The ABC-transporter hutCD genes of Photobacterium damselae subsp. piscicida are essential for haem utilization as iron source and are expressed during infection in fish.

The marine fish pathogen Photobacterium damselae subsp. piscicida utilizes haem compounds as the sole iron source. In a previous work, we characterized a gene cluster with ten potential haem uptake and utilization genes. Two of these genes, hutC and hutD, which are iron-regulated, conform a putative inner membrane haem ABC transporter. In this study, we constructed an insertional mutant, leading to the inactivation of hutCD genes. Reverse transcriptase-PCR analyses demonstrated that an insertion between the hutB and hutC genes abolished transcription of the downstream hutC and hutD genes. The hutCD mutant was unable to utilize haem as the sole iron source, demonstrating that the putative ABC-transporter proteins HutC and HutD are essential for haem utilization as an iron source in P. damselae subsp. piscicida. In addition, reverse transcriptase-PCR assays conducted with RNA samples isolated from experimentally infected fish revealed the presence of hutCD transcripts. The results demonstrate for the first time that haem uptake genes of a fish pathogen are expressed during the infective process in fish.

Evaluation of the AQUARAPID-Va, AQUAEIA-Va and dot-blot assays for the detection of Vibrio anguillarum in fish tissues.

The comparative accuracy of the serological assays AQUARAPID-Va, AQUAEIA-Va (BIONOR AS), and dot-blot for a rapid diagnosis of vibriosis in fish was evaluated. Twenty-one Vibrio anguillarum strains, representative of pathogenic and environmental serotypes, and 13 strains of other fish pathogenic bacteria were used to assess the sensitivity and specificity of the detection methods. The serological assays tested detected all the strains of V. anguillarum serotypes O1 and O2. The dot-blot assay was the most specific and sensitive method, detecting almost all isolates from serotypes O1, O2 and O3, with an average sensitivity of 1 x 10(6) bacteria g(-1) of fish tissue. The AQUARAPID-Va and the AQUAEIA-Va systems were able to detect 5 x 10(6) and 5 x 10(7) bacteria g(-1) of fish tissue, respectively. The simplicity, effectiveness and speed of the AQUARAPID-Va system confirmed this method as the most suitable serological test for the detection of V. anguillarum in field analysis and small-scale laboratory studies.

Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish pasteurellosis.

The accuracy of the magnetic beads-EIA based BIONOR AQUAEIA-Pp kit for the rapid diagnosis of pasteurellosis was evaluated. The kit reacted with all the Photobacterium damselae subsp. piscicida strains included in this study, with a detection limit of 10(4) bacteria/ml. However, non-specific reactions were observed with isolates of Ph. damselae subsp. damselae or Ph. histaminum when the bacterial concentration was high (10(9)-10(10) bacteria/ml). Similar findings in specificity and sensitivity were observed when the kit was applied to experimentally infected fish tissues. However, since those bacterial species are not usually found in the fish species susceptible to pasteurellosis, the AQUAEIA kit appears applicable for a rapid screening of the disease. In addition, when the kit was utilized to analyze cultured populations of seabream, it allowed the detection of the pathogen, not only in individuals affected by the disease, but also in asymptomatic carrier fish. Furthermore, the positive detection of Ph. damselae subsp. piscicida in broodstock gonads, seminal, and ovaric fluids, and also in eggs indicated the possibility of vertical transmission of pasteurellosis.