Inter-Research > DAO > v48 > n3 > p187-195  
Diseases of Aquatic Organisms

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DAO 48:187-195 (2002)  -  doi:10.3354/dao048187

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP)

H. Kvitt1,*, M. Ucko1, A. Colorni1, C. Batargias1,**, A. Zlotkin2, W. Knibb1,***

1Israel Oceanographic & Limnological Research Ltd., National Center for Mariculture, PO Box 1212, Eilat 88112, Israel
2Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
*E-mail: Present addresses: **Nireus S.A., Group of Companies, R&D Dept. Hiliadou Doridos, 330 56 Hiliadou, Greece ***Bribie Island Aquaculture Research Centre, 144 North Street, Woorim, PO Box 2066, Bribie Island, Queensland 4507, Australia

ABSTRACT: A PCR protocol for the rapid diagnosis of fish Œpasteurellosis¹ based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64°C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.

KEY WORDS: AFLP · Dicentrarchus labrax · PCR · Photobacterium damselae · 16S rRNA gene · Sparus aurata

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