Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation
IntroductionDetection 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.MethodsThis st...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-08-01
|
| Series: | Frontiers in Veterinary Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2025.1609897/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849233468813213696 |
|---|---|
| author | Mònica Sagrera Mònica Sagrera Mònica Sagrera Àlex Cobos Àlex Cobos Àlex Cobos Àlex Cobos Laura Garza-Moreno Mónica Pérez Mónica Pérez Mónica Pérez Gema García-Buendía Gema García-Buendía Gema García-Buendía Eva Huerta Eva Huerta Eva Huerta Anna Maria Llorens Anna Maria Llorens Anna Maria Llorens David Espigares Marina Sibila Marina Sibila Marina Sibila Joaquim Segalés Joaquim Segalés Joaquim Segalés |
| author_facet | Mònica Sagrera Mònica Sagrera Mònica Sagrera Àlex Cobos Àlex Cobos Àlex Cobos Àlex Cobos Laura Garza-Moreno Mónica Pérez Mónica Pérez Mónica Pérez Gema García-Buendía Gema García-Buendía Gema García-Buendía Eva Huerta Eva Huerta Eva Huerta Anna Maria Llorens Anna Maria Llorens Anna Maria Llorens David Espigares Marina Sibila Marina Sibila Marina Sibila Joaquim Segalés Joaquim Segalés Joaquim Segalés |
| author_sort | Mònica Sagrera |
| collection | DOAJ |
| description | IntroductionDetection 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.MethodsThis 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).ResultsA 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).DiscussionThese 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. |
| format | Article |
| id | doaj-art-52c72c9f27b54b4b83098a10d851ae0c |
| institution | Kabale University |
| issn | 2297-1769 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj-art-52c72c9f27b54b4b83098a10d851ae0c2025-08-20T05:32:26ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692025-08-011210.3389/fvets.2025.16098971609897Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisationMònica Sagrera0Mònica Sagrera1Mònica Sagrera2Àlex Cobos3Àlex Cobos4Àlex Cobos5Àlex Cobos6Laura Garza-Moreno7Mónica Pérez8Mónica Pérez9Mónica Pérez10Gema García-Buendía11Gema García-Buendía12Gema García-Buendía13Eva Huerta14Eva Huerta15Eva Huerta16Anna Maria Llorens17Anna Maria Llorens18Anna Maria Llorens19David Espigares20Marina Sibila21Marina Sibila22Marina Sibila23Joaquim Segalés24Joaquim Segalés25Joaquim Segalés26IRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainCeva Salud Animal, Avenida Diagonal, Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainDepartament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, Barcelona, SpainCeva Salud Animal, Avenida Diagonal, Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainCeva Salud Animal, Avenida Diagonal, Barcelona, SpainIRTA, Animal Health, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainUnitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, SpainWOAH Collaborating Center for Research and Control of Emerging and Re-emerging Pig Diseases (IRTA-CReSA), Barcelona, SpainDepartament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, Barcelona, SpainIntroductionDetection 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.MethodsThis 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).ResultsA 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).DiscussionThese 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.https://www.frontiersin.org/articles/10.3389/fvets.2025.1609897/fullporcine circovirus 2in situ hybridisationqPCRRNAScope®digital pathology |
| spellingShingle | Mònica Sagrera Mònica Sagrera Mònica Sagrera Àlex Cobos Àlex Cobos Àlex Cobos Àlex Cobos Laura Garza-Moreno Mónica Pérez Mónica Pérez Mónica Pérez Gema García-Buendía Gema García-Buendía Gema García-Buendía Eva Huerta Eva Huerta Eva Huerta Anna Maria Llorens Anna Maria Llorens Anna Maria Llorens David Espigares Marina Sibila Marina Sibila Marina Sibila Joaquim Segalés Joaquim Segalés Joaquim Segalés Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation Frontiers in Veterinary Science porcine circovirus 2 in situ hybridisation qPCR RNAScope® digital pathology |
| title | Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation |
| title_full | Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation |
| title_fullStr | Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation |
| title_full_unstemmed | Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation |
| title_short | Automated pixel-based quantification of porcine circovirus 2 genome in formalin-fixed, paraffin-embedded tissues using in situ hybridisation |
| title_sort | automated pixel based quantification of porcine circovirus 2 genome in formalin fixed paraffin embedded tissues using in situ hybridisation |
| topic | porcine circovirus 2 in situ hybridisation qPCR RNAScope® digital pathology |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2025.1609897/full |
| work_keys_str_mv | AT monicasagrera automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT monicasagrera automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT monicasagrera automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT alexcobos automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT alexcobos automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT alexcobos automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT alexcobos automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT lauragarzamoreno automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT monicaperez automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT monicaperez automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT monicaperez automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT gemagarciabuendia automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT gemagarciabuendia automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT gemagarciabuendia automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT evahuerta automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT evahuerta automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT evahuerta automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT annamariallorens automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT annamariallorens automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT annamariallorens automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT davidespigares automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT marinasibila automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT marinasibila automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT marinasibila automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT joaquimsegales automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT joaquimsegales automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation AT joaquimsegales automatedpixelbasedquantificationofporcinecircovirus2genomeinformalinfixedparaffinembeddedtissuesusinginsituhybridisation |