Polymerase chain reaction and indirect fluorescent antibody technique for the detection of Kudoa amamiensis (Multivalvulida: Myxozoa) in yellowtail seriola quinqueradiata

Abstract

Polymerase chain reaction (PCR) and indirect fluorescent antibody technique (IFAT) were developed for the detection of Kudoa amamiensis (Multivalvulida: Myxozoa), which produces pseudocysts in the skeletal musculature of yellowtail Seriola quinqueradiata. A nested PCR derived from the 18SrDNA sequences of K. amamiensis proved to be highly specific and sensitive, detecting a single spore in a 20 μL reaction mixture. In a periodic monitoring of yellowtail, the single-round and nested PCRs were conducted as well as conventional diagnostic techniques (visual inspection, wet-mount observation and histological examination). After one month culture of yellowtail in an endemic area, neither pseudocysts nor spores could be detected visually, whereas 53% of the examined fish were positive for infection by the nested PCR. After 3 and 5 months, macroscopic pseudocysts were detected in 27% and 60% of the fish, respectively, while the nested PCR was more sensitive (93% and 70% were positive, respectively). Histologically, intracellular organisms with several internal cells were detected only in the muscle fibers of the samples which were positive by PCR tests. By the IFAT using a rabbit antiserum to sonicated K. amamiensis spores, both pre-spore and spore stages reacted, and intracellular plasmodia were positively identified as presporogonic stages of K. amamiensis. In conclusion, we developed the IFAT and the nested PCR for the early diagnosis of K. amamiensis, and described for the first time its intracellular presporogonic stage in the muscle fiber of yellowtail.