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. 2025 Aug 20:12:1609897.
doi: 10.3389/fvets.2025.1609897. eCollection 2025.

Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation

Mònica Sagrera #  1   2   3 , Àlex Cobos #  1   2   4   5 , Laura Garza-Moreno  3 , Mónica Pérez  1   2   4 , Gema García-Buendía  1   2   4 , Eva Huerta  1   2   4 , Anna Maria Llorens  1   2   4 , David Espigares  3 , Marina Sibila  1   2   4 , Joaquim Segalés  2   4   5
Affiliations

Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation

Mònica Sagrera et al. Front Vet Sci. .

Abstract

Introduction: Detection of porcine circovirus 2 (PCV2) in lymphoid tissues is essential for diagnostic and research purposes. In situ hybridisation (ISH) enables the localisation of viral genomes in tissue sections but is traditionally assessed visually, which may introduce subjectivity.

Methods: This study developed an automated pixel classifier to quantify the PCV2 genome using RNAscope® ISH technology. Four lymphoid tissues (tonsils and tracheobronchial, mesenteric, and superficial inguinal lymph nodes) from 66 experimentally inoculated pigs were analysed. PCV2 labelling was assessed both visually (scores 0-3) and digitally (percentage of labelled area).

Results: A strong correlation was observed between visual and digital ISH scoring (ρ = 0.96), allowing the definition of digital thresholds corresponding to visual scores. Among all tissues, TBLN exhibited the highest PCV2 labelling. This tissue was further evaluated by PCV2 quantitative polymerase chain reaction (qPCR), showing a high correlation with digital ISH results (ρ = 0.85).

Discussion: These findings demonstrate the reliability of digital pathology tools for objective quantification of PCV2 in lymphoid tissues. Automated scoring enhances consistency, reduces observer bias, and improves diagnostic efficiency in PCV2 research and surveillance.

Keywords: RNAScope®; digital pathology; in situ hybridisation; porcine circovirus 2; qPCR.

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Conflict of interest statement

MoS, LG-M, and DE were employees of Ceva Salud Animal. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Representative images of PCV2 ISH staining (left column) and the corresponding automated pixel classification analysis (right column). Examples of ISH visual scores (0–3) are shown alongside their respective digital scores (% of labelled area). Each pink dot (left) and red dot (right) represents a PCV2 genomic copy. ISH: in situ hybridisation; PCV2: porcine circovirus 2.
Figure 2
Figure 2
Spearman’s rank correlation between PCV2 in situ hybridisation visual score (0–3) and digital score (labelled area expressed in percentage) for (A) tonsil (TO), (B) tracheobronchial lymph node (TBLN), (C) mesenteric lymph node (MSLN), and (D) inguinal superficial lymph node (ISLN). A single asterisk (*) indicates that Spearman’s correlation coefficient is statistically significant (p < 0.05). The colour of the points corresponds to the visual score, ranging from light blue (0) to dark blue (3), to facilitate the visual interpretation of the graph. PCV2: porcine circovirus 2.
See this image and copyright information in PMC

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