The WuHan strain of PCV2 (GenBank: FJ 598044) was used to prepare both the vaccine and the challenge inoculum for the animals. The virus was propagated in porcine kidney cells (PK-15) free of PCV1 contamination and grown in Minimal Essential Medium (MEM) containing 10% fetal bovine serum. The titer of the virus stock was 107 TCID50/ml.
The PCV2 was produced according to the methods described by Tischer et al.
, with minor modifications. Briefly, the PCV2 WuHan strain was inoculated into PK-15 cells by one-step inoculation and incubated at 37 °C with 5% CO2 in MEM containing 10% fetal bovine serum. One day after seeding, the medium was removed, and the cultures were incubated with 300 mM D-glucosamine in Hank’s salt solution (HSS) for 30 min at 37°C in a volume sufficient to cover the monolayer. Thereafter, the treated PK-15 cell cultures were incubated in growth medium for two additional days in MEM containing 2% fetal bovine serum at 37°C with 5% CO2. The inoculated cells were frozen and thawed three times. After centrifugation at 5000 × g for 20 min, the supernatant was titered and then inactivated by incubation with formalin at a 0.3% final concentration for 48 hours at 37°C. Virus inactivation was confirmed by the inoculation of the formalin-treated samples into PK-15 cells. The formalin in the samples was then neutralized by the addition of one part 0.2% (w/v) sodium metabisulfite to 100 parts of vaccine. The titer of the virus suspension prior to inactivation was 107 TCID50/ml. The inactivated viral suspension was then mixed with an oil adjuvant (MARCOL 52, EXXonMobile, USA) at an appropriate ratio.
Animals and housing
Ten healthy, weaned, three-week-old crossbred piglets were obtained from a pig farm that was negative for PCV2 and PRRSV infections. The selected animals were transported to the animal facilities at the Huazhong Agricultural University and allowed to acclimatize for seven days before the PCV2 vaccination. All experimental protocols were approved by the Research Ethics Committee of College of Veterinary Medicine, Huazhong Agricultural University, Hubei, China (No. 2009–0012). The animals were randomized into two groups with five pigs each and raised separately in two isolation rooms with individual ventilation. The pigs were provided commercial diets and water ad libitum. During that period, the animals were weighed and bled to assess their serological and virological status to PCV2, and all piglets were confirmed to have neither serological nor virological evidence of previous exposure to PCV2 prior to the vaccination (data not shown).
Experimental design and sample collection
After arriving at the animal facilities, the piglets were given an identity and distributed into two groups with stratification by their weight, namely Group 1 and Group 2. The animal care was provided under an Institutional Animal Care and Use Committee approved protocol. At 4 weeks of age, each piglet in Group 1 was intramuscularly injected with 2.0 ml of prepared inactivated PCV2 vaccine on the right side of the neck, whereas each piglet in Group 2 received 2.0 ml of sterile PBS as a placebo. Next, four weeks after the immunization (8 weeks of age), all pigs were challenged with 5 ml (2.5 ml intranasally and 2.5 ml intramuscularly) of the WuHan strain of PCV2 at a dose of 107 TCID50/ml. After the inoculation, the pigs were monitored for 28 days. During this period, the pigs were clinically examined, and rectal temperatures were recorded on a daily basis. Body weight was measured before the immunization, at challenge and at the time of necropsy. The relative daily weight gains (expressed as daily weight gains/primary body weight, RDWG) were determined. Blood samples were collected from the vena cava at the time of the vaccination and at weeks 1, 2 and 3 post-vaccination (PV), at the time of challenge and on a weekly basis thereafter. Sera were obtained and stored at −80°C until the serological and virological test were performed. Twenty-eight days after the challenge, the animals were euthanized with an intravenous overdose of sodium pentobarbital, and a complete necropsy was performed. All tissue collection procedures were performed according to the protocols approved by the Hubei Province PR China for Biological Studies Animal Care and Use Committee. Macroscopic and microscopic lesions were compared between the groups. The amount of PCV2 antigen in the lymphoid tissues was determined by immunohistochemistry (IHC).
Quantitative real-time PCR for evaluation of viremia
DNA extraction from the serum samples collected on the day of challenge and on DPC 7, 14, 21 and 28 was performed using the E.Z.N.A.TM Viral DNA Kit (OMEGA, USA) according to the manufacturer’s instructions. The DNA was used to quantify the PCV2 genomic DNA copy numbers by real-time PCR. GenBank entry FJ598044 was used for the primer and probe design. The Cap gene region (corresponding to nucleotides 1033–1734 bp of the whole PCV2 genome)
[19, 29, 30] was chosen for the primer and probe design because it has a lower nucleotide homology with porcine circovirus type 1 (PCV1) than ORF1 (~65%)
. The forward (5'-CCAGGAGGGCGTTCTGACT-3') and reverse (5'-CGTTACCGCTGGAGAAGGAA-3') primers and probe (5'-AATGGCATCTTCAACACCCGCCTCT-3') were designed using the Primer Express v.1.5 software (ABI Prism 7500 User’s Guide, Applied Biosystems, Foster City, CA, USA). The primers and probe were selected to work under universal conditions (ABI Prism 7500 User’s Guide, Applied Biosystems, USA). The probe was labeled on the 5' end with FAMTM (6-carboxyfluorescein) and on the 3' end with TAMRATM (6-carboxytetramethylrhodamine). The PCR reaction contained a final concentration of 1× THUNDERBIRD Probe qPCR Mix (TOYOBO, Japan), 0.3 μM each primer, 0.2 μM Taqman Probe, 0.04 μl of 50× ROX reference dye and DNA equivalent to that of 1 ml serum as the template. Autoclaved nanopure water was added to bring the final volume to 25 μl. All reactions were conducted in triplicate on an ABI7500 (Applied Biosystems). The PCR program consisted of one cycle of 95°C for 1 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. For a standard curve, serial dilutions of plasmid pORF2 (the ORF2 gene cloned into the pMDTM 18-T Vector) were used to quantify the virus genomic copy number. The numbers of virus copies for each sample were presented as the mean value of triplicate reactions.
Cap protein-specific antibodies were determined with an endpoint ELISA using the recombinant Cap protein antigen as described previously
. The titers were expressed as the reciprocal of the highest dilution of sera producing ratio values of 2:1.
Formalin-fixed, paraffin-embedded tissue samples were cut into 4 μm thick slices, stained with hematoxylin-eosin and examined for lesions compatible with PMWS. The sections for histopathological examination were taken from lung and lymphoid tissues, including the lymph nodes (mesenteric and superficial inguinal), tonsil, and spleen. The tissues were examined in a blinded fashion and given a subjective score for the severity of the lesions. The lung scores ranged from 0 (normal) to 3 (severe lymphohistiocytic interstitial pneumonia). Lymphoid tissues were evaluated for the presence of lymphoid depletion, ranging from 0 (normal) to 3 (severe), and for histiocytic inflammation and the replacement of follicles, ranging from 0 (normal) to 3 (severe)
. The overall microscopic lymphoid lesion score was the average of the five studied tissues (lung, tonsil, spleen, and the mesenteric and superficial inguinal lymph nodes).
IHC for the detection of PCV2-specific antigens was performed on lymphoid tissues, including lymph nodes (mesenteric and superficial inguinal), tonsil, and spleen collected during the necropsy at 28 DPC. A rabbit polyclonal antiserum against PCV2 was used for IHC following procedures described previously
. The amount of PCV2 antigen distributed in the tissues was scored in a blinded fashion by assigning a score ranging from 0 for no signal to 3 for a strong positive signal. The mean group score was determined for each tissue and compared between groups.
The statistical analysis was performed by one-way analysis of variance using SPSS version 17.0 (SPSS). The results were considered to be statistically significant for P < 0.05.