Tissues collected during necropsy were

analyzed by IHC for the presence of PCV2 antigen. All pigs were weighed on the day of arrival, vaccination and challenge and at necropsy. The average daily weight gain was calculated before (−28 to 0 dpc), after challenge (0 to 21 dpc), and for the entire study period (−28 to 21 dpc). In addition, all animals were examined daily for signs of illness such as: lethargy, respiratory signs, inappetance and lameness. The pigs were vaccinated at −28 dpc with 2 mL of an experimental live-attenuated chimeric PCV2 vaccine with an ORF2 based on the PCV2a subtype (PCV1-2a) as previously described (37, 39) at a titer of 1.6 × 103 TCID50 per mL.

This is the same titer as was used for the inactivated version of the chimeric PCV2 vaccine (Suvaxyn PCV, Fort Dodge Animal Health). For the IDH inhibitor IM route of vaccination, 2 mL of the experimental PCV1-2a vaccine was injected into the right side of the neck using a 0.7 mm × 25.4 mm needle and a 3 mL syringe. For the PO route of vaccination, each pig was held in an upright position and the experimental vaccine administered by slowly dripping Ibrutinib purchase 2 mL into their mouths using a 3 mL syringe. The volume of vaccine dose for both IM and PO routes (2 mL) was chosen on the basis of what is routinely used and convenient for vaccinating pigs in the field. The PCV2b isolate NC-16845 was propagated on PK-15 cells to produce a virus stock at an infectious dose of 2.5 × 103.0 TCID50 per mL, which was used to challenge the pigs. At dpc 0, each pig in the PCV2-challenged

groups (Table 1) received 1 mL of the virus inoculum IM into the right neck area and 3 mL (1.5 mL per nostril) intranasally by holding the pig in the upright position and administering the inoculum by slowly dripping 1.5 mL into each nostril using a 3 mL syringe. Porcine reproductive and respiratory syndrome virus isolate ATCC VR2385 (44, 45) was propagated on MARC-145 cells to produce an infectious stock with a titer of 1 × 105.0 TCID50 /mL. At dpc 0, each pig in the PRRSV-challenged groups (Table 1) received 2.5 mL of the PRRSV challenge virus inoculum intranasally Glycogen branching enzyme in a similar fashion to that described for PCV2 inoculation. All serum samples from all groups were tested for anti-PCV2-antibodies using the SERELISA PCV2 Ab Mono Blocking kit (Synbiotics Europe, Lyon, France) according to the manufacturers’ instructions. The results were expressed as a SNc ratio, samples being considered negative if the SNc ratio was > 0.50 and positive if it was ≤ 0.50. Serum samples collected at −28, 0 and 21 dpc were tested for the presence of anti-PRRSV antibodies by ELISA (HerdChek PRRS virus antibody test kit 2XR, IDEXX Laboratories).