Abstract

Aeromonas salmonicida, the etiologic agent of furunculosis, is recognized as one the most important fish pathogens due to its significant economic impact on the salmonid farming industry. More recently, an atypical form of Aeromonas salmonicida has been identified as the causative agent for erythrodermatitis of carp (Cyprinus carpio L.).² This atypical form of the bacteria has also been implicated as the causative agent in a similar clinical condition in Koi (Cyprinus carpio), commonly known as Koi ulcer disease.¹ Infected Koi initially present with small, reddened, inflamed lesions primarily along the body wall and head. The inflamed areas develop into ulcers and predispose the fish to secondary bacterial infections, osmotic imbalances, and death. Fish that survive the initial infection are permanently scarred and are commonly stunted in their growth, which lowers the value for these highly prized ornamental fish. Additionally, it has been theorized that some fish may have subclinical infections, and therefore these carrier fish act as a continual source of new outbreaks of the disease. Although a variety of identification methods have been employed, such as culturing, phenotyping, and PCR-based assays, rapid identification and differentiation of Aeromonas to the species level has proven to be difficult. Atypical strains are particularly difficult to identify by biochemical means since most are slow-growing, fastidious organisms which require long incubation periods. The purpose of the present study was to investigate the use of a PCR-based assay for the rapid identification of atypical Aeromonas salmonicida isolated from Koi. Previous studies have shown that PCR amplification of the exeF-exeG intergenic region of different hybridization groups of Aeromonas can be used for the differentiation of Aeromonas species.³ The goal of this research is to develop a rapid, non-lethal diagnostic test for the detection of Aeromonas salmonicida in Koi. Five atypical Aeromonas salmonicida strains and thirteen typical strains, archived at The University of Georgia College of Veterinary Medicine were used in this study. The isolates were grown on blood agar plates at 25-30° for 48-72 hr for atypical isolates and 24-48 hr for typical isolates. Several 1-2 mm colonies were then inoculated into 3 ml of LB broth and incubated at 30° until an optic density of 0.5 was reached at 600 nm. DNA template was extracted from these cultures using the boiling method and then stored at -20°C. Alternatively, two to three 1-2-mm colonies for each isolate were resuspended in distilled water, and DNA template was extracted from these isolates using the boiling method and then stored at -20°C. Bacterial DNA was analyzed using PCR amplification of the exeF-exeG intergenic region, and the PCR products were subsequently separated by a 1.5% agarose gel containing 0.1 µ/ml of ethidium bromide. Escherichia coli DH5-α and Aeromonas hydrophila were used as negative controls. Four of the five atypical isolates and twelve of the thirteen typical isolates gave a single 160 base pair product. Of the thirteen hybridization groups identified by this method in the genus Aeromonas, a single 160 bp product size is unique to Aeromonas salmonicida. Other hybridization groups have multiple product sizes ranging from 360 to 160 base pairs. These data and preliminary experiments using clinical samples from ulcerated Koi suggest that this PCR-based assay may serve as the basis for the development of a rapid, non-lethal diagnostic test for the detection of Aeromonas salmonicida in Koi. Subsequent investigations are focused on the use of this PCR assay on clinical samples taken from Koi exhibiting dermal ulcers.

References

1.  Anders BB, VV Burnley, B Ritchie, SE Poet. 1999. Identification of the etiologic agent for ulcerative disease in koi. Proceedings of the International Association for Aquatic Animal Medicine 30:2.

2.  Bootsma R, N Fijan, J Bloomaert. 1977. Isolation and identification of the causative agent of carp erythrodermatitis. Veterinarski Archiv. 47:291-302.

3.  Karlyshev AV, S MacIntyre. 1996. Study of the intergenic exeF-exeG region and its application as a simple preliminary test for Aeromonas spp. FEMS Microbiology Letters 137:37-44.