The use of filamentous hemagglutinin adhesin to detect immune responses to Campylobacter hepaticus infections in layer hens

Abstract

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA_1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA_1,628-1,899 was used as antigen coating on ELISA plates to capture FHA_1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.

Keywords: Campylobacter hepaticus; ELISA; Spotty Liver Disease; filamentous hemagglutinin adhesion; immunoassay; sera.

Figure 1

Bioinformatics workflow used in the prediction of C. hepaticus specific immunogenic proteins.

Figure 2

Western blot of C. hepaticus, C. jejuni, and C. coli total proteins probed with pooled SLD positive sera. (A) Coomassie stained gel, (B) PVDF membrane probed with SLD positive pooled sera that showed specific bands at 220 and 25 kDa regions, and (C) PVDF membrane probed with pre-absorbed SLD positive pooled sera that still showed the specific bands at 220 and 25 kDa. Lane M–Precision plus protein ladder (Bio-Rad). Total protein profiles of C. hepaticus–H, C. jejuni–J, and C. coli–C.

Figure 3

Determination of protein expression in E. coli cell lysates by Coomassie staining and anti-his western blotting. (A) Coomassie stained gel containing expressed E. coli cell lysates that confirmed the expression of 17 out of 19 proteins, highlighted in blue boxes and (B) PVDF membrane blotted with expressed E. coli cell lysates probed with anti-his antibody that confirmed the expression of 16 out of 19 proteins, highlighted in blue boxes. M–Precision plus protein ladder (Bio-Rad), U–uninduced E. coli BL21 (DE3) cell lysate, P–an unrelated his-tagged protein used as a positive control.

Figure 4

Determination of C. hepaticus specific immunogenicity of the recombinant proteins coded by PEGs 407, 433, 486, and 1,316 by western blotting using individual SLD positive and negative sera. (A) PVDF membrane blotted with E. coli cell lysates (PEGs 407, 486, and 1,316) and probed with sera from four SLD positive birds, P1, P2, P3, and P4 and four negative control birds, N1, N2, N3, and N4. (B) PVDF membrane blotted with E. coli cell lysate (PEG 433) and probed with sera from three SLD positive birds, P1, P2, and P3, and two negative control birds, N1 and N2. Only the recombinant protein encoded by PEG 1,316 showed C. hepaticus specific immunogenicity as it was recognized by SLD positive sera and not by the negative control sera. The recombinant proteins encoded by PEGs 433, 407, and 486 cross reacted with antibodies in several SLD negative sera. M–Precision plus protein ladder (Bio-Rad).

Figure 5

Optimisation of SLD-ELISA2 parameters. (A) Sera dilution and (B) Protein coating. The red arrows indicate the optimal values.

Figure 6

Absorbance produced by anti-FHA antibodies present in the bird sera used in SLD-ELISA2. Ordinary one-way ANOVA pairwise comparison results were highly significant with ****p < 0.0001 for both naturally and experimentally infected groups compared to the negative control group.