Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome¿affected ascidian Halocynthia roretzi using real-time polymerase chain reaction

Abstract

Background: The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR). Findings: The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm

Seasonal variation in Azumiobodo hoyamushi infection among benthic organisms in the southern coast of Korea

“…In particular, the inhalent tissue of ascidians was removed and used for quantification of the pathogen. Shin et al [ 11 ] examined the infection intensity of A. hoyamushi in various parts of the tunic, including inhalent siphon, exhalent siphon, and three different parts of the tunic; they found that the inhalent siphon was the most sensitive organ for the diagnosis of the pathogen. They also reported that infection intensity of A. hoyamushi increased as STS developed and the pathogens spread to other parts of the tunic from the inhalent siphon.…”

“…After pulverization of 150 mg of each sample, DNA was extracted using DNA blood kit (Qiagen) and A. hoyamushi was quantified using the Taq-Man probe technique [ 11 ]. The primer used in the qPCR was F: 5′-GCC TCT GTG GTT TGC TCC TT-3′, R: 5′-TAC TGG GCG GCT TGG ATC TCG T-3′ and the probe was 5′ FAM-CCG CTC AAA GAC GAA CTA CAG CGA -BHQ1 3′.…”

“…The qPCR conditions were as follows: pre-denaturation at 94 °C for 5 min, followed by 45 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, after which the reaction results were observed. Infection intensity was tested according to reported methodology [ 11 ]. Briefly, in vitro cultured A. hoyamushi was diluted to 1, 10, 100, 1000, and 100,000 cells and the Ct value for each parasite concentration was obtained using real-time PCR.…”