Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues

Diseases of Aquatic Organisms

Holly L. Mendonca¹  Kristen D. Arkush¹ 

关键词: Sphaerothecum destruens;    Rosette agent;    Polymerase chain reaction;    Salmonids;    Detection method;   

DOI  :  10.3354/dao061187

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, anintracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pgfor purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stagesof the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from thekidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over thecourse of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captivebroodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from othertissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of theparasite.

【 授权许可】

Unknown