Studies on the Epidemiology, Vaccination, Susceptibility, and Treatment of Columnaris Disease in Fishes

Abstract

In this dissertation, I explored the intraspecies genetic heterogeneity within Flavobacterium columnare isolates collected during particularly severe columnaris outbreaks observed at the E.W. Shell Fisheries Center, Aquatic Experiment Research Station at North Auburn, AL, USA. These outbreaks affected both farmed and sport fish species that were raised in ponds located under the same hydrologic unit from spring 2010 throughout summer 2012. All the recovered isolates were ascribed to genomovar II of F. columnare following restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene. Fingerprinting using amplified fragment length polymorphism (AFLP) revealed genetic diversity among the genomovar II isolates and persistence of certain clones throughout the watershed. Antimicrobial susceptibility testing of the isolates demonstrated resistance to some of the commonly used antimicrobial agents in aquaculture. I also compared the vaccine efficacy of newly generated stable and safe rifampicin resistant genomovar II mutants to that of the attenuated mutant strain in the commercial vaccine (AQUAVAC-COLTM, Merk), which is derived from a genomovar I (the less virulent genomovar) F. columnare strain. Based on relative percent survival rates in channel catfish, zebrafish, and Nile tilapia, both genomovar I and II mutants were equally protective against columnaris disease when a genomovar I strain of F. columnare was used to challenge the vaccinated fish. However, administration of the new genomovar II attenuated mutant as vaccine conferred greater protection against columnaris disease caused by a genomovar II strain than that provided by the genomovar I mutant. Because healthy microbiome on the skin and gills of fish was reported to benefit the hosts and prevent infections by opportunistic pathogens by hindering their invasion and/or stimulating the fish’s immune system, I investigated the effect of disrupting the surface microbiome of channel catfish by short term exposure to one of the commonly used surface-acting disinfectants in aquaculture: potassium permanganate (PP) (KMnO4). I tested the susceptibility of fish treated with PP to columnaris disease. Dysbiosis of the surface microbiome was analyzed by ribosomal intergenic spacer analysis (RISA) and pyrosequencing. Chemical treatment with PP altered the composition of the external microbiome and increased catfish mortality following experimental challenge with F. columnare. Finally, I tested the in vitro and in vivo efficacy of Nigella sativa, a medicinal plant commonly used in folk medicine by many nations to treat a wide range of diseases, against columnaris disease. I found that N. sativa oil possess a potent antibacterial activity against all genomovars of F. columnare by disc diffusion method. Moreover, supplementation of fish diets with N. sativa seeds or oil were protective to zebrafish and channel catfish against columnaris disease in controlled laboratory challenges. In summary, data obtained during my dissertation emphasized the importance of genomovar II isolates as causative agents of high mortality-columnaris disease in aquaculture and recreationally valued fish species. I tested two new promising methodologies to prevent and control columnaris disease and proved that caution should be used when treating external diseases in fish to avoid damaging the external microbiome.