You are a researcher working for the National Wildlife Health division of the U. S. Geological Survey. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from fish that is responsible for several major outbreaks of fish deaths along the coasts and in the Great Lakes. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the spirochete Cristispira clone, which is thus far noncultivatable, and you have named it Cristispira fisherii. You have subsequently determined that C. fisherii has an unusual polysaccharide capsule and that it is sensitive to chloramphenicol, tetracycline, and erythromycin but is resistant to ampicillin, penicillin, and gentamicin. You have also determined that it contains two similar circular chromosomes and three endogenous plasmids, one of which has some homology with plasmids from gram-negative bacteria. You have used this plasmid to construct an E. coli shuttle vector for C. fisherii, which you can use for genetic manipulation in E. coli and transfer into C. fisherii by electroporation. You have also determined that zebra fish, which have good innate immune systems, are an excellent animal model for the disease, which results primarily from uptake through the gills into the fish lungs, followed by invasion and dissemination through the body, and then death.
A. You are interested in identifying virulence factors associated with infection in fish. Considering all of the above information, describe in detail a strategy that you might use to identify these virulence factors. Be sure to provide a rationale for your choice of strategy, the appropriate reagents that you will need to use, the overall experimental design, and how you will determine the identities of the virulence factors.
B. Describe how you will verify that the putative virulence factors identified by your method are indeed involved in pathogenesis.
C. From your screening, you identified two genes encoding putative virulence factors, which you have named cff1 and cff2 for C. fisherii factors 1 and 2. You have created mutant strains with deletions in these two genes. The wild-type bacterium has 50% infectious dose (ID50) and 50% lethal dose (LD50) values of 10. When administered to the zebra fish via the water, both mutant strains, the
and
strains, have ID50 and LD50 values of 107 . However, when injected into the dorsal muscle of the fish, the
strain has an ID50 value of 102 and an LD50 value of 104 , while the
strain has an ID50 value of 102 and an LD50 value of 10. Provide an explanation for these findings.