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

Genes Genomics. 2019 May;41(5):567-572. doi: 10.1007/s13258-019-00792-z. Epub 2019 Mar 4.

Abstract

Introduction: 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.

Objectives: 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.

Methods: Each fish was injected subcutaneously with 2.75 × 103 CFU M. avidus/ fish. Organs infected with M. avidus were obtained after 7 and 25 days. Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene.

Results: Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene. The levels of 28 s 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 7 days 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.

Conclusion: 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.

Keywords: Miamiensis avidus; Olive flounder; Real-time PCR; Scuticociliate; Target organ.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animal Structures / parasitology
  • Animals
  • Ciliophora Infections / genetics*
  • Fish Diseases / genetics
  • Flounder / genetics
  • Flounder / microbiology
  • Flounder / parasitology*
  • Oligohymenophorea / genetics*
  • Oligohymenophorea / pathogenicity
  • Organ Specificity
  • RNA, Ribosomal, 28S / analysis
  • RNA, Ribosomal, 28S / genetics
  • Real-Time Polymerase Chain Reaction / methods

Substances

  • RNA, Ribosomal, 28S