Porcine Circovirus

Healthy, 6-week-old, landrace piglets that were free of PRRSV, classic swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2) and M. hyopneumoniae infections were obtained from Beijing Center for SPF Swine Breeding & Management. All piglets were confirmed to be negative for PRRSV and PCV2 antibodies, as determined using commercial ELISA kits and using RT-PCR or PCR for viral nucleic acid detection in sera. The animals were raised in the animal facilities at China Agricultural University (CAU). For the first batch of animal trials, forty-five piglets were randomly divided into nine groups. The animals in each group (n = 5) were separately raised in different isolation rooms. Each piglet in each infection group was intranasally administered with 2 ml of each virus containing 2×10⁵ TCID₅₀ (RvJXwn, RvJH1a, RvJH1b, RvJHSP, RvHB-1/3.9, RvHJ1a, RvHJ1b or RvHJSP). Each piglet in the control group was mock-inoculated with the same dose of MARC-145 cell culture supernatant. For the second batch of animal trials, fifty-five piglets were randomly allotted to eleven groups (n = 5). Each piglet in each infection group was intranasally inoculated with 2 ml of each virus containing 2×10⁵ TCID₅₀ (RvJHn9, RvJHn10, RvJHn9n10, RvJHn9n10n11, RvHJn9, RvHJn10, RvHJn9n10 or RvHJn9n10n11). The piglets in the control group were mock inoculated with 2 ml of MARC-145 cell culture supernatant. All the survived piglets were euthanized and necropsied on day 21 post-inoculation (pi).

Thirty-nine PRRSV strains were used in this study (Table 1). This set of strains included genotype-I (European) isolates from 1991 to 2006 of which some were retrieved from a viral collection (n = 15) and others were freshly isolated from frozen (-80 C) serum (n = 9) or lung tissue (n = 15) that have yielded positive results for PRRSV by RT-PCR. Freshly isolated viruses were from Spain and Portugal and archive viruses were from different countries of Western Europe. No epidemiological relationship was known to exist between the different isolates. Isolation was done in porcine alveolar macrophages (PAM) obtained from two healthy pigs free from all major diseases including PRRSV, pseudorabies virus and classical swine fever virus. Additionally, all PAM batches were tested for porcine circovirus type 2 (PCV2), hepatitis E virus and torque-tenovirus (TTV) according to previously described PCR protocols [14 (link)-16 (link)]. Viral stocks were also tested for mycoplasma by PCR. Viral stocks were produced from passages n = 2, n = 3 or n = 4 in PAM and, for each strain, batches of virus were larger enough to assure that the same batch could be used in all the experiments performed with that isolate, avoiding thus the use of different viral batches of the same strain for different experiments or replicas. Viral titrations were performed by inoculation of serial dilutions of viral stocks in PAM and readings were done by means of the immunoperoxidase monolayer assay using monoclonal antibodies for ORF5 (clon 3AH9, Ingenasa, Madrid, Spain) and ORF7 (clon 1CH5, Ingenasa, Madrid, Spain) using a method reported before with minor modifications [17 (link)].
In order to examine the need for virus viability for the induction of cytokines, three of the isolates were re-tested in parallel before and after inactivation by heat (60°C, 60 min). Complete inactivation was verified by inoculation of the heat-treated viral suspensions in PAM, which were examined at 72 h post-inoculation for the cytopathic effect and presence of PRRSV by IPMA. Untreated viable virus was used to assess the adequateness of the PAM batches for titrations.

Primary porcine airway epithelial cells were isolated from pigs of a local slaughterhouse. PTEC and PBEC were obtained from swine trachea and bronchi, respectively. Primary cells were harvested as previously described18 (link) and were seeded on type I collagen (Sigma)-coated flasks with bronchial epithelial cell serum-free growth medium (BEGM). The BEGM was modified from previous studies28 29 (link) and contained the BEBM basal medium (Lonza) supplemented with the required additives. PTEC and PBEC were transferred to type IV collagen-coated Transwell^® polycarbonate membrane (24 well, 0.4 μm pore size, Corning Costar) at a density of 2.5 × 10⁵ cells per filter support with the air-liquid interface (ALI) medium consisting of a mixture of DMEM (Gibco) and BEBM basal medium (1:1) with additives described previously28 . After PTEC and PBEC reached confluence, the cells were maintained under ALI conditions for at least 4 weeks at 37 °C in a humidified 5% CO₂ atmosphere. Both cultures were validated for porcine specific respiratory tract pathogens including porcine circovirus-2, porcine reproductive and respiratory syndrome virus, porcine cytomegalovirus, porcine influenza A virus, porcine respiratory coronavirus, Mycoplasma hyorhinis and Mycoplasma hyopneumoniae by multiplex Polymerase Chain Reaction (PCR)30 . All PTEC and PBEC used in this study were free from the above mentioned pathogens.

Two commercial farrow-to-finish farms were included in the study based on the willingness of the farmer to participate. On both farms, circulation of M. hyopneumoniae was assumed based on the presence of the pathogen in tracheobronchial swabs (TBS) taken from fattening pigs in the past. Danbred breeding gilts were reared on farm A and purchased on farm B. On farm A, the breeding gilts were vaccinated once against M. hyopneumoniae four weeks prior to first insemination with Ingelvac MycoFLEX^® (Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany). On farm B, breeding gilts were vaccinated with Stellamune^®Mycoplasma (Elanco, Utrecht, The Netherlands) at six months of age upon arrival at the farm and a second time four weeks later. Furthermore, on farm B gilts were also booster vaccinated twice shortly before farrowing. On both farms, sows were not vaccinated against M. hyopneumoniae.
Farm A practiced a 5-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. The piglets were vaccinated at 16 days of age against M. hyopneumoniae with an inactivated whole cell J strain-based bacterin (Ingelvac MycoFLEX^®, Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany), porcine circovirus type 2 (PCV2) (Ingelvac CircoFLEX^®, Boehringer Ingelheim Vetmedica GmbH) and porcine reproductive and respiratory syndrome virus (PRRSV) (UNISTRAIN^® PRRS, HIPRA, Amer, Spain). Pigs were moved to the fattening unit at 9 weeks of age.
Farm B worked in a 4-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. They were vaccinated at 16 days of age against M. hyopneumoniae with the same vaccine as in farm A (Ingelvac MycoFLEX^®, Boehringer Ingelheim Vetmedica GmbH) and PRRSV (UNISTRAIN^® PRRS, HIPRA). The piglets were moved to the fattening unit at 10 weeks of age. On both farms, five breeding animals (two gilts and three sows of mixed parity) were included in the study. The farrowing process was monitored by the main investigator and from each litter, five healthy piglets (birth weight >1 kg) were selected, ear notched and followed up monthly from birth till slaughter (n = 25 piglets / farm). Cross-fostering of the ear notched piglets was not allowed and pigs did not receive antibiotics active against M. hyopneumoniae on both farms during the entire trial.

The presence of PRRSv-specific antibodies (Abs) was assessed by means of two commercially available ELISA kits. The IDEXX PRRS X3 Ab test (ELISA 1) (IDEXX Laboratories, Westbrook, ME, USA) was used to analyze the presence of PRRSv-specific Abs directed against recombinant PRRSv-1 and PRRSv-2 ORF7 antigens and is considered the gold standard for PRRSv Ab testing [4 ]. Additionally, Abs directed against a specific PRRSv-1 antigen-gylcoprotein-rich extract were analyzed using the CIVTEST SUIS PRRS E/S test (ELISA 2) (HIPRA, Amer, Spain) [18 (link)]. The presence of porcine circovirus type 2 (PCV2)-specific Abs was assessed using the Biochek PCV2 Antibody Test (Biochek, Reeuwijk, The Netherlands). All ELISA tests were performed according to the manufacturers’ guidelines, without modifications. All washing steps were performed using the WellWash Versa Microplate Washer (Thermo Fisher Scientific, Waltham, MA, USA). Absorbance at 650 nm (ELISA 1), 450 nm (ELISA 2) and 405 nm (PCV2 ELISA) were measured using the MultiSkan FC MicroPlate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples with a sample-to-positive (S/p) ratio ≥ 0.4 or ≥ 0.5 were considered to be seropositive in ELISA 1 or the PCV2 ELISA, respectively. Samples with a relative index percent (IRPC value) > 20 were considered to be seropositive in ELISA 2.