Development of diagnostic test methods for detecting key wildlife pathogens in bacteria-containing commercial biodegradation products

Abstract

Bacteria-containing commercial products, sold to facilitate biodegradation of human and animal wastes, consist of complex mixtures of bacteria. These mixtures are often of undetermined composition and grown in batch cultures from diverse bacteria-rich inocula of proprietary origins. In order to provide a means of testing for the presence of small numbers of microorganisms, pathogenic to terrestrial or avian wildlife, in bacteria-containing biodegradation products, five DNA extraction protocols were tested for their ability to purify total genomic DNA from nine biodegradation products of different formulations. A diatomaceous earth and guanidine thiocyanate-based DNA extraction method was found to be the most reliable. Fourteen microorganism-specific polymerase chain reaction (PCR)-based assays were developed. Each PCR assay was demonstrated to be specific using DNA from 61 other species of microorganisms (105 different isolates). The mean detection limit for 10 assays using cultured organisms spiked into biodegradation products was assessed. The mean was 1 × 102.62 ± 1 × 101.58 c.f.u.g−1 (bacteria) and 1 × 103.88 ± 1 × 101.14 cells g−1 (protozoa). There were no target wildlife pathogens detected by the 14 PCR assays in unspiked biodegradation products. This study has demonstrated that molecular diagnostic means can be used to detect small numbers of selected wildlife pathogens in complex biodegradation products.