You are a researcher working for the USDA. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of deaths in mammalian wildlife from contaminated pond water in the South. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the gram-negative bacterium V. cholerae, and you have named the new strain Vibrio birdsii. You have subsequently determined that V. birdsii is sensitive to tetracycline (i.e., it cannot grow in its presence) but is resistant to ampicillin. You obtained a Vibrio plasmid that you then used to construct a suitable E. coli shuttle vector for V. birdsii, which you could use for genetic manipulation in E. coli and then transfer into V. birdsii by transformation. You have also determined that guinea pigs, which have good innate immune systems, are an excellent animal model for the disease, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, and then by invasion of intestinal cells with spreading to lymph nodes and spleen, and finally death by dehydration and/or organ failure. You are interested in identifying virulence factors associated with disease in guinea pigs. You have decided to use IVET as a strategy to identify these virulence factors. After infection and harvesting of the intestine, spleen, and lymph nodes of the guinea pigs, you have isolated a number of colonies on MacConkey agar plates from your initial screen: 10 white colonies, 15 pink colonies, and 75 red colonies.
A. Draw a diagram of the shuttle vector that you constructed. Be sure to label all the key features (genes, promoters, etc.) that are necessary for it to work with IVET for V. birdsii.
B. How do you interpret the results from the initial screen? (Be sure to explain why there are three different colony colors.) What would you do with the pink colonies?
C. Assuming that you have successfully identified several potential genes from the IVET screen that may encode virulence factors, describe how you would verify that the putative virulence factors identified by your method are indeed involved in pathogenesis. Be sure to state what specific criteria must be satisfied.