A method to diagnose the carrier state of Vibrio vulnificus serovar E in eels: Development and field studies

The pathogen Vibrio vulnificus serovar E (VSE) has been related to both human infections and to epizootics causing high mortality in brackish water eel farms. To control the spread of the eel vibriosis and prevent VSE transmission to humans we designed and tested a protocol to detect carriers, which involves isolating the pathogen. To identify the organs where VSE persists in survivors we infected eels with different degrees of immunity against the pathogen (non-immune [NI], immune [I, eels vaccinated 1 year before] and freshly vaccinated [V]) by bath challenge. Then, we followed the pathogen survival in selected external and internal organs for 72 h post-infection. VSE was isolated by using previously described protocols [Sanjuan, E., Amaro, C., 2004. Protocol for specific isolation of virulent strains of V. vulnificus serovar E (biotype 2) from environmental samples. Appl. Environ. Microbiol. 70, 7024-7032.] and identified by a new specific PCR methodology [Lee C.-T., Amaro, C., Sanjuan, E., Hor, L-I., 2005. Identification of DNA sequences specific for V. vulnificus biotype 2 strains by suppression subtractive hybridization. Appl. Environ. Microbiol. 71: 5593-5597.]. The pathogen survival depended on the immunological status of the animal. Thus, VSE persisted in the gills and spleen of NI and I eels but was eliminated by the immune system of V eels in less than 9 h post-infection. We selected gills as the organ to be sampled and designed a whole protocol for carrier detection. The feasibility of the protocol was demonstrated in field studies after the positive isolation of the pathogen from asymptomatic carriers without killing the animals.