Development of selective agars for the isolation of Tenacibaculum spp. from ulcerative diseases in aquaculture, with emphasis on Norwegian salmon farming

Abstract

Ulcerative diseases have become a significant concern in the aquaculture industry on a global scale, including in Norway, where ulcerative diseases have posed increasing challenges for fish welfare, resulting in direct mortalities and downgrading at harvest. The primary ulcerative diseases in Norway are winter ulcer disease and tenacibaculosis. While Moritella viscosa was initially identified as the causative agent for winter ulcer disease, recent bacteriological investigations using Marine Agar (MA) have revealed the presence of Tenacibaculum spp., which also contributes to outbreaks of winter ulcer disease. Furthermore, Tenacibaculum spp. also induces the ulcerative disease called tenacibaculosis. Despite the identification of Tenacibaculum spp., difficulties have been encountered in isolating the bacteria due to the lack of sufficient agar, which may have resulted in the underdiagnosis of the bacteria.

This study aimed to create and test two new optimized media for the detection and isolation of Tenacibaculum spp. The first medium, BAMA, contains blood and sea salts, while the second, KABAMA, also includes 50 g/ml kanamycin. These agar mediums were tested and compared to commonly used agar media for bacteriological investigations of diseased fish in Norwegian aquaculture (i.e., Blood Agar with 2% NaCl (BAS) and MA) and Flexibacter Maritimus Medium (FMM), which is used in other countries for isolating Tenacibaculum spp. The ability of the new agar media to grow Tenacibaculum spp. and differentiate the bacteria was tested in both lab and field settings, with additional molecular analyses conducted to support the findings.

This study concluded that BAMA and KABAMA were the most effective agars for differentiating bacteria grown on agar media. This is due to their ability to induce gliding motility of the bacteria, as well as the appearance of different colony coloring and hemolytic activity, making it easier to identify bacterial colonies compared to MA and BAS. While FMM also induced gliding motility in the growth experiments, the coloring of the colonies was not as distinct as BAMA and KABAMA. In the field, KABAMA was found to be the most suitable agar medium for the recovery and differentiation of Tenacibaculum spp. due to the addition of 50 g/ml kanamycin. This study suggests using the agar media KABAMA and BAS for bacterial investigations of winter ulcer disease to recover and differentiate the relevant bacteria Tenacibaculum spp., M. viscosa and Aliivibrio wodanis.