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. 2025 Jun 6;15(12):1682.
doi: 10.3390/ani15121682.

Bright-Field Multiplex Immunohistochemistry in Swine PCV2 and PRRSV Lymphadenopathies

Giulia D'Annunzio  1 , Luisa Vera Muscatello  2 , Chiara Tugnoli  2 , Stefano Pesaro  3 , Andrea Luppi  1 , Michelangelo Fiorentino  4 , Tania Franceschini  4 , Alessia Grillini  4 , Gianluca Rugna  1 , Giuseppe Sarli  2 , Luciana Mandrioli  2
Affiliations

Bright-Field Multiplex Immunohistochemistry in Swine PCV2 and PRRSV Lymphadenopathies

Giulia D'Annunzio et al. Animals (Basel). .

Abstract

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.

Keywords: CD20; CD3; IBA1; IHC; PCV2; PRRSV.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Swine. Inguinal lymph node. Reactive hyperplasia. Multiplex immunohistochemistry for CD3 (brown), CD20 (blue) and IBA1 (fuchsia). (a) Multiple reactive follicles (higher magnification in (b) containing mainly CD20+ lymphocytes with scattered CD3 lymphocytes and IBA1+ cells; (c) interfollicular areas containing CD3+ lymphocytes and few IBA1+ cells; (d) the medulla-like tissue shows a prevalence of IBA1+ reticular cells. Scale bar: (a) 800 μm; (b–d) 100 μm.
Figure 2
Figure 2
Swine. Inguinal lymph node. PCV2 systemic infection. Multiplex immunohistochemistry for CD3 (brown), CD20 (blue) and IBA1 (fuchsia). (a) Sparse and fading follicles show a reduction of CD20+ lymphocytes (b) and the presence of IBA1+ follicular epithelioid cells (c). In the interfollicular area, CD3+ lymphocytes are severely reduced (b,c), while IBA1+ macrophages are increased (b). IBA1 staining is also detectable in epithelioid and giant cells (d). Scale bar: (a) 800 μm; (b) 200 μm; (c) 150 μm; (d) 100 μm.
Figure 3
Figure 3
Swine. Mediastinal lymph node. PRRSV infection. Multiplex immunohistochemistry for CD3 (brown), CD20 (blue) and IBA1 (fuchsia). (a) Multiple reactive follicles rich in CD20+ lymphocytes (higher magnification in (b). (b–d) The interfollicular area shows an increase of CD20+ lymphocytes, associated with a decrease of CD3+ lymphocytes. Scale bar: (a) 800 μm; (b) 200 μm; (c) 150 μm; (d) 100 μm.
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