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Avian - 2003
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| Project Contact: | Douglas Foster | Funding: | $65,000 |
| David Halvorson |
District:
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Unknown |
| • The Problem • Background • Objectives • Mid-Year Progress Report • Final Report • |
Currently there is no reproducible way to propagate APV to a high enough concentration, or titer, for reliable vaccine production. Previous work analyzed a number of different cell types propagated in culture from a variety of turkey embryonic tissues and found turkey kidney and turbinate epithelial cells produced the highest APV titers. The life span of such cells has also been successfully extended. These cell substrates now need to be used to produce large volumes of very high APV titers for testing against APV in turkeys and for eventual vaccine production.
A summary of Avian Respiratory Disease Research Progress (1998-2001) is available. See also the project topic page for 2002.
Update – February 2003
Since Avian Pneumovirus (APV) is such an important pathogen to the turkey industry, it is expected that the propagation of high amounts (known as "titers") of APV in turkey cells will be invaluable for use as a vaccine. This project is propagating APV to high titers using turkey cell substrates. The turkey-specific high titer virus will soon be tested in turkeys and compared to monkey-specific virus propagated to equivalent titers.
Primary turkey turbinate and kidney cells isolated from 17 to 20-day-old embryos were used to incubate APV strain MN2a. As that virus strain is of a non-turkey cell line, it was also propagated in monkey Vero cells. The amounts of APV from all three cell types (turbinate, kidney and monkey) were analyzed by several means, after using both a new hypotonic burst method and a conventional freeze-thaw method to liberate the virus particles from the host cells.
These methods have produced about 100 ml of APV in turkey turbinate and kidney cells and in monkey Vero cells at a titer of approximately 109 particles per milliliter. Further refinement of the processes are expected to produce at least 100 ml of APV at greater than 1010 particles per milliliter. This will be used for in vivo testing scheduled for this spring.
Primary turkey turbinate and kidney cells isolated from17-20 day old embryos (kindly supplied by the Jenny-O Turkey Store, Barron, WI) were used to passage avian pneumovirus (APV) strain MN2a. Researchers also propagated the virus in monkey Vero cells. APV titers (reported as plaque forming units/ml) from all three cell types were determined using Vero cell substrates in multi-well plates and analyzed by either immunofluorescent staining or plaque formation. Researchers also used a new hypotonic burst method to liberate APV particles from host cells and compared this to the conventional freeze-thaw method.
Using the above isolation methods researchers produced about 100 ml of APV in turkey turbinate and kidney cells and in monkey Vero cells at a titer of approximately 106-7/ml. The APV levels for the hypotonic burst method were not significantly different than the freeze-thaw method.
The researchers have succeeded in one of the long-term goals of the project-the development of the world’s first and only non-virally immortalized continuously growing turkey cell line (T T-1) that was derived from turkey turbinate epithelial tissue. They have also ascertained that this new turkey-specific cell line supports the propagation of APV. The researchers consistently have been able to achieve APV titers as good or better than the currently used standard substrate, monkey Vero cells.
They have made major progress with the immortalization of a turkey-specific cell line that originated from the tissue that is one of the first lines of defense against APV. They have further shown that this homologous turkey cell line propagates APV at least as well as monkey Vero cells. They also are using their newly established T T-1 cells to attenuate the APV MN2a virus for eventual use as a vaccine in the field. For this somewhat time consuming side-project they are propagating APV for at least 50 passages in T T-1 cells (something that previously could not be accomplished in primary, non-immortalized turkey turbinate cells). This attenuated APV will be the substrate for future in vivo testing using their infectious model.
Impacts Resulting from the Research:
The researchers have shown that turkey epithelial cells can produce APV titers equal to or greater than the best currently available cell substrate (which is not even avian). Using either the conventional freeze-thaw or the newly developed hypotonic burst method, they have been able to produce at least 100 ml of APV in turkey turbinate and kidney cells as well as monkey Vero cells for subsequent in vivo testing. They have been successful in developing the world’s only immortal turkey turbinate epithelial cell substrate for the continuous propagation of high titer APV.