[논문]Molecular detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in sea water and marine invertebrates

Paari, Alagesan; Jeon, Chan-Hyeok; Choi, Hye-Sung; Jung, Sung-Hee; Kim, Jeong-Ho

doi:10.1186/s41240-017-0062-z

Molecular detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in sea water and marine invertebrates 원문보기

Fisheries and aquatic sciences, v.20 no.8, 2017년, pp.16.1 - 16.8

Paari, Alagesan (Department of Marine Bioscience, Gangneung-Wonju National University) , Jeon, Chan-Hyeok (Department of Marine Bioscience, Gangneung-Wonju National University) , Choi, Hye-Sung (Pathology Division, National Institute of Fisheries Science (NIFS)) , Jung, Sung-Hee (Pathology Division, National Institute of Fisheries Science (NIFS)) , Kim, Jeong-Ho (Department of Marine Bioscience, Gangneung-Wonju National University)

Abstract ▼ AI-Helper

The exportation of cultured olive flounder (Paralichthys olivaceus) in Korea has been recently decreasing due to the infections with a myxozoan parasite Kudoa septempunctata, and there is a strong demand for strict food safety management because the food poisoning associated with consumption of raw olive flounder harbouring K. septempunctata has been frequently reported in Japan. The life cycle and infection dynamics of K. septempunctata in aquatic environment are currently unknown, which hamper establishment of effective control methods. We investigated sea water and marine invertebrates collected from olive flounder farms for detecting K. septempunctata by DNA-based analysis, to elucidate infection dynamics of K. septempunctata in aquaculture farms. In addition, live marine polychaetes were collected and maintained in well plates to find any possible actinosporean state of K. septempunctata. The level of K. septempunctata DNA in rearing water fluctuated during the sampling period but the DNA was not detected in summer (June-July in farm A and August in farm B). K. septempunctata DNA was also detected in the polychaetes Naineris laevigata intestinal samples, showing decreased pattern of 40 to 0%. No actinosporean stage of K. septempunctata was observed in the polychaetes by microscopy. The absence of K. septempunctata DNA in rearing water of fish farm and the polychaetes N. laevigata intestinal samples during late spring and early summer indicate that the infection may not occur during this period. N. laevigata was suspected as the possible alternate invertebrate host of K. septempunctata, but the actinosporean stage was not found by well plate method and further studies will be necessary. This research provides important baseline information for understanding the infection dynamics of K. septempunctata in olive flounder farms and further establishment of control strategies.

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제안 방법

septempunctata infection. As knowledge about the infection dynamics of this parasite is scarce, we carried out a monthly inspection of water samples to study the occurrence pattern of K. septempunctata in aquatic environment by molecular analysis. We also investigated the prevalence of K.
Cycling parameters were preheating at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 1 min and the analyses were conducted twice.
DNA was extracted by using a QIAamp DNA Mini Kit (QIAGEN, USA) according to the manufacturer’s instructions with slight modifications.
septempunctata in marine environment. In this study, we analysed rearing water samples from fish farms on a monthly basis for the presence of K. septempunctata by molecular methods.

성능/효과

All of the live polychaetes from fish farm sediments were identified as Naineris laevigata (Polychaeta, Orbiniidae) by PCR amplification of mt COI gene and maintained in 12- well plates for approximately 2 weeks, but no actinosporean stage were observed. Freshwater actinospores from oligochaetes have been successfully observed by well plate method (Yokoyama et al.
All of the polychaetes were identified using PCR. Primers targeting mt COI gene amplified a PCR product of size 710 bp, and the amplified sequences represent the species Naineris laevigata (Polychaeta, Orbiniidae) with 99.0% homology (data not shown).
The amount of K. septempunctata DNA was inversely proportional to the Ct value obtained in this study, and the Ct value of the highest standard (108 copies/μl) was 14.8 and the lowest standard (101 copies/μl) was 37.1.
2009, 2011). Thus, it would be helpful to detect any seasonal patterns in prevalence of actinosporean infection in polychaetes hosts by molecular method, but successful microscopic observation should be accompanied, which is also indispensable to make a clear conclusion whether N. laevigata is the alternate host of K. septempunctata.

후속연구

septempunctata in olive flounder farms as because it has been suggested that the actinosporean stages are generally fragile and easy to be destroyed by strong water flow (Kerans and Zale 2002; Hoz Franco and Budy 2004; Hallet and Bartholomew 2007). Nevertheless, considering the sea water volume flowing into the fish tanks, the parasite density calculated in this study cannot be negligible and should be considered for elucidating infection dynamics of K. septempunctata in olive flounder farms.

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표제어: PCR

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