Abstract
BackgroundSlaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved.ResultsTo enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated.Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection.ConclusionsA suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.
Highlights
Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology
Other signals ranged from 3 to 17 median fluorescence intensity (MFI), i.e. signals of agglutination sera on beads carrying LPS with disparate O antigens, as well as signals of negative control samples on all five bead sets, namely agglutination serum against O:20 or no serum at all. These results indicate that the antigenic properties of LPS are retained when a triazine is used to immobilize LPS on carboxylated beads, and that the resulting LPS-conjugated beads can be used to detect O antigen specific antibodies
Detection of anti-Salmonella antibodies in experimental pig sera The results of the suspension array with experimental pig sera showed that each serum is recognized by LPS of the corresponding serogroup, i.e. B sera are recognized by B LPS, C1 sera by the two C1 LPS variants, C2 sera by C2 LPS, and D1 sera by D1 LPS (Fig. 2)
Summary
Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Most Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). Based on the presence of somatic O antigens and flagellar H antigens, a large number of serovars have been described (White-Kauffman-Le Minor scheme) [5] These are distributed over ~ 30 serogroups as determined by the composition of O antigenic polysaccharides, i.e. the outermost component of LPS. LPS can be used as antigen to probe for anti-Salmonella antibodies in animal sera, which is the basis for determining the Salmonella status of pig herds using serological ELISAs
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