Development of PCR method for detecting Kudoa iwatai (Myxozoa: Multivalvulida) from rock bream Oplegnathus fasciatus

Abstract

We developed a PCR assay targeting the 28S rDNA of Kudoa iwatai (Multivalvulida: Myxozoa) and investigated the prevalence of infection in rock bream Oplegnathus fasciatus, which is commercially an important aquaculture species in Korea, with this assay. Detection limit of the PCR assay was 2.5 fg/μl with plasmid DNA and 8.6 × 10 spores/ml with purified spores, respectively. This PCR assay did not amplify DNA of other Kudoa species (Kudoa septempunctata, Kudoa lateolabracis, Kudoa thyrsites) tested. Sliced muscles of whole body from 318 rock bream (wild and cultured) were examined by this PCR assay and also with the naked eyes. All of the wild fish did not produce amplicons nor did harbor visible Kudoa cysts (0/70). Three of the cultured fish were PCR-positive and also harbored visible Kudoa cysts (3/248, 1.2%). The sequences of amplicons (574 bp) were 100% identical with those of the K. iwatai already registered in Genbank. When the visceral organs of these three fish were examined, visible cysts were not found, but one stomach sample was found to be PCR-positive. There was no difference in the prevalence of infection estimated by PCR assay and the presence of visible Kudoa cysts in our samples. This is thought to be because the development of K. iwatai is already completed and only mature Kudoa cysts existed in our samples.

Molecular Detection, Histopathology, and Scanning Electron Microscopy of Myxobolus koi Infecting Cyprinus carpio Koi

“…148 Molecular Detection, Histopathology and SEM of Myxobolus koi (Soelistyoadi, et al) histology, morphology, and physiology), it is important to look for alternative parameters such as molecular biomarkers [21,22,23]. The genome strategy is revolutionizing scientific research in understanding fish physiology and gene evolution because it is relatively easy to isolate novel genes and homologs using public databases [24,25]. The aim of this research is to investigate the clinical symptoms that occur in the gills of Koi carp (C. carpio) infected by Myxobolus sp.…”

“…The results of electrophoresis can be detected at 2000 bp. DNA-based examination with molecular techniques has several advantages over microscopic observation because it is very specific and sensitive, and thus it can detect infections at an early stage and very mild infections [25]. In Figure 4, the appearance of the band in lane 2 shows the copy of Myxobolus koi genome in the target organ sample, whereas in lane 3, the band does not appear, presumably due to the absence of a copy of the Myxobolus koi genome or the very small volume of the genome that is undetectable with the nested PCR method.…”