TDK conference 2011

The aim of the study was to characterize immunohistochemically the lung lesions after a challenge with a recently isolated PRRSV field strain in growing pigs. Methods: 9-week-old PRRSV negative pigs were challenged with 2.2 × 105 TCID50 of a Type 1, subtype 1 virulent PRRSV field isolate. Negative control pigs were inoculated with virus free cell culture supernatant. Animals were euthanized on 10 DPI (n=7) and 21 DPI (n=5). Lung lesions were compared to age matched pigs of the non-infected control group.

In the first phase of the study lung lesions were evaluated on routine HE stained slides. The microscopic evaluation of the lung lesions was performed as a blinded analysis and the lesions were scored based on the following criteria: (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intraalveolar necrotic debris, (4) intraalveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation.

For further characterization of the lung lesions, immunohistochemical stainings were performed using anti-cytokeratin, anti-Ki67, anti-TTF-1 (Thyroid Transcription Factor-1) and anti-myeloid receptor (MAC387) antibodies to identify alveolar epithelial cells, proliferating cells, type II pneumocytes, and macrophages, respectively. In case of Ki67, TTF-1 and MAC387 the labelled cells were counted in 50 non-overlapping and consecutively selected high magnification fields of 0.20 mm2. R software was applied to carry out statistical analyses.

The evaluation of the immunohistochemical stainings revealed that humanized anti TTF-1 antibodies can succesfully identify type II pneumocytes in porcine lung tissues. Marked proliferation of these cells was confirmed by a significant (p<0.05) increase of TTF-1 positive cells in acute cases compared to the lungs of control pigs. Upregulation of Ki67 and MAC387 positive cells was also observed, however due to the relative low number of the sample animals and high values of standard deviation, the increase of these values were found not to be statistically significant. Cytokeratin labelling marked the type I, and type II pneumocytes as well as bronchial epithelial cells, however this staining was not suitable for cell counting purposes.

When the routine histological scores were compared to the number of immunohistochemically positive cells, Ki67 cell counts were found to show positive correlation (p<0.05) with the overall severity of the lesions.