Investigation of the heterogeneity among Flavobacterium psychrophilum strains devastating salmonid stocks in the laurentian Great Lakes basin.

摘要

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD), threatens wild and propagated salmonid populations and is considered one of the most important salmonid pathogens worldwide, yet a substantial lack of epidemiological knowledge exists. In particular, the heterogeneity among F. psychrophilum strains in the United States is largely unknown, which has hindered our understanding of the pathogen tracking dynamics as well as development of vaccines based on fish host species and/or highly virulent strains. I have focused my research on understanding the diversity of F. psychrophilum, particularly as it pertains to Great Lakes basin (GLB) salmonid populations. Because of the variable fish host susceptibility to F. psychrophilum and its vertical transmission, I hypothesized the pathogen prevalence among different salmonid broodstocks in the GLB will vary by species and demonstrate a link to the incidence of BCWD in resultant progeny. To test these hypotheses, a multiyear study was performed involving 7 species spanning 11 broodstock populations and their resultant progeny. Chinook salmon (Oncorhynchus tshawytscha) broodstock had the highest infection prevalence (63.2%); however, steelhead (O. mykiss) progeny had the highest incidence of BCWD. Building on these results, a thorough understanding of the genetic heterogeneity of F. psychrophilum in the GLB may influence the development of vaccines against this pathogen, particularly if dominant, highly virulent, and/or species specific strains are identified. Herein, I hypothesized the use of alternative loci, such as gyrB, murG, and tuf will demonstrate a robust intraspecific phylogeny of GLB F. psychrophilum. My results indicated a genetically diverse F. psychrophilum population, identified dominant and highly virulent strains, and demonstrated an association between sequence variation and fish host species within the GLB. To further the understanding of this genetic diversity nationwide, and allow for larger, more robust epidemiological conclusions, a multilocus typing approach was used on 96 U.S. F. psychrophilum isolates. I hypothesized that some sequence types (STs) would be unique to the GLB, while others would be widespread in their distribution. This study resulted in the identification of 28 novel STs, including ST78 which has been linked to mass mortalities in multiple states. Furthermore, these results confirm the broad distribution of ST10 within the U.S. and lend evidence to the global dissemination of F. psychrophilum. Historically, the use of antimicrobials, such as oxytetracycline and florfenicol, has been the main method of F. psychrophilum control in the U.S. In this context, I hypothesized that GLB isolates will show resistance to these antimicrobials due to their frequency of use. The phenotypic diversity among these isolates as it pertains to antimicrobial susceptibility was demonstrated using a standardized microbroth dilution assay. My results show the occurrence of oxytetracycline resistance among F. psychrophilum isolates in Michigan, although no resistance to florfenicol has been detected at this point. The demonstrated widespread distribution of F. psychrophilum and its genetic and phenotypic heterogeneity, along with the frequent BCWD outbreaks within the GLB, identifies the need for improved F. psychrophilum control measures. The documentation of strains that are fish host species specific, highly virulent, and/or resistant to commonly used antimicrobials provides a platform for development of targeted control measures including vaccination, biosecurity, and chemotherapeutic strategies.