Concurrent bacterial infections and bacterial characterization from columnaris diseased fish

Abstract

Columnaris disease, a bacterial infection caused by Flavobacterium columnare, has been reported in various freshwater fish species worldwide. Disease outbreaks usually result in mass mortality of cultured fish and severe financial losses for aquaculture producers. In aquaculture system, where the fish naturally exposed to various potential pathogens, the reality of dead-loss due to dual or multiple infections is highly predictable and probably outweighs single infection. This study aims to investigate concurrent bacterial infections and pathogen characterization from columnaris diseased fish with emphasis on tilapia and striped catfish. From two outbreaks in Nile tilapia farms in Thailand where fish exhibiting external clinical signs of columnaris disease, each naturally diseased fish was found to be concurrently infected by 2 to 4 out of the 5 identified pathogens including F. columnare, Aeromonas veronii, Streptococcus agalactiae, Plesiomonas shigeloides and Vibrio cholera. Upon experimental challenge tests, fish exposed to A. veronii or F. columnare mimicked major internal and external clinical signs of naturally infected fish, respectively. This suggested that A. veronii and F. columnare are two main pathogens co-responsible for the dead-loss of the outbreak cases reported in the present study, whereas remaining pathogens might serve as opportunistic agents in the disease outbreaks. Similarly, natural co-infections of F. columnare and Edwardsiella ictaluri were first reported in the striped catfish exhibiting external clinical signs of columnaris disease. Co-infection challenge by both intraperitoneal and intramuscular routes successfully mimicked typical signs and histopathological manifestations of natural co-infections thus fulfilling Koch’s postulates. Genetic diversity of F. columnare revealed that isolates originated from tilapia and striped catfish were phylogenetically different based on 16S rRNA. It was also found that the majority of F. columnare isolates from Thailand belongs to Genomovar II and formed rhizoid morphotype on AOA medium. The virulence of two morphotypes (rhizoid and non-rhizoid colonies) of F. columnare from tilapia collection was determined in vivo. The typical rhizoid isolate (CUVET1214) was a highly virulent isolate whereas the non-rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Adherence and persistence of both F. columnare morphotypes to tilapia fry were determined by whole fish bacterial loads. The results suggested that an inability of the non-rhizoid morphotype to persist in tilapia fry may explain lack of virulence. Additionally, diversity of non-F. columnare bacteria associated with columnaris diseased fish was characterized and proven to be merely opportunistic bacteria in this study.