Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp

Abstract

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Keywords: 18S rRNA; Cyprinus carpio nudus; Thelohanellus kitauei; identification; intestinal giant-cystic disease; quantitative PCR (qPCR).

Fig. 1

Characterization of spores. (A) Various sizes of giant cysts (arrows) containing mature spores found in the intestine of an Israel carp infected with Thelohanellus kitauei. (B) Spores of T. kitauei isolated from the intestine of a diseased fish. Scale bar: 20 µm.

Fig. 2

Determination of the detection limit of conventional PCR using AccuPower™ PCR PreMix system. Four µl of PCR products were loaded on 1% agarose gels and ethidium bromide-stained specific PCR bands were visualized using a UV-transilluminator. PCR was carried out using various concentrations of the full-length 18S rRNA gene-harboring plasmid pKitauei (A), the suspension containing different number of spores (B), genomic DNA purified from isolated spores (C) and genomic DNA from the various numbers of spore-spiked tissues (D) as templates. M: 100 bp ladder from Bioneer.co.kr

Fig. 3

Linear standard curves derived from 10-fold serially diluted pKitauei plasmid (A) and DNA purified using known numbers of T. kitauei spores (B). x-axis and y-axis represent a log₁₀ of number of spore used in DNA preparation and mean (± SD) real-time qPCR Cp value, respectively. PCR efficiency calculated using slope of the standard curve was 99.2%.