{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T16:07:17Z","timestamp":1785773237280,"version":"3.56.0"},"reference-count":50,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T00:00:00Z","timestamp":1749168000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>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\u2013cell interactions, proving to be a valuable tool for studying and understanding disease dynamics.<\/jats:p>","DOI":"10.3390\/ani15121682","type":"journal-article","created":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T06:11:08Z","timestamp":1749190268000},"page":"1682","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Bright-Field Multiplex Immunohistochemistry in Swine PCV2 and PRRSV Lymphadenopathies"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0931-8421","authenticated-orcid":false,"given":"Giulia","family":"D\u2019Annunzio","sequence":"first","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Lombardia e dell\u2019Emilia-Romagna \u201cBruno Ubertini\u201d, 25124 Brescia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4653-0624","authenticated-orcid":false,"given":"Luisa","family":"Muscatello","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche Veterinarie, Universit\u00e0 di Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Tugnoli","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche Veterinarie, Universit\u00e0 di Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefano","family":"Pesaro","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Agroalimentari, Ambientali e Animali, 33100 Udine, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1457-3518","authenticated-orcid":false,"given":"Andrea","family":"Luppi","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Lombardia e dell\u2019Emilia-Romagna \u201cBruno Ubertini\u201d, 25124 Brescia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1749-150X","authenticated-orcid":false,"given":"Michelangelo","family":"Fiorentino","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche e Chirurgiche, Universit\u00e0 di Bologna, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2897-7756","authenticated-orcid":false,"given":"Tania","family":"Franceschini","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche e Chirurgiche, Universit\u00e0 di Bologna, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessia","family":"Grillini","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche e Chirurgiche, Universit\u00e0 di Bologna, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7437-1929","authenticated-orcid":false,"given":"Gianluca","family":"Rugna","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Lombardia e dell\u2019Emilia-Romagna \u201cBruno Ubertini\u201d, 25124 Brescia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6405-3484","authenticated-orcid":false,"given":"Giuseppe","family":"Sarli","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche Veterinarie, Universit\u00e0 di Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0579-9136","authenticated-orcid":false,"given":"Luciana","family":"Mandrioli","sequence":"additional","affiliation":[{"name":"Dipartimento di Scienze Mediche Veterinarie, Universit\u00e0 di Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1369\/jhc.2010.957191","article-title":"Antigen retrieval immunohistochemistry: Review and future prospects in research and diagnosis over two decades","volume":"59","author":"Shi","year":"2011","journal-title":"J. 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