my hypothesis

You might know that I work for a veterinary research hospital. In short, the major goal of our department (veterinary microbiology and pathology) is to apply our scientific understanding to find ways of better controlling disease. Bovine babesiosis (often referred to as Texas cattle fever in the US) is the disease I’m after. It is caused by Babesia bovis a single celled eukaryote (protozoan) parasite that infects the red blood cell of cattle.  Symptoms of this disease can be fever, pneumonia, cerebral/nervous degeneration, and death; and constitutes for enormous economical loss in the cattle industry. This parasite is only endemic where its tick vector (Boophilus microplus) persists, which is in the temperate regions of South America, Europe, Africa, Asia, and Aulstralia. B. microplus had once been eradicated from the US, but is now creeping into Texas putting ranchers, polititions, and scientists at the edge of their seat. Currently the most effective form of control for this disease is live vaccination. Cattle go through two phases of disease when infected with B. bovis: acute and persistent. The acute phase is the life threatening period lasting up to 30 days after initial infection, however if the cattle survive this phase they are persistently infected and “immune” to further exposures (like chicken pocks..sorta). Cattle before the age of 9 months show particular resistance to the effects from the acute phase of the disease. A B. bovis live vaccination is the intentional inoculation of the parasites into young cows in order to establish a persistent infection. This does protect further disease in the vaccinated cow, however does not help to control the spread of the parasite through the cattle population. In fact, you can imagine this method perpetuating the spread of B. bovis. There has been a lot of research into the development of antibabesial non-living vaccines, which are the specific pieces (recombinant DNA, protein, extracts, killed parasites, ect.) that may induce and prime antibodies against the parasite. Unfortunately, no non-living vaccine has been found that can protect as well as a live vaccine. There has been some development on an anti-tick vaccine, which in theory would keep ticks off cattle and cut the life cycle of B. bovis. The problem with this non-living vaccine is that it continually needs to be “boosted” in the cow, and continually giving multiple vaccinations with regular introvoles would be a costly and logistical nightmare for large cattle operations.

In the last decade the major work of my lab has been to develop a system for expressing foreign genes in the genome of B. bovis. In other words, we want to cut the DNA of the parasite and insert DNA that we “made” in the lab. As you know, DNA codes for everything your body makes. With this system we could make the parasite make stuff that it other wise wouldn’t. Why would we want to do this? Remember that the best protection of babesiosis is live vaccination and live parasites will persist through the life of the cattle, but does nothing to stop the spread of the parasites. And, remember that the problem with the anti-tick vaccine is that it continually has to be boosted. What if we make the B. bovis parasite express the anti-tick vaccine? Now, this is still a few years down the road, but this modified parasite could the silver bullet vaccine candidate. It will protect against disease, kill ticks, and cattle will only need to be vaccinated once.

Well, where are we at? We have developed a transfection system that is able to incorporate selectable markers into the genome of B. bovis. Selectable markers are DNA tools to see if there has been a stable incorporation resulting in the parasite expressing the exogenous (foreign) genes. We have used a gfp-bsd fusion protein. I will tell you how they work with the following figures:

BSD stands for blasticidin-S-deaminase an enzyme that breaks down the antibiotic blaticidin. So, if we grow the gfp-bsd transfected parasites in the drug we will select for the ones that are resistant to the drug. This method alows us to weed out any parasites that may not have properly incorporated the forgin gene. As you can see in the graphs above, the transfected parasites grow with the same rate with or with out the precence of the drug. This can only happen if the parasites are expressing BSD, and, as you can see, the wild type (not transfected) can not grow in the drug.

GFP stands for green fluorescence protein. This protein will emit light at 450nm (green) when subjected to UV light. So, if the parasite glows green then we can assume that the transfection system worked. The picture above seems to indicate that the parasites glow.

These transfected B. bovis parasites have been shown to be very viable growing in a culture system but have not been introduced in to cattle. This leads me to my hypothesis which I will be testing over the next two years that a transfected T2Bo Babesia bovis parasite line expressing an exogenous protein is able to establish acute clinical infection and induce an antibody response against the exogenouse protein in a bovine host.

stay tuned…