Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia.

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 10(4) CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the Chelex method could be used to detect fewer than 20 cultured Vibrio cells in sea-water or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.

Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle.

Trypsin is the most abundant protease in Crustacea. This enzyme was purified from the digestive gland of Penaeus vannamei, revealing three major isoforms (molecular weights 31-32 kDa) and several minor components. Five cDNAs encoding five isoforms of trypsin were detected by two successive screenings of an amplified cDNA library from the digestive gland of P. vannamei. The longest isolated and sequenced cDNA encoded a preproenzyme of 255 amino acids containing a putative precursor peptide of 14 residues and a highly hydrophobic signal sequence of 14 amino acids. Amino acid sequence alignments revealed a high degree of identity between the trypsin from P. vannamei and that from crayfish (74%) and an equal level of sequence similarity to that from mammals and insects (approximately 40). Dot blot hybridization and subsequent analysis of the variation in trypsin-specific activities revealed that mRNA expression is at a maximum during early premoult (D1), declining sharply in late premoult (D2-D3). The specific activity of trypsin also followed this pattern, suggesting the regulation of trypsin biosynthesis is, at least in part, transcriptional. The characterization of trypsin cDNA from P. vannamei provides the first description of a putative zymogen sequence in a crustacean species, enabling us to elucidate the regulatory mechanism of trypsin synthesis in these important marine organisms.