Bacillus licheniformis isolated during a fishkill is non-pathogenic

Authors: Pasnik, David; Evans, Joyce; Klesius, Phillip

Submitted to: Fisheries Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2008
Publication Date: 12/1/2008
Citation: Pasnik, D.J., Evans, J.J., Klesius, P.H. 2008. Bacillus licheniformis isolated during a fishkill is non-pathogenic. Fisheries Sciences. 74(6):1351-1353.

Interpretive Summary: When a fish kill occurred in October 2006 in the Corsica River, a tributary of the Chesapeake Bay located near Centreville, Maryland, USA, the event affected mostly white perch (Morone americana) and Atlantic menhaden (Brevoortia tyrannus), and a study was performed to determine whether pathogenic organisms caused significant mortalities among the menhaden. A cast net was used to sample Atlantic menhaden at fish kill sites in the river, and the fish were transported in a cooler with ambient river water and necropsied at the Aquatic Animal Health Research Laboratory in Chestertown, Maryland, USA. None of the sampled wild menhaden exhibited gross external or internal lesions. Samples for microbiologic examination were aseptically obtained from the nares, brain, head kidney, intestine, and posterior kidney of all menhaden. Twelve bacterial isolates were obtained from five of the seven menhaden, with isolation of bacteria from the intestines (7 isolates), nares (4), and liver (1). Four of these isolates formed flat, irregularly-shaped, white vegetative colonies that were variably mucoid and rough, and the colonies were consisted of spore-forming Gram-positive, oxidase-positive, catalase-positive rods. These isolates were identified as Bacillus licheniformis by fatty acid methyl ester (FAME) profiles of the isolates was analyzed with the Microbial Identification System (MIDI, Newark, DE, USA). A challenge experiment with one B. licheniformis isolate was conducted using Nile tilapia (Oreochromis niloticus) given their availability at the AAHRL and given the difficulties holding menhaden in aquaria. The fish were challenged by intraperitoneal injection with 1.0 x 108, 107, 106, 105, or 104 colony-forming units of B. licheniformis/fish or sham-challenged with TSB, sequestered as 10 fish/tank according to dose group, and observed for 7 d. None of the tilapia in the challenge study exhibited clinical signs of disease, and no external or internal lesions were noted. There were no mortalities after challenge, and samples for microbiologic examination taken from the nares, brain, head kidney, intestine, and posterior kidney were negative. Given that no morbidity or mortality was elicited with the experimental challenge, the findings in this study do not suggest that B. licheniformis was associated with the Corsica River fish kill and cannot provide evidence that B. licheniformis is pathogenic to fish.

Technical Abstract: When a fish kill occurred in October 2006 in the Corsica River, a tributary of the Chesapeake Bay located near Centreville, Maryland, USA, the event affected mostly white perch (Morone americana) and Atlantic menhaden (Brevoortia tyrannus), and a study was performed to determine whether pathogenic organisms caused significant mortalities among the menhaden. A cast net was used to sample Atlantic menhaden at fish kill sites in the river, and the fish were transported in a cooler with ambient river water and necropsied at the Aquatic Animal Health Research Laboratory in Chestertown, Maryland, USA. None of the sampled wild menhaden exhibited gross external or internal lesions. Samples for microbiologic examination were aseptically obtained from the nares, brain, head kidney, intestine, and posterior kidney of all menhaden. Twelve bacterial isolates were obtained from five of the seven menhaden, with isolation of bacteria from the intestines (7 isolates), nares (4), and liver (1). Four of these isolates formed flat, irregularly-shaped, white vegetative colonies that were variably mucoid and rough, and the colonies were consisted of spore-forming Gram-positive, oxidase-positive, catalase-positive rods. These isolates were identified as Bacillus licheniformis by fatty acid methyl ester (FAME) profiles of the isolates was analyzed with the Microbial Identification System (MIDI, Newark, DE, USA). A challenge experiment with one B. licheniformis isolate was conducted using Nile tilapia (Oreochromis niloticus) given their availability at the AAHRL and given the difficulties holding menhaden in aquaria. The fish were challenged by intraperitoneal injection with 1.0 x 108, 107, 106, 105, or 104 colony-forming units of B. licheniformis/fish or sham-challenged with TSB, sequestered as 10 fish/tank according to dose group, and observed for 7 d. None of the tilapia in the challenge study exhibited clinical signs of disease, and no external or internal lesions were noted. There were no mortalities after challenge, and samples for microbiologic examination taken from the nares, brain, head kidney, intestine, and posterior kidney were negative. Given that no morbidity or mortality was elicited with the experimental challenge, the findings in this study do not suggest that B. licheniformis was associated with the Corsica River fish kill and cannot provide evidence that B. licheniformis is pathogenic to fish.