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

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.

Figure 1

Quick protocol of flow cytometry antibody test for Lawsonia intracellularis. The brief protocol of immunostaining and cytometric analysis for the detection of porcine anti-L. intracellularis (Li) IgG. MFI, Mean Fluorescence Intensity. This figure was created with BioRender.com.

Figure 2

Antigen stability analysis. An aliquot of Enterisol^® Ileitis vaccine reconstituted and stored at 4-8°C was analyzed daily by flow cytometry. The aliquot diluted 1:1,000 was analyzed in triplicate and the mean of L. intracellularis absolute count plus standard deviation is shown in “(A)”. The coefficient of variation of each quantification is represented at the foot of the bars. The morphological characteristic of the bacterial population at 4 different times is illustrated in “(B)”. The subpopulation of L. intracellularis with greatest size and complexity is indicated with the arrowhead highlighted in red. Statistical comparison was performed using one-way ANOVA with Tukey’s multiple comparisons test and no significant (ns) differences were found between the moments compared.

Figure 3

Antigenicity analysis of the reconstituted antigen. This analysis was performed by flow cytometry using sera (n=3) from pigs immunized with the Porcilis^® Ileitis vaccine. The results express the percentage of L. intracellularis bacteria (total population containing 10⁶ bacteria) containing antibodies (IgGs) surface. Statistical comparison was performed using one-way ANOVA with Tukey’s multiple comparisons, and statistical differences are represented in the figure. Dot plots showing antigenic differences between time D1 and D30 are highlighted in zoom format within the figure.

Figure 4

Impact of antigen concentration on anti-L. intracellularis IgG detection. In this analysis 3 different concentrations of antigens (10⁵, 10⁶ and 10⁷) and 3 different categories of sera (weakly, moderately, and highly positive for L. intracellularis) were analyzed by flow cytometry after immunostaining. Statistical comparison was performed using two-way ANOVA with Tukey’s multiple comparisons. Statistical differences are indicated in the figure.

Figure 5

Primary antibody dilution and its impact on serology interpretation. In this analysis 3 different dilution (1:50, 1:100 and 1:200) of primary antibody (pig sera) classified in 3 different categories (weakly, moderately, and highly positive for L. intracellularis) were analyzed by flow cytometry after immunostaining. Statistical comparison was performed using two-way ANOVA with Tukey’s multiple comparisons. Statistical differences are indicated in the figure.

Figure 6

Outcome of incubation time on the detection of anti-L. intracellularis IgG. A total of 10 pig sera positive for IgG anti-L. intracellularis were included in this analysis. Pig sera were incubated for 20, 30 and 60 minutes with 10⁶ L. intracellularis at 37°C. Subsequently, the samples were incubated with the secondary antibody for the same times. Statistical comparison was performed using one-way ANOVA with Tukey’s multiple comparisons. No statistical differences (ns) were observed between the different times analyzed.

Figure 7

Basic immunological characteristics of the FCAT method. (A) Receiver Operating Characteristic (ROC) analysis. (B) Representation of the percentage values (dispersion) of L. intracellularis recognition of the samples used to define the cut-off of the FCAT test. The sensitivity and specificity of the test using the cut-off of > 15.15% are described in the figure. (C) FCAT specificity analysis using sera from pigs immunized with other pathogens. (D) Analysis of clinical samples collected from pigs with different immunological backgrounds against L. intracellularis.

Figure 8

Illustration of different serological diagnostic tests for Lawsonia intracellularis. (A) Flow Cytometry Antibody Test. In this assay, porcine antibodies interact with L. intracellularis floating in the well of the plate. The binding of specific antibodies to L. intracellularis is demonstrated with a phycoerythrin-conjugated antibody specific for porcine IgG. The test is read on the flow cytometer. (B) Immunoperoxidase Monolayer Assay (IPMA) & Indirect Fluorescent Antibody Test (IFAT). In these assays, cells (i.e., McCoy cells) infected with L. intracellularis are chemically fixed with acetone-methanol, which promotes a structural change in the cell (cytoplasmic membrane damage, as indicated in figure). This damage is essential for porcine antibodies to interact with L. intracellularis. The presence of antibodies bound to L. intracellularis is demonstrated by a peroxidase conjugate antibody plus chromogen (3-amino-9-ethyl-carbazole) solution (IPMA). In the case of IFAT, a fluorescein isothiocyanate conjugated antibody is used to reveal the presence of porcine IgG bound to the pathogen. Plates are read under an inverted microscope. (C) Blocking ELISA. In this ELISA, a monoclonal antibody bound to the ELISA plate is used to capture L. intracellularis. Porcine antibodies compete with the peroxidase-conjugated monoclonal antibody for a specific epitope present on a surface antigen of L. intracellularis. The presence of porcine antibodies to L. intracellularis is determined by the potential to reduce or inhibit binding of the monoclonal antibody and, therefore, the enzymatic reaction associated with the competing conjugated antibody. (D) ELISA Indirect. In this ELISA, L. intracellularis is immobilized directly on the ELISA plate; and for this reason, a considerable surface area of the antigen is not accessible to antibodies (physical masking of antigens). Porcine antibodies that recognize L. intracellularis are detected with a peroxidase-conjugated anti-porcine IgG antibody. This figure was created with BioRender.com.