Progress 09/01/06 to 08/31/09
Outputs
OUTPUTS: The following projects have been carried out in the course of this study. Establishment of A. marginale in both commercial and primary endothelial cell lines. Primary bovine endothelial cell cultures were prepared from bovine testicular vein endothelial cells and verified immunochemically with factor VIII antigen antibody as endothelial cells. The Virginia strain of A. marginale was established in these primary cell lines as stable long-term cultures. In addition to the primary endothelial cell lines, we used commercial RF/6A cells from American Type Cell Culture (ATCC) for propagation of the Bacteria. Determination of infection kinetics of A. marginale in endothelial cell lines. Real-time PCR assays using the single-copy opag2 gene and live-dead fluorescence assays were developed to monitor the kinetics of infection and the production of A. marginale suitable for infection of cattle. These assays accurately determine the presence of between 102 to 108 live organisms in a sample and have shown that production of A. marginale in cultured primary bovine endothelial cell lines and RF/6A cells reaches a maximum after two-four weeks and is evident by cytopathic effect. In some cases; however, we were able to establish and propagate low-level, minimally cytopathic long-term cultures. Interestingly these cultures cycled in vitro approximately every 9-14 days. Sample DNA collected at the time of the cyclic peaks was sequenced and analyzed to see if the compliment of variants changed from peak to peak. Two predominant variants were identified in each of the peaks representing from 65 to 89% of the variants. Cytotoxic T cell assays. Cytotoxic T cell release assays were done utilizing whole, half matched, and unmatched primary bovine endothelial cell cultures as target cells and whole PBMC from immunized and infected steers as effector cells. Significant cytotoxicity was not seen in the samples assayed. However, in order to increase MHC expression on the endothelial cells we may need to add IFN-gamma to the cultures. Inoculation of cattle with endothelial cell culture derived A. marginale. In four different trials, four young steers were inoculated with endothelial cell derived A. marginale bacteria. Trial 1 used A. marginale grown in high passaged (100 plus) RF6A Rhesus macaque endothelial cells in 75 cm2 flasks. The A. marginale Virginia strain used in this study has been in culture for approximately 4 years. A cell free fraction of the bacteria was isolated via shearing (10 passages through a 25 gauge needle) the cultured cells, low speed centrifuge to pellet intact cells, and filtered with a 3 um filter. 1x108 live bacteria were inoculated into the jugular vein of a 4-month-old mixed breed steer. Trial 2 used 2.5x109 live bacteria inoculated into the jugular vein of a second 4-month-old Holstein steer. Trial 3 used A. marginale grown in low passaged (<20) primary bovine endothelial cells derived from testicular veins. The calf was inoculated with 1x107 live bacteria and endothelial cells into the jugular vein. Trial 4 used a splenectomized steer inoculated intravenously with 1.3 x 106 A. marginale-infected endothelial cells. PARTICIPANTS: Heather Wamsley, Graduate student during this project. Jeffrey Abbott, Assistant Professor Anthony F. Barbet, Professor. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results of the above trials: Infection with A. marginale was not detected in any of the above trials. This suggests that A. marginale organisms (cultured Virginia strain) grown in mammalian endothelial cells are not infective to cattle. Vaccination with endothelial cell derived A. marginale and challenge with blood stabilate. To discover if vaccination with killed endothelial cell derived A. marginale organisms could protect against challenge, a pilot study was done. Two of the animals employed in the previous trials were subsequently given 3 inoculations separated over a 2 month period of time. The inoculum consisted of whole A. marginale Virginia strain cultured in RF6A Rhesus macaque fetal endothelial cells. The cultured bacteria were inactivated with beta-propriolactone in 0.5 ml and mixed with an equal volume 0.5 ml of ISA 50 (montinide) from Seppic Inc Fairfield NJ. The inoculum was 1 ml total volume injected subcutaneously in the left side of the neck/shoulder. The immune response was measured with lymphocyte proliferation before the time of each inoculation and 2 weeks following the last inoculation. Both animals showed significant responses to A. marginale antigen in a titrated manner after the second inoculation that continued after the third inoculum. Two months following the third inoculation, the steers were boosted a fourth time. The 2 inoculated steers and a naive control were simultaneously challenged 2 weeks following the fourth boost. The animals were inoculated IV with 20 ml of A. marginale infected blood stabilate estimated to contain approximately 8 x 1011 organisms (both live and dead). All three of the animals became bacteremic concurrent with fever and anemia. The control and one of the vaccinated steers peaked at 14 days post infection. The second vaccinated steer had a delayed peak of bacteremia, peaking at 29 DPI. Although the number of animals is small, there may be some potential for using cultured organisms as vaccine material to overcome limitations of blood-derived products. However, as with many other A. marginale vaccines, sterile immunity was not induced by these cultured organisms.
Publications
- Wamsley, HL and Barbet, AF 2008. In situ detection of Anaplasma spp. by DNA target-primed rolling-circle amplification of a padlock probe and intracellular colocalization with immunofluorescently labeled host cell von Willebrand factor. J. Clinical Microbiology 46:2314-2319.
|
Progress 09/01/06 to 08/31/07
Outputs
We have begun this project by establishing A. marginale infected and uninfected endothelial cell lines, real-time PCR assays for monitoring infection of cultures and cattle and testing infectivity of cultured A. marginale for cattle in a pilot trial. The St. Maries and Virginia strains of A. marginale were obtained from our collaborators, growing in ISE6 tick cells (St. Maries) or RF6A endothelial cells (Virginia). Primary bovine endothelial cell cultures were also prepared from bovine testicular vein endothelial cells and verified immunochemically with factor VIII antigen antibody as endothelial cells. The Virginia strain of A. marginale established in these primary cell lines as stable long term cultures. The St. Maries strain has only established in the tick cell line. Real-time PCR assays using the single-copy opag2 gene and live-dead fluorescence assays were developed to monitor the kinetics of infection and the production of A. marginale suitable for infection of
cattle. These assays accurately determine the presence of between 102 to 108 live organisms in a sample and have shown that production of A. marginale in cultured RF6A cells reaches a maximum after two-four weeks. 1 X 108 live Virginia strain A. marginale were prepared from cultures by needle disruption followed by differential centrifugation and used to intravenously inoculate a steer. Serial sampling of temperature, attitude, and blood were used to monitor the course of infection. The blood sampling included parameters of packed cell volume, fibrinogen, and total and individual leukocyte numbers. In addition to clinical monitoring microscopy of Giemsa-stained smears, real-time PCR assay of extracted DNA and seroconversion were used. Seroconversion was determined by a competitive ELISA, using sera to block reactivity of a monoclonal antibody with recombinant MSP5. Additionally, an aliquot of the inoculated organisms was used to infect new cultured endothelial cells, to verify the
infectivity of cultured A. marginale.
Impacts
All blood samples from the infected steer were negative for A. marginale up to 60 days post-infection by microscopy and real-time PCR. Similarly, only one sample tested was positive for seroconversion by cELISA and this sample was very close to the cut-off value for seropositivity. Likewise, there was no reduction in packed cell volume of the blood and no clinical symptoms indicative of infection. The inoculated organisms were confirmed to be infective in culture, as A. marginale was recovered by culture of an aliquot of the inoculum with uninfected endothelial cells. We are currently intending to immunosuppress this steer to determine if the steer was not infected or had a subclinical infection.
Publications
- No publications reported this period
|
|