Abstract

This presentation will review the basic design and application of an assay which utilizes inhibition of Escherichia coli D31 growth as an indicator of exogenous antibiotic activity in the channel catfish, Ictalurus punctatus. Aliquots of plasma or tissue extracts are dispensed into agar wells of plates inoculated with the test organism. The size of zones of inhibition surrounding the wells is used to quantitate antimicrobial activity when compared against known standards. Standards are formulated based on a range of antibiotic concentrations over which gradations of bacterial inhibition occur. The procedure is relatively straight forward and does not require extensive sample preparation. This assay has the advantage of measuring levels of biologically active drug in the target tissue in contrast to traditional biochemical methods which give no indication of drug activity.

For the plasma assay, a standard curve is prepared by inoculating pooled plasma from drug-free control fish with antibiotic doses which produce zones of inhibition ranging from 15 to 23 mm in diameter. Control spike samples are prepared to determine the efficiency of recovery by inoculating pooled control plasma with antibiotic at 2 ug/ml increments over four doses. Plasma from each experimental fish is inoculated with a low dose of antibiotic based on those used in the standard curve to insure the resulting zones of inhibition fall within the curve. Each well is loaded with 100 ul of plasma from the standard preparations, control spikes and experimental fish.

For a liver assay, standard, control and experimental samples are prepared in a similar manner to the plasma samples. Two gram aliquots of liver are homogenized in an equal volume of control plasma. The homogenate is centrifuged for 10 minutes at 3500 xg and the supemate recovered. The standard curve samples are prepared by inoculating the super mate as described for the plasma samples. For control spike samples, livers are inoculated with antibiotic at 2 ug/nil increments prior to homogenization. For experimental fish samples, the supemate is inoculated with an antibiotic dose which insures the zones of inhibition will fall within the standard curve. Each well is loaded with 100 ul of supemate.

Examples of plasma assay results obtained during method development are included below. All zones are measured with a caliper accurate to ± 0.05 mm. This method was developed to detect activity of romet in catfish plasma. Romet is a potentiated sulfonamide consisting of one part ormetoprim and five parts sulfadimethoxine. It is one of only two antibiotics currently approved for use in channel catfish. Recoveries of romet activity from plasma are very good ranging from 95% to 100%. In liver samples, recoveries are lower and refinement of the liver assay is continuing.

The biological assays have potential application in other species for which antibiotic activity in plasma or tissue sites has not been established. This information would enable the clinician to better design treatment schedules for an antibiotic in a particular species. Knowing effect and duration of dose is critical to maximize efficacy and discourage the onset of antibiotic resistance associated with improper dosing. Consistency is critical to insure accurate results with biological assays, however, their cost is low and they can be reliably performed in most clinical laboratories.

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