Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues
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 h...
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| Format: | Article |
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Veterinary World
2023-07-01
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| Series: | Veterinary World |
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| Online Access: | https://www.veterinaryworld.org/Vol.16/July-2023/10.pdf |
| _version_ | 1827878115253157888 |
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| author | Chew Yee Tan Kah Chun Lee Ming-Tang Chiou Chao-Nan Lin Peck Toung Ooi |
| author_facet | Chew Yee Tan Kah Chun Lee Ming-Tang Chiou Chao-Nan Lin Peck Toung Ooi |
| author_sort | Chew Yee Tan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T17:47:36Z |
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| id | doaj.art-80e4845664624bb2afc3cf3169332335 |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-03-12T17:47:36Z |
| publishDate | 2023-07-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj.art-80e4845664624bb2afc3cf31693323352023-08-03T13:28:18ZengVeterinary WorldVeterinary World0972-89882231-09162023-07-011671444145010.14202/vetworld.2023.1444-1450Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissuesChew Yee Tan0https://orcid.org/0000-0002-2674-3959Kah Chun Lee1https://orcid.org/0009-0006-3780-1524Ming-Tang Chiou2https://orcid.org/0000-0002-6500-8367Chao-Nan Lin3https://orcid.org/0000-0003-1911-7535Peck Toung Ooi4https://orcid.org/0000-0002-1174-0288Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.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.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.Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.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.https://www.veterinaryworld.org/Vol.16/July-2023/10.pdfchromogenic in situ hybridizationin situ hybridizationporcine circovirus type 3porcine circovirusespigs |
| spellingShingle | Chew Yee Tan Kah Chun Lee Ming-Tang Chiou Chao-Nan Lin Peck Toung Ooi Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues Veterinary World chromogenic in situ hybridization in situ hybridization porcine circovirus type 3 porcine circoviruses pigs |
| title | Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| title_full | Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| title_fullStr | Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| title_full_unstemmed | Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| title_short | Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| title_sort | chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues |
| topic | chromogenic in situ hybridization in situ hybridization porcine circovirus type 3 porcine circoviruses pigs |
| url | https://www.veterinaryworld.org/Vol.16/July-2023/10.pdf |
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