Establishment of an ELISA for the detection of antibodies against Stenotrophomonas maltophilia

Abstract

Background: Stenotrophomonas maltophilia (S. maltophilia) is a conditionally pathogenic bacterium around the world. In humans, it mainly harms patients with immunodeficient or chronic diseases, leading to high mortality rate of patients with pneumonia and bacteremia. In the veterinary medicine, it caused respiratory infections or mixed infections with other common pathogens in pigs. However, infection of S. maltophilia has been overlooked and the prevalence of S. maltophilia in porcine remains unknown due to a lack of diagnosis method.

Methods: In this study, an indirect ELISA was established using the purified Phospholipase C (PLC) protein of S. maltophilia SMFZ-01 strain as the coating antigen. Checkerboard titration was performed for the optimization of the coating concentration of the recombinant PLC (rPLC) protein, the dilution and incubation time of serum and the IgG-HRP secondary antibody. The specificity, repeatability and sensitivity of established ELISA was also optimized. The positive rate of S. maltophilia antibodies was calculated using 305 clinical porcine sera samples by either established indirect ELISA or a nested PCR.

Results: rPLC was successfully purified and identified by Western blot. The optimal coating concentration of rPLC was 2.5 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:400 and 1:2500, respectively. The analytical sensitivity was 1:1600, with no cross-reaction with the positive sera of other common porcine pathogens. The intra-assay and inter-assay reproducibility coefficients of variation was less than 10%. Detection of 305 clinical porcine sera samples revealed a high positive rate by established ELISA (12.01%) and nested PCR (29.87%).

Conclusions: To our knowledge, this is the first study to develop an indirect ELISA for the detection and epidemiological analysis of S. maltophilia in porcine. Our findings indicate that S. maltophilia may be more prevalent in porcine populations than previously recognized, though further studies are needed to confirm this. The established indirect ELISA enhances our understanding of S. maltophilia infections in pig herds and provides valuable insights for designing future prevention and control strategies, along with considerations regarding the health and welfare of porcine.

Keywords: Stenotrophomonas maltophilia; Indirect ELISA; PRDC; Phospholipase C; Pig.