Identification, virulence, and mass spectrometry of toxic ECP fractions of West Alabama isolates of Aeromonas hydrophila obtained from a 2010 disease outbreak

Authors: Wei Pridgeon, Yuping; Klesius, Phillip; SONG, LIN - Auburn University; Zhang, Dunhua; KOJIMA, KYOKO - University Of Alabama; MOBLEY, JAMES A. - University Of Alabama

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2013
Publication Date: 2/28/2013
Publication URL: http://handle.nal.usda.gov/10113/56860
Citation: Wei Pridgeon, Y., Klesius, P.H., Song, L., Zhang, D., Kojima, K., Mobley, J. 2013. Identification, virulence, and mass spectrometry of toxic ECP fractions of West Alabama isolates of Aeromonas hydrophila obtained from a 2010 disease outbreak. Veterinary Microbiology. 164:336-343.

Interpretive Summary: In West Alabama, disease outbreaks in 2009 caused by Aeromonas hydrophila have led to an estimated loss of more than $3 million. In 2010, disease outbreak occurred again in West Alabama, causing losses of hundreds of thousands of pounds of market size channel catfish. During the 2010 disease outbreak in West Alabama, four isolates of Aeromonas hydrophila were cultured from the kidney tissues of diseased channel catfish. Virulence studies revealed that the four isolates were highly virulent to channel catfish. The extracellular proteins of the four 2010 isolates were found to be toxic to channel catfish fingerlings. Thirty fractions were obtained from the ECPs of the 2010 isolates of A. hydrophila by cation-exchange chromatography, of which nine were found to be toxic to catfish gill cells and channel catfish fingerlings. Mass spectrometry identified 228 proteins from the nine toxic fractions, of which 23 were shared by toxic fractions, including well known virulence factors such as hemolysin, aerolysin, elastase (metalloprotease), nuclease, and 5’-nucleotidase. Hemolytic activity, protease activity, and nuclease activity of the four isolates were found to be significantly higher than that of a reference A. hydrophila strain AL98-C1B. Our results might shed light on the possible virulence factors of the highly virulent West Alabama isolates of Aeromonas hydrophila.

Technical Abstract: In West Alabama, disease outbreaks in 2009 caused by Aeromonas hydrophila have led to an estimated loss of more than $3 million. In 2010, disease outbreak occurred again in West Alabama, causing losses of hundreds of thousands of pounds of market size channel catfish. During the 2010 disease outbreak in West Alabama, four isolates of Aeromonas hydrophila were cultured from the kidney tissues of diseased channel catfish. Both analytical profile index (API) 20 E biochemical tests and 16S-23S rRNA sequencing results confirmed the four isolates as Aeromonas hydrophila. Virulence studies revealed that the four isolates were highly virulent to channel catfish by intraperitoneal injection, with LD50 value of ~ 1.3 × 105 CFU/fish. Extracellular proteins (ECPs) of A. hydrophila are well known to be toxic to fish. Therefore, ECPs of the four 2010 west Alabama isolates of A. hydrophila were characterized in this study. The ECPs of the four 2010 isolates were found to be toxic to channel catfish fingerlings, with LD50 value of 16 µg/fish. Thirty ECP fractions were obtained from the ECPs of the 2010 isolates of A. hydrophila by cation-exchange chromatography, of which nine fractions were found to be toxic to catfish gill cells and channel catfish fingerlings. Mass spectrometry identified 228 proteins from the nine toxic fractions, of which 23 were shared by toxic fractions, including well known virulence factors such as hemolysin, aerolysin, elastase (metalloprotease), nuclease, and 5’-nucleotidase. Hemolytic activity, protease activity, and nuclease activity of the four isolates were found to be significantly (P<0.05) higher than that of a reference A. hydrophila strain AL98-C1B. Our results might shed light on the possible virulence factors of the highly virulent West Alabama isolates of Aeromonas hydrophila.