Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues

Research (Published online: 09-07-2023)

10. Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues

Chew Yee Tan, Kah Chun Lee, Ming-Tang Chiou, Chao-Nan Lin, and Peck Toung Ooi

Veterinary World, 16(7): 1444-1450

Chew Yee Tan: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

Kah Chun Lee: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

Ming-Tang Chiou: Animal Disease Diagnostic Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Chao-Nan Lin: Animal Disease Diagnostic Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Peck Toung Ooi: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

doi: 10.14202/vetworld.2023.1444-1450

Article history: Received: 04-03-2023, Accepted: 29-05-2023, Published online: 09-07-2023

Corresponding author: Peck Toung Ooi

E-mail: ooi@upm.edu.my

Citation: Tan CY, Lee KC, Chiou M, Lin C, and Ooi PT (2023) Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues, Veterinary World, 16(7): 1444-1450.

Abstract

Background and Aim: Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)- labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens.

Materials and Methods: Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282–350).

Results: Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples.

Conclusion: This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Keywords: chromogenic in situ hybridization, in situ hybridization, porcine circovirus type 3, porcine circoviruses, pigs.