The development, validation and application of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Streptococcus dysgalactiae subspecies dysgalactiae in lamb and ewe sera

Abstract

Background: Neonatal infectious arthritis (NIA) is a bacterial disease of lambs caused by Streptococcus dysgalactiae sub species dysgalactiae (SDSD). NIA negatively impacts animal welfare; whilst its control entails widespread antibiotic use. The aims of this study are to develop, validate and apply an indirect, ELISA assay to inform SDSD vaccine development and epidemiology studies.

Methods: The antigen (inactivated isolate of SDSD) was incubated with sera from SDSD infected sheep, negative control sheep and serum from sheep flocks with outbreaks of SDSD NIA. SDSD antibody was detected with rabbit anti-sheep IgG conjugated with horseradish peroxidase. Antibody levels were estimated using peroxidase substrate and detection of colour change. Test performance was determined by receiver operating characteristic (ROC) curve analysis and by comparison with the animals' concurrent infection status, as determined by a Real Time PCR assay for SDSD bacterial DNA.

Results: Using ROC curve analysis, a median test sensitivity of 85.71 % (95 % CI, 77.51-93.91) and a median test specificity of 73.68 % (95 % CI, 53.89-93.48), with a positive predictive value of 92.31 % (95 % CI, 86.07-98.55), and a negative predictive value of 58.33 % (95 % CI, 36.16-80.50). ELISA test performance was reduced when serum ELISA status (n = 362 sheep) was compared with concurrent SDSD infection status. The SDSD ELISA sensitivity was 39.74 % (95 % CI, 28.83-51.46), the specificity 60.92 % (95 % CI, 54.97-66.63), the PPV 21.83 % (95 % CI, 15.34-29.53), and the NPV was 78.64 % (95 % CI, 72.62-83.86).

Conclusion: This ELISA assay could be applied to estimate flock sero-prevalences for SDSD and changing SDSD antibody titres in vaccine trials.

Keywords: ELISA; Joint ill; Lambs; Neonatal infectious arthritis; ROC curve; Streptococcus dysgalactiae sub species dysgalactiae; Test performance.