Genome based quantification of Miamiensis avidus in multiple organs of infected olive flounder (Paralichthys olivaceus) by real-time PCR.

Abstract

Miamiensis avidus is the major parasitic pathogen affecting the olive flounder, Paralichthys olivaceus. Recent epidemiological studies have shown that M. avidus infections are becoming increasingly severe and frequent in the olive flounder farming industry. This study aimed to evaluate the infection density of M. avidus in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm. Each fish was injected subcutaneously with 2.75 × 103CFU M. avidus/ fish. Organs infected with M. avidus were obtained after 7 and 25days. Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28s in M. avidus, which was used as a target gene. Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28s in M. avidus, which was used as a target gene. The levels of 28s rRNA were used to calculate quantitative gene copy number. Real-time PCR of brain (60.58 ± 38.41), heart (64.03 ± 62.40), muscle (6.10 ± 3.12), gill (5.06 ± 4.56), intestine (2.38 ± 1.69), esophagus (4.22 ± 3.72), stomach (3.25 ± 2.68), kidney (0.81 ± 0.15), liver (0.63 ± 0.15), and spleen (11.18 ± 4.08) was performed at 3 days post-infection. At 7days post-infection, heart (754.15 ± 160.85), brain (247.90 ± 62.91), spleen (38.81 ± 17.52), liver (7.47 ± 4.54), kidney (10.90 ± 3.41), stomach (19.50 ± 8.86), esophagus (39.37 ± 14.10), intestine (17.54 ± 12.63), gill (38.27 ± 20.20), and muscle (33.62 ± 15.07) were measured. The present study, together with previous data, demonstrated that the gill, intestine, and brain are the major target organs of M. avidus in olive flounder. However, this does not mean that tiny amounts of DNA extracted from those tissues of fish during the early stages of infection can guarantee successful detection and/or quantification of M. avidus. Our data suggest that the brain might be the best organ for detection in the early stage.