Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis

Abstract

Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis. KEY WORDS: Photobacterium damselae subsp. piscicida · Fish pasteurellosis · PCR-RFLP · Diagnostic tool Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [53][54][55][56][57][58][59][60][61] 2005 a better prevention of the disease through control of the presence of the pathogen in the water and/or carrier fishes. Thus far, a broad variety of methods has been evaluated for highly sensitive, species-(subspecies) identification of the pathogen without prior isolation as pure culture. Tested methods include enzyme-linked immunosorbent assays (ELISA) and enzyme immuno-assay (EIA) techniques (Bakopoulos et al. 1997, plasmid DNA PCR (Aoki et al. 1997), 16S rRNA gene sequencing , and primers designed following random amplified polymorphic DNA (RAPD) (Dalla Valle et al. 2002). All these methods, however, could not reliably discriminate between the 2 subspecies. A method based on duplex PCR of ureC and 16S rRNA genes was proposed by Osorio et al. (2000), due to the lack of the ureC gene in the subspecies piscicida, but subspecies discrimination was based on indirect evidence (lack of amplification of ureC). Lately, a combined approach of PCR technique and plating method has been proposed (Rajan et al. 2003). Other methods such as ribotyping (Magariños et al. 1997), RAPD (Random Amplified Polymorphic DNA) (Magariños et al. 2000), and amplified fragment length polymorphism (AFLP) (Thyssen et al. 2000, Kvitt et al. 2002 showed the ability to differentiate the 2 subspecies as well as to identify clusters of P. damselae subsp. piscicida strains. However, these methods require DNA extraction from pure cultures and refe...

Development of a molecular methodology for fast detection of Photobacterium damselae subspecies in water samples

“…In the last two decades efforts have been made to develop rapid and specific protocols for early detection of fish pathogens and prevent potential disease outbreaks (del Cerro et al, 2002;Romalde et al, 1999;Zappulli et al, 2005). Although these approaches proved to be appropriate for positive identification of the subspecies of P. damselae, none of them were able to perform direct simultaneous detection of both subspecies in water samples from natural or artificial systems, which limit their application in the process of disease outbreak control.…”

“…However, these techniques are often laborious, time-consuming and lack sensitivity. In the last years, despite the high homology in the DNA sequence of both subspecies (Gauthier et al, 1995;Osorio et al, 1999), several studies developed protocols using molecular techniques to overcome the limitations of the conventional microbiological techniques and to allow the detection of P. damselae (Kvitt et al, 2002;Osorio et al, 1999;Zhao and Aoki, 1989) and distinguish the two subspecies (Amagliani et al, 2009;Zappulli et al, 2005). Previous studies showed that different genotypes of P. damselae subspecies piscicida and damselae can have different phenotypic characteristics and pathogenicity potential (Hawke et al, 2003;Takahashi et al, 2008).…”

Full-length sequence and expression analysis of Toll-like receptor 9 in the gilthead seabream (Sparus aurata L.)

“…Individuals of group 1 were challenged by immersion with Photobacterium damselae subspecies piscicida (3 × 10 5 bacteria l − 1 ). Group 1 was challenged with a low virulence strain (DI21, Zappulli et al, 2005) to obtain sub-lethal infection. Group 2 was mock-infected by immersion in clean aerated seawater.…”

“…Tissue samples from these fishes were collected and stored as described above. A pathogen-specific test was performed on DNA extracted from infected tissues (Zappulli et al, 2005). All group 1 individuals were strongly positive for the presence of the pathogen.…”

“…Details of the experimental infection are reported elsewhere (Zappulli et al, 2005). Briefly, sea breams were divided in two groups.…”