Source of virus isolate
In January 2016, organ samples of 10 week-old piglets were submitted for diagnostic investigation to our lab from a 1500 sow farrow-to-finish farm, having a clinical history of ill thrift and uneven condition. Necropsy was performed at the farm which revealed wasting, bronchopneumonia and chronic pleurisy. PCV2 was suspected as predisposing agent for subsequent secondary infections. To detect and isolate the virus, tissue samples of lungs and lymph nodes were collected.
For virus isolation, 10% v/w tissue homogenate of the mediastinal lymph nodes was prepared in PBS. After clarification by low speed centrifugation (1000 rpm for 10 min), 100 μL of the supernatant was inoculated onto 106cell/T25 PK-IV-D56 cells. The inoculated cells were incubated at 37 °C with 5% CO2 and passaged at a 1 to 4 dilution rate four times, at 4–5 days intervals. After the last passage, the cell cultures were frozen at -20 °C. After three freeze-thaw cycles, the culture medium was collected and centrifuged at 3000 rpm for 10 min. The resulting supernatants were aliquoted. The identity of the isolate was confirmed during passages by comparing the nucleotide sequence of the passaged virus to the nucleotide sequence of the original virus detected in the lymph nodes.
The lymph node samples were tested for the presence of PCV2 , PCV3 , PRRSV (ADI132–100-ADIAVET PRRSV EU/NA REAL TIME PCR kit) and M. hyopneumoniae . The purity of the PCV2 isolate after the passages was tested for the presence of Mycoplasmas, porcine parvoviruses, PCV-1 and PRRSV (EU1, EU2 and NA subtypes) by Vet-Med-Labor Ltd. (Budapest, Hungary).
The presence and level of virus in the tissue culture supernatant during the isolation process was monitored by qPCR .
Challenge inoculum preparation
To prepare inoculum for the challenge experiment, the virus was passaged nine times as described above.
Nucleotide sequencing, phylogenetic analysis
The whole genome of isolate D3276/5/16HU; (GenBank accession number: MG833033) was amplified using CBB1, CBB2, CBB3, and CSZ2 primers . The nucleotide sequence was determined by Biomi Ltd. (Gödöllő, Hungary). Related PCV2 DNA sequences were obtained from NCBI GenBank using BLAST. DNA sequences were aligned and phylogenetic analyses were performed using MEGA 7.0 software .
Pre-screening of the piglet source herd
The piglets (Seghers breed) originated from a farrow-to-finish farm in Szabolcs-Szatmár-Bereg county (Hungary). No PCV2 vaccination was used at the facility because it was considered a “low PCV2 pressure” farm. Furthermore, the farm was free of PRRSV, M. hyopneumoniae, and influenza A viruses according to regularly performed monitoring by PCR and serology. Before selecting the piglets for the study (at two weeks of age), sows and their litters were tested for viremia and fecal shedding of PCV2 (see details below).
Piglets and housing
Twenty-two, 15–17 day old piglets of either gender, originating from sows found negative for PCV2 viremia and fecal shedding were transported to the trial facilities and kept on straw bedding in a pen. The piglets were fed according to their age and provided water ad libitum throughout the trial.
Experimental challenge infection and sampling
Serological testing was performed at 3 weeks of age to check the level of residual MDA to PCV2, then right before challenge, and at weekly intervals for 4 weeks post-challenge. The pigs were challenged at ten weeks of age by administering 3 mL of challenge virus (103.1TCID50/100 μL) into each nostril (6 mL/pig).
The applied challenge dose (1X103.1TCID50/100 μL; 6 ml/pig) was in line with those published in the literature, i.e., 3 mL (1X103.55TCID50/pig) and 3 mL (1X104.7TCID50/pig), ; 2 mL of 104.5TCID50/mL/pig .
The pigs were observed for 4 weeks for clinical signs and sampled weekly for fecal shedding, viremia, and isotype specific humoral immune responses.
At the end of the trial, the pigs were slaughtered at an abattoir. Swab and serum samples were collected. At the same time, mesenteric and mediastinal lymph nodes were collected for viral load measurements. Approximately 10 mg of the lymph nodes were processed for qPCR.
The identity of the challenge virus was checked by nucleotide sequence analysis of the detected PCV2 originating from separate pigs and comprising of pre-challenge serum samples, mediastinal and mesenteric lymph node samples collected at the slaughterhouse at the end of the trial.
For the screening of the sows providing the piglets for the study, SERELISA PCV2 Ab Mono Blocking kit (Zoetis) was used according to the manufacturer’s instructions. The immune response of the pigs to the challenge infection was measured by using the following assays:
1) SERELISA PCV2 Ab Mono Blocking kit; 2) BioChek Porcine Circovirus type 2 Antibody Test kit; 3) INGEZIM CIRCO IgG 11.PCV.K1; 4) INGEZIM Circovirus IgM/IgG ELISA kit (INGENASA), and 5) a modified virus neutralization assay (see below). The first three ELISAs and the VN assa were used on serum samples collected at 3, 10 (pre-challenge), and 14 weeks of age (4 weeks post challenge) while the INGEZIM IgM/IgG ELISA was used only to test the serum samples collected during the post-infection period in order to monitor the Ig isotype specific responses upon challenge.
In the VN assay the above-mentioned sera were checked for their ability to neutralize the PCV2b Rm strain (Ceva-Phylaxia Ltd.), which replicates more readily on cell culture than the PCV2d-2 isolate. Serial two-fold dilutions (in the range of 10–20,480X) were prepared from the sera and 500 μL of each serum sample was incubated with 500 MOI of PCV2/well on a 96-well plate (Falcon) for 1 h at 37 °C. Then, 105 PI-IV-D56 cells (a PK-15 derived cell line) were added to each well and incubated for 5 days at 37 °C. The plates were frozen at -20 °C overnight before reading. The reading was performed by an antigen detecting sandwich ELISA. Briefly, the plates were coated with monoclonal antibody 36F1 (INGENASA, specific to VP2 of PCV2), incubated overnight at 4 °C, and then washed with PBS. Next, 50 μL of supernatant from each well of the neutralization test plates and 50 μL of PBTN (PBS with 0.05% Tween 20 and 0.02% sodium azide) was added and incubated for 1.5 h at 37 °C. Then the conjugate (INGENASA Anti PCV2 36F1 conjugated with 60 M biotin) was added and incubated for 1 h at 37 °C. Next, streptavidin peroxidase was added, followed by adding a mixture of tetramethyl-benzidine and hydrogen peroxide as substrate. The reaction was stopped by the addition of sulphuric acid and the absorbance was measured at 450 nm. Samples with an optical density (OD) > 2X the mean OD of a cell control were considered positive. The titer of the tested serum sample was determined by using the Spearman-Kärber method .
Quantitative real-time PCR
Sera, rectal swabs, mediastinal and mesenteric lymph nodes of the experimentally infected pigs were tested by quantitative PCR (qPCR). Viral DNA was extracted by the QIAamp 96 DNA QIACube HT Kit with QIACube HT device (Qiagen) according to the manufacturer’s instructions. The qPCR assays were carried out according to Brunborg et al.  using QuantiNova Probe PCR Kit in a Rotor-Gene Q instrument (Qiagen). PCV2 virus suspension of known titer and a reference plasmid containing PCV2 sequences were used to quantify PCV2 virus load. For each reaction, the base-line and cycle threshold (Ct) number was determined automatically. A positive cut-off value of 36.7 Ct was determined. The PCV2 copy number of the samples was calculated using a 10-fold dilution series of a stock solution of a plasmid DNA containing PCV2 DNA sequences.
The sensitivity and specificity of each ELISA kit used were calculated by comparing the results to the VN results, considered as reference of true positivity.
Regression analysis was performed between the VN and ELISA titers, the serological data and post-challenge viremia or virus excretion values (Ct), and the virus content of the mesenteric and mediastinal lymph nodes by using the STATGRAPHICS Centurion XVI (version 16.2.04) and Microsoft Excel software programs.