Development and validation of a flow cytometry antibody test for Lawsonia intracellularis

Débora Zini Baldasso; João Antônio Guizzo; Cláudia Cerutti Dazzi; Gabriela Carolina Paraboni Frandoloso; César Feronato; Stephan von Berg; Roberto Maurício Carvalho Guedes; Heather Lynne Wilson; Luiz Carlos Kreutz; Rafael Frandoloso

doi:10.3389/fimmu.2023.1145072

Development and validation of a flow cytometry antibody test for Lawsonia intracellularis

Front Immunol. 2023 Mar 21:14:1145072. doi: 10.3389/fimmu.2023.1145072. eCollection 2023.

Affiliations

¹ Laboratory of Microbiology and Advanced Immunology, Faculty of Veterinary Medicine, University of Passo Fundo, Passo Fundo, Brazil.
² Section of Immunology, AFK Imunotech, Passo Fundo, Brazil.
³ Swine Technical Department, Merck Sharp & Dohme (MSD), São Paulo, Brazil.
⁴ Global Marketing Swine, Merck Sharp & Dohme (MSD), Animal Health, Munich, Germany.
⁵ Department of Veterinary Clinic and Surgery, Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
⁶ Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, Canada.

Abstract

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (10⁶ bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Keywords: Ileitis; Lawsonia intracellularis; diagnostic test; flow cytometry assay; pig; serological antibody detection.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Desulfovibrionaceae Infections* / diagnosis
Desulfovibrionaceae Infections* / microbiology
Desulfovibrionaceae Infections* / veterinary
Flow Cytometry
Inflammatory Bowel Diseases*
Lawsonia Bacteria*
Reproducibility of Results
Swine

Grants and funding

This study was funded by MSD Animal Health. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.