# Bright-Field Multiplex Immunohistochemistry in Swine PCV2 and PRRSV Lymphadenopathies

## TL;DR

This study uses multiplex immunohistochemistry to examine lymph node changes in swine infected with PCV2 and PRRSV, revealing distinct patterns compared to normal lymphoid tissue.

## Contribution

The study introduces bright-field multiplex immunohistochemistry as a novel method to visualize and compare lymphocyte distributions in swine lymphadenopathies caused by PCV2 and PRRSV.

## Key findings

- PCV2-affected lymph nodes showed reduced follicles and fewer CD20+ and CD3+ cells in follicles and interfollicular areas.
- PRRSV-associated lymphadenopathy showed increased CD20+ cells in follicles and interfollicular regions.
- Multiplex immunohistochemistry preserves spatial information and enables detailed analysis of cellular interactions in disease states.

## Abstract

Single-label immunohistochemistry (sIHC) is used in infectious disease studies to detect etiological agents, and to quantify and spatially localize antigens in lesions. While sIHC is limited to staining one antigen per tissue section, multiplex immunohistochemistry (mIHC) offers significantly more detailed information and is more cost- and time-efficient than repeated sIHC. Given that swine lymph node pathology involves variations in T and B lymphocytes and macrophages, this study aims to apply mIHC to visualize simultaneously and on the same slide their spatial distribution and variation across the three main regions of the swine lymph node: follicles, interfollicular areas and medulla-like tissue. We demonstrated the use of bright-field mIHC for in situ analysis of spatial cellular distribution in swine lymph nodes during PCV2-SD and PRRSV lymphadenopathy, revealing distinct lymphocyte distributions compared to reactive lymphoid hyperplasia. The study highlights the potential of mIHC as a valuable tool for understanding and studying disease dynamics.

Multiplex immunostaining (mIHC) allows the simultaneous detection of multiple antigenic targets within the same tissue section, providing a deeper understanding of spatial variation in cellular distribution. The aim of the present study is to apply this technique to examine the spatial variation of lymphocyte populations in swine lymph nodes during PCV2-SD and PRRSV lymphadenopathy compared with reactive lymphoid hyperplasia. A triple immunohistochemical stain with CD3, CD20 and IBA1 antibodies for the concurrent detection of T lymphocytes, B lymphocytes and macrophages, respectively, was performed. Multiplex immunohistochemistry (mIHC) revealed that, compared to reactive hyperplasia, the most significant changes in lymph node cell populations occurred in the follicles for both PCV2 and PRRSV infections. Additionally, in PCV2 cases, notable alterations were also observed in the interfollicular areas. In PCV2-affected lymph nodes, follicles not only significantly decreased in number but also showed a marked significant reduction in CD20+ and CD3+ cells. The interfollicular region in these cases also exhibited a significant reduction in CD3+ cells. In contrast, PRRSV-associated lymphadenopathy showed significantly increased CD20+ cells in the follicles, with a similar trend noted in the interfollicular region. mIHC provides more informative results on a single tissue section, thus preserving the topographical information of the tissue and allowing a comprehensive study of cellular composition, cellular functionality and cell–cell interactions, proving to be a valuable tool for studying and understanding disease dynamics.