Enzyme-liked immunosorbent assay for the detection of pathogenic spiroplasma in commercially exploited crustaceans from China

Spiroplasmas are emerging pathogens in commercially exploited freshwater crustaceans from China. The bacteria infect Chinese mitten crab (Eriocheir sinensis), crayfish (Procambarus clarkii) and pacific white shrimp (Litopenaeus vannamei) in Jiangsu and Zhejiang provinces of China. A rabbit antiserum was prepared against the spiroplasma isolated from a Chinese mitten crab that had obvious signs of tremor disease. An indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of pathogen was developed. The titer of antibody against the pathogen was 1:31,250. The lowest detectable antigen protein was 0.573 μg/mL (3 × 105 CCU·ml− 1 pathogens) in pure culture. No color reaction was observed with seven other bacteria (Acinetobacter spp, Bacillus subtilis, Streptomyces sp., Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Aeromonas hydrophila). Very weak color reaction was observed in tests with spiroplasma species from plants and honeybees. The coefficient of variability of the assay was less than 10%, with an excellent reliability in moderate and heavy infections. The whole test procedure was rapid, being completed within 3 h. The results showed that the assay is a simple and applied method for diagnosis of spiroplasma diseases in crustaceans.