Genetic characterization of the lobster pathogen Aerococcus viridans var. homari by 16S rRNA gene sequence and RAPD

Abstract

A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.

Identification of Vibrio campbellii isolated from diseased farm-shrimps from south India and establishment of its pathogenic potential in an Artemia model

“…Greenwood et al, 2005). However, V. harveyi and V. campbellii share more than 97 % similarity in their 16S rRNA gene sequences and show a 69 % match in DNA-DNA hybridization (Gomez-Gil et al, 2004).…”

“…Misidentification of bacterial pathogens in fish/shrimp farm settings may create problems in selecting appropriate prophylactic measures. In recent years, sequencing and comparison of the 16S rRNA gene has become an important tool for identification of bacterial species (Greenwood et al, 2005). However, V. harveyi and V. campbellii share more than 97 % similarity in their 16S rRNA gene sequences and show a 69 % match in DNA-DNA hybridization (Gomez-Gil et al, 2004).…”

Limited prevalance of gaffkaemia (Aerococcus viridans var. homari) isolated from wild-caught European lobsters Homarus gammarus in England and Wales

“…The partial 16S rRNA gene se quence data from isolates confirmed as Aero coccus sp. were also directly compared with the partial 16S rRNA gene sequences lodged in GenBank that were derived from the study by Greenwood et al (2005).…”

“…In addition, the nearly full-length 16S rRNA gene sequences were also ob tained for 2 of the isolates recovered from the holding facility in South Wales, and these were amplified using protocols previously described by Green wood et al (2005) with minor modifications. For this, approximately 10 ng of genomic DNA was amplified in 20 µl reactions containing 1× Green GoTaq Flexi Buffer (Promega), 1.5 mM MgCl 2 , 0.25 mM of each dNTP, 1 µM of each primer (BSF-8/20 and BSR-1541/20, see Greenwood et al 2005) and 1 U Taq polymerase. Amplifications were performed with an initial denaturation of 94°C for 2.5 min, followed by 35 cycles at 94°C for 1 min, 45°C for 1 min and 72°C for 1.5 min, with a final elongation step at 72°C for 5 min.…”

“…To help determine the origins and relatedness of the Aerococcus viridans isolates recovered from wild Homarus gammarus and those held in storage, they were typed by using pulsedfield gel electrophoresis (PFGE) and compared with a range of isolates previously characterised by random amplified polymorphic DNA (RAPD) (Greenwood et al 2005). PFGE is one of the most powerful molecular typing methods available and has been successfully applied to a range of different bacterial pathogens (Arbeit 1995), including some that affect fish (Vela et al 2000, Nawawi et al 2008, Wheeler et al 2009); however, it had not previously been used for typing A. viridans.…”