PCV2b and PRRSV experimental challenge, data, and sample collection

Animals were challenged with PCV2b (strain UNL2014001) following weaning and passive PCV2 IgG antibody decay, via intramuscular injection (1 ml) and intranasal spray (1 ml in both nostrils) with a titer of 10^4.0 TCID50/ml as previously described (McKnite et al., 2014). After 7 d, the animals were subsequently co-infected with PRRSV (strain FL12) via intramuscular injection (2 ml) with a titer of 10^5.0 TCID50/2 ml. The virulence of these strains has been previously described (Truong et al., 2004; Engle et al., 2014; McKnite et al., 2014).

Animal weights were recorded at 0, 7, and 17 d, and blood was collected at 0, 4, 7, 11, 14, and 17 d after PCV2b challenge. Serum was obtained through centrifugation of whole blood samples at 2,400 × g for 30 min at 4 °C. Viral DNA was isolated from serum for the detection of PCV2b using the QIAamp 96 DNA Blood Kit (Qiagen) according to the manufacturer’s protocol with minor modifications for enhanced binding of viral nucleic acids. Viral RNA was isolated from serum for the detection of PRRSV utilizing the MagMAX-96 Viral RNA Isolation kit (Applied Biosystems) and MagMAX Express Magnetic Particle Processor (Applied Biosystems) according to the manufacturer’s protocol. PRRSV cDNA was obtained from isolated RNA using the First-Strand cDNA Synthesis Kit (Cytiva) according to the manufacturer’s protocol.

At the end of the experiment, 54 piglets, equally representing both SYNGR2 homozygote genotypes, (Arg/Cys 27/27) were euthanized for necropsy. The right cranial lung lobe, right middle lobe, right caudal lobe, accessory lobe of right lung, cranial aspect of the left cranial lobe, caudal aspect of the left cranial lobe, and left caudal lobe were grossly scored for discoloration and consolidation. The scores represent the percentage, by volume, of each lung lobe grossly affected following infection. Three samples of lung from the same region of each pig and a tracheobronchiolar lymph node were fixed in 10% neutral buffered formalin and routinely processed overnight on a Tissue-Tek VIP 5 Tissue Processor. The resultant tissue pieces were embedded in paraffin blocks and cut to 5 µm thickness using a Leica RM2255 microtome and stained with hematoxylin and eosin on a Leica ST5020 H&E stainer, and coverslipped with a Leica CV5030 coverslipper.

Lung and tracheobronchiolar lymph node lesions were histologically graded by a board certified veterinary anatomic pathologist using a previously described grading scale ­(Krakowka et al., 2005). Lung scoring was done using a subjective grading scale from 0 to 4 to assess the severity of interstitial inflammation in the lung sections. A grade of 0 was assigned to normal lungs, a grade of 1 was assigned to lungs with minimal interstitial pneumonia, a grade 2 was assigned to lungs with moderate interstitial pneumonia in multifocal regions, a grade 3 was assigned to lungs with severe interstitial pneumonia that had a multifocal to coalescing pattern, and a grade 4 was assigned to lungs with severe interstitial pneumonia that diffusely affected the tissue. Tracheobronchiolar lymph node sections were scored with a grading scale from 0 to 4 in severity as described in Krakowka et al. (2005). A grade of 0 was assigned to normal lymph nodes, a grade of 1 was assigned to minimal lesions, a grade 2 was assigned to moderate lesions, a grade 3 was assigned to severe lesions, and a grade 4 was assigned to very severe lesions that were diffuse. Lesions in the lymph node were assessed for lymphoid depletion, lymphoid hyperplasia, germinal center development, reticuloendothelial hyperplasia, and histiocytic and giant cell infiltration.