, nasal secretion was collected using Puritan swabs (Guilford, United States) from dogs with respiratory disease. The swab was placed in 1% (v/v) sterile phosphate buffer saline (PBS) and processed for genomic extraction using the Viral DNA/RNA extraction Kit (Geneaid Biotech Ltd., Taiwan) according to the manufacturer’s protocol. The extracted nucleic acid was initially tested for CanineCV Rep and Cap genes. Subsequently, the full-length genome of CanineCV was amplified using primers based on the acquired Rep and Cap gene sequences. The primers and conditions of cPCR were described in the previous study (11).

To construct the recombinant Cap gene, the primers were designed based on the Cap gene sequence deposited in the GenBank database (accession number ON863358). The truncated Cap gene was amplified using the primer that skipped the first 120 base pairs of the Cap gene, resulting in the removal of 40 amino acids from the N-terminal region. The forward primer was 5’-CAC CTT GAC AGC TGA TTG GC-3′, and the reverse primer was 5’TTA CAA CTG TCG ACC AGT TTC A − 3′. The reaction mixture consisted of 50 ng of DNA template, 0.5 μM of each primer, 200 μM of each deoxynucleotide triphosphate (dNTPs), 1x Phusion GC buffer, nuclease-free water, and 0.4 U Phusion® High-Fidelity DNA Polymerase (New England Biolabs, United Kingdom) to a final volume of 20 μL. The thermocycling condition was composed of initial denaturation at 98°C for 30 s, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 63°C for 20 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The amplified PCR products were stained with FluoroStain™ DNA Fluorescent Staining Dye (SMOBIO, Taiwan) and run in 1.5% agarose gel electrophoresis at 100 V, for 40 min. A 100-bp DNA Ladder M (MolBio™ HIMEDIA, India) was used as a marker. The specific band of PCR product was 693 bp in length of truncated Cap gene. Subsequently, the target amplicons in agarose gel were cut and purified using NucleoSpin Extract II (Macherey-Nagel, Germany) and submitted for sequencing to confirm the target sequences.

For ligation, a mixture of 4 μL of purified PCR product, 1 μL of salt solutions, and 1 μL of the pET100/D-TOPO^® vector was conducted according to the manufacturer’s protocol. The ligation mixture was transformed into chemically competent E. coli strain Top10 cells (Invitrogen) using the heat shock method. Then, 200 μL of transformed E. coli culture was spread on the Luria-Bertani (LB) agar plates containing 100 μg/mL of ampicillin and incubated at 37°C overnight. The positive bacterial clones were selected and grown in an LB medium containing ampicillin at 37°C overnight. The bacterial cultures were then extracted using a PureDireX Plasmid miniPREP Kit (Bio-Helix, Taiwan) according to the manufacturer’s protocol and submitted for sequencing before subcloning into the expression host cells.

The extracted pET100/D-TOPO^® vector containing the Cap gene was transformed into the Rosetta (DE3) pLysS chemically competent E. coli cells using the heat shock method. In total, 50 μL of transformed E. coli culture was grown on the LB agar plates containing 100 μg/mL of ampicillin and 25 μg/mL of chloramphenicol and incubated overnight at 37°C. The transformants were subsequently incubated at 37°C for 16 h. After that, 1% of overnight bacterial culture was added into the culture media and incubated at 37°C until the optical density (OD₆₀₀) reached 0.5–0.8. Then, the Histidine (His)-tagged rCap protein was induced for expression by isopropyl β-D-1-thiogalactopyranoside (IPTG) (Merck, India) at a final concentration of 1 mM and further incubated for 4 h. The bacterial cell pellets were collected by centrifugation at 9,000 g for 10 min at 4°C and resuspended with 1:20 volume of lysis buffer (50 mM sodium phosphate buffer, 300 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride or PMSF, 20 μg/mL DNAse, 0.2 mg/mL lysozyme, and 0.2% Triton X-100, pH 8.0). The cells were mechanically lysed by sonication at 5 s each with 5 s intervals on ice and centrifuged at 13,000 g for 30 min at 4°C. Thereafter, the pellets were resuspended with 1:10 volume of binding buffer (50 mM sodium phosphate buffer, 300 mM sodium chloride, and 8 M urea, pH 8.0) and centrifuged at 13,000 g for 30 min at 4°C. The supernatant was collected and run through the HisTrap™ high-performance columns (GE Healthcare Life Science, United States) following the manufacturer’s protocol. Finally, the bound protein was eluted with the elution buffer (50 mM sodium phosphate buffer, 300 mM sodium chloride, 300 mM imidazole, and 8 M urea, pH 8.0). The protein concentration in each fraction was measured using Bradford’s assay (17) with bovine serum albumin (BSA) as a standard protein.

The purified CanineCV rCap protein was run on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12% SDS-PAGE). The bands corresponding to the target protein were excised and sent for mass spectrometry to confirm the obtained protein by the NCBIprot database. Proteins with scores over 65 were considered to be statistically significant (p < 0.05). The verification of proteins included assessing peptide matches and the percentage of sequence coverage.

Polyclonal anti-rCap protein was produced with the method described by Watthanadirek et al. (18, 19) and Junsiri et al. (20). In brief, a female New Zealand White rabbit was injected with 500 μg of purified rCap protein mixed with 500 μL of complete Freund’s adjuvant to a final volume of 1,000 μL via a subcutaneous route. Three boosters of 250 μg of purified rCap protein mixed with 500 μL incomplete Freund’s adjuvant to a final volume of 1,000 μL were injected at 2-week intervals via the same route. The whole blood was collected and transferred to clotted blood sterile tubes (BD Vacutainer^®, United States) and then centrifuged at 3,000 g at 4°C for 15 min. The anti-sera against rCap protein were purified by ammonium sulfate precipitation followed by HiTrap Protein G HP columns purification (Sigma-Aldrich, United States) and stored at −20°C until further used. Pre-immunized rabbit sera collected prior to antigen injection served as negative controls in ELISA and Western blot assays.

The ELISA method was assessed rigorously as described by Watthanadirek et al. (18, 19). Briefly, the 96-well plates (Nunc A/S, Denmark) were coated with 50 μL of 2.5 μg/mL of rCap protein diluted in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) at 4°C overnight. The wells were washed three times with washing buffer (0.05% Tween-20 in PBS buffer, PBST) and blocked with 100 μL/well of blocking buffer (1% bovine serum albumin (BSA) in PBST, pH 7.4) for 1 h at 37°C. After washing with the same buffer, 50 μL of serially diluted sera (5-fold dilution, from 1:5 to 1:9,765,625) from rCap protein-immunized rabbit were added triplicate into the wells and incubated at 37°C for 2 h. The plates were washed before adding 50 μL/well of horseradish peroxidase (HRP)-conjugated swine anti-rabbit IgG secondary antibody (Dako, Denmark) at 1:6,000 dilution in blocking buffer and incubated at 37°C for 1 h. After washing, the reaction was detected by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, United State) for 10 min at room temperature, followed by adding 1 N HCl to stop the reaction. The OD value at 450 nm was measured using a microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland). The OD cut-off values were calculated by the mean OD of negative samples plus 3 standard deviations (SD). An OD above this cutoff value was considered positive.

The Western blot was performed as described by Watthanadirek et al. (18, 19). In brief, the purified rCap proteins were run separately by 12% SDS-PAGE and transferred onto nitrocellulose (NC) membranes (GE Healthcare Life Science, United States). The membranes were blocked with blocking buffer (5% BSA in PBST, pH 7.4) for 1 h at room temperature. The NC membranes were incubated in mouse anti-6xHis antibody (Abcam, United Kingdom) at 1:3,000 dilution or purified rabbit anti-CanineCV rCap protein sera at 1:10,000 dilution at 4°C overnight. The blotted membranes were washed with PBST buffer for 5 min at room temperature. Thereafter, the membranes were incubated in HRP-conjugated goat anti-mouse IgG (Agilent Dako, United States) at 1:5,000 dilution or HRP-conjugated mouse anti-rabbit IgG (Santa Cruz Biotechnology, United States) at 1:6,000 dilution for 1 h at room temperature. After washing with the same buffer, the enzymatic reaction was detected by the addition of a substrate 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich, United States) for 5 min at room temperature. Finally, the reaction was stopped by adding distilled water. The CanineCV-infected dog sera (diluted at 1:100) and rabbit pre-immunized sera or CanineCV-uninfected dog sera (diluted at 1:10,000) were used as positive and negative controls, respectively. The HRP-conjugated goat anti-dog IgG (Abcam, United Kingdom) at 1:3,000 dilution and HRP-conjugated mouse anti-rabbit IgG (Santa Cruz Biotechnology, United States) at 1:6,000 dilution were also used.

The cross-reactivity of rabbit anti-CanineCV rCap protein PoAb was assessed with the recombinant Cap proteins of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) received courtesy of Associate Professor Dr. Manakorn Sukmak, Thailand. The proteins were run through 12% SDS-PAGE and subjected to Western blot analysis as mentioned above.

The PCR-positive CanineCV tissue samples of dogs obtained from the previous study (11) were initially tested negative for PCV2 and PCV3 using cPCR. These tissue samples were fixed with 10% buffered formalin and processed into paraffin blocks. The formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut at 4 μm thickness and placed onto positive-charged slides. Then, the sections were deparaffinized and rehydrated with a series of xylene and alcohol concentrations followed by sequential washing with distilled water and PBS. The antigen retrieval was conducted using a citrate buffer (10 mM citrate buffer, pH 6.0) in a microwave for 10 min, followed by another wash with PBS. The endogenous peroxidase within the tissue was neutralized by immersion in 3% hydrogen peroxide for 30 min at 37°C. After a PBS wash, non-specific antigen binding was diminished by incubating with 5% skim milk in PBS at 37°C for 2 h. The sections were then incubated with rabbit anti-rCap IgG PoAb (diluted at 1:3,000) at 4°C overnight in a moist chamber. After washing, the sections were incubated with rabbit-specific Envision polymer (Dako, Germany) for 45 min at room temperature, followed by adding 3,3′-diamenobenzidine (DAB). Finally, the sections were counterstained with Mayer’s hematoxylin.

In this process, a spleen section from a PCR-positive CanineCV-infected dog (10) was used as the positive control, while a lymph node section from a PCR-negative CanineCV but PCR-positive for PCV3 from a pig was employed as the negative control. The IHC universal IgG-negative control (Enzo, ADI-950-231, United States) was used as substitution control, instead of rabbit anti-rCap IgG PoAb. The brown precipitates observed within the cellular structure were interpreted as positive results.

The truncated CanineCV Cap gene with a CACC overhang was primarily amplified by cPCR and then cloned into the pET100/D-TOPO^® vector to construct the recombinant plasmid. The recombinants were successfully transformed into E. coli Top10 cells confirmed by colony PCR and sequencing. As shown in Figure 1, the amplified PCR products from the positive samples were 693 bp in length, which was consistent with the sequencing results. There have not been any bands observed in the negative control.

The CanineCV rCap protein was successfully expressed in the insoluble fraction with a molecular weight (MW) of approximately 31 kDa as shown in Figure 2. Subsequently, it was purified and exhibited a single protein band of the same size. The purified protein was confirmed through mass spectrophotometry. The result revealed that the protein band corresponded to the CanineCV rCap protein (Supplementary Table 1 and Supplementary Figure 1).

To assess the application of the rabbit anti-rCap protein PoAb, the immunoperoxidase technique was employed to detect the distribution of Cap protein in tissues from a PCR-positive CanineCV-infected dog. As presented in Figure 5, specific immunostaining was observed in the white and red pulps of the spleen (Figure 5A). In white pulps, the intense staining was mainly found in the cytoplasm of lymphocytes and macrophages (Figures 5B,C). In addition, brownish staining was observed in the cytoplasm of lymphocytes and macrophages distributed in the splenic red pulps. There was no immunostaining in the substitution control (Figure 5D) and negative control section (Supplementary Figure 2). Further identification of the CanineCV antigen in respiratory-associated organs was conducted. Positive immunostaining was shown in the lung and tracheobronchial lymph nodes (Figure 6). Intense brownish staining was detected in the cytoplasm of pulmonary alveolar macrophages residing in the lung parenchyma (Figures 6A,B), as well as in areas of suppurative inflammation (Figure 6C). Positive stainings were also apparent in the cytoplasm of type 2 pneumocytes (Figure 6D). In the tracheobronchial lymph nodes, intense immunostainings were distinctly found in the cytoplasm of macrophages (Figures 6E,F). In addition to the organ-related respiratory system, positive immunostainings were observed in the small intestine and kidney. In the small intestine section, the brownish stainings were mainly observed within the cytoplasm of mononuclear cells residing in the germinal center of the Peyer’s patch lymphoid follicle in the ileum (Figures 7A,B). A small number of positively stained macrophages and plasma cells were also found in the lamina propria layer (Figures 7C,D). Regarding the kidney section, positive stainings were detected in the renal tubular epithelial cells (Figures 8A–C). No positive signal was observed in the other tissue sections. No immunostaining was observed in the substitution control experiment in lung (Figure 6G), tracheobronchial lymph node (Figure 6H), small intestine (Figure 7E), and kidney (Figure 8D).