Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes

https://doi.org/10.1016/j.bbrc.2003.11.093Get rights and content

Abstract

The channel catfish (Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment. Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila. The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities. Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish. Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid. The granule extracts were further purified by gel filtration chromatography. Bactericidal assays against the two fish pathogens and SDS–PAGE analysis were done on the isolated antimicrobial factor. Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1. Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor.

Section snippets

Materials and methods

Maintenance of channel catfish. Adult catfish were maintained in tanks at 27 °C in a recirculating water-reuse culture system at the Ecological Research Center (Dr. Bill Simco, Director), Department of Biology, University of Memphis [11], [12], [13]. Channel catfish were provided by Dr. William Wolters, USDA-ARS, Catfish Genetics Research Unit, Stoneville, MS.

Separation of leucocytes from channel catfish blood. Fifty milliliters of heparinized blood was obtained by bleeding from the caudal sinus

Results

A blood cell fraction of leucocytes was isolated from channel catfish peripheral blood by density gradient centrifugation. A procedure that specifically extracts a granular-rich sediment from human neutrophils was used for the catfish leucocytes [15]. The leucocytes were first homogenized to obtain granule-rich supernatants by low-speed centrifugation. The supernatants were then combined and subjected to high-speed centrifugation to obtain a pellet of granules. A 10% acetic acid extract of the

Discussion

We have extensively investigated immunity in the channel catfish concentrating on the complement system [5], [11], [12], [13], [28], [30], [31], [32], [33], [34], [35]. It was found that both the classical and alternative complement pathways are activated and can have a bactericidal effect against catfish pathogens. Bactericidal activity by catfish serum, mediated by the alternative complement pathway, was an important means of defense against many Gram-negative bacteria. The alternative

Acknowledgements

The authors thank the following individuals for their assistance: Dr. William Lewis, Center for Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN, for doing Edman degradation for amino acid sequence analysis; Dr. Weixing Zhang, Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, for doing NMR structural analysis; Dr. Edward Umstot, Stout Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, TN, for doing

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    Present address: Department of Life Science and Biotechnology, Dong Eui University, Pusan, Korea.

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