Abstract

In order to complement the ongoing preventive medicine efforts, which include immunization and surveillance, the Navy Marine Mammal Program (NMMP) is also pursuing the expansion of in-house pathodiagnosis. The ability to rapidly identify microbes of clinical relevance has been central to successful disease prevention (prophylaxis) and mitigation (therapeutics). Historically, the identification of etiologic agents has involved the synergism of several complimentary approaches: clinical presentation, pathogen-specific serology, microbiological characterization, and pathology. And indeed, NMMP currently employs all of these methods when determining etiology. However, in the past decade molecular microbiology has benefited enormously from the explosive growth of molecular techniques. A multitude of assays now exist that when used in combination with genomic information available through databases like GeneBank, can provide powerful tools for pathodiagnosis. Perhaps most significantly, these molecular applications can greatly enhance the speed, sensitivity and specificity of an etiologic diagnosis for researchers and clinicians. Applied Biosystems has recently developed the MicroSeq® Microbial Identification System. This system utilizes PCR-based DNA analysis for the amplification of unknown nucleic acid from clinical samples. RNA or DNA can be directly isolated from suspect clinical samples and then without the need for culture, used as a template from which pathogen-specific primers (targeting 16SrRNA) amplify short segments of DNA from the putative microbial agent (if present). The amplified products are visualized using agarose gel electrophoresis, purified and then sequenced (either in-house or commercially). Identification and diagnosis are then made by comparison of the sequence data to comprehensive bacterial and fungal sequence libraries provided by AB using MicroSeq® software. NMMP is in the process of adapting this new system in-house to establish proof-of-concept and feasibility of use. When used in concert with standard biochemical methods and pathogen-specific serology, MicroSeq® should greatly improve the ability of NMMP veterinarians to identify common and as yet undefined microbes of clinical significance.