Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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A standardized method to study immune responses using porcine whole blood

  • Sameer-ul-Salam Mattoo (Korea Zoonosis Research Institute) ;
  • Ram Prasad Aganja (College of Veterinary Medicine, Jeonbuk National University) ;
  • Seung-Chai Kim (College of Veterinary Medicine, Jeonbuk National University) ;
  • Chang-Gi Jeong (College of Veterinary Medicine, Jeonbuk National University) ;
  • Salik Nazki (The Pirbright Institute) ;
  • Amina Khatun (Department of Veterinary Pathology, Faculty of Animal Science and Veterinary Medicine, Sher-e-Bangla Agricultural University) ;
  • Won-Il Kim (College of Veterinary Medicine, Jeonbuk National University) ;
  • Sang-Myeong Lee (College of Veterinary Medicine, Chungbuk National University)
  • Received : 2022.08.20
  • Accepted : 2022.11.08
  • Published : 2023.01.31
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Abstract

Background: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment. Objectives: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses. Methods: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV). Results: The frequency of IFN-γ+CD3+ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ+ CD4-CD8+ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples. Conclusions: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

Keywords

Acknowledgement

This research was funded by a grant from "BioGreen21 Agri-Tech Innovation Program (Award number: PJ01561102, Recipient: Won-Il Kim)" of Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs of Korea and National Research Foundation of Korea (NRF) grant (Award number: 2019R1A2C2010432, Recipient: Sang-Myeong Lee) funded by the Korea Government (MIST).

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