Flow cytometry to evaluate Anaplasma marginale parasitemiausing a fluorescent nucleic acid stain

MORETTA R.; RUYBAL ; MESPLET M.; PETRIGH R.; NUÑEZ P.; GIL G.; WILKOWSKY S.; GARBOSSA G.; FARBER M.

Merida, Yucatán

Conferencia; 9th Bienal Conference of the Society for Tropical Veterinary Medicine. Animal Biodiversity and Emerging Diseases; 2007

Society for Tropical Veterinary Medicine

Anaplasma marginale, an intracellular rickettsiae, is the most prevalent tick-borne pathogen of cattle with a world-wide distribution. Replication of A. marginale inside mature erythrocytes takes place in membrane-bound vauoles, called inclusion bodies that contain two to ten organisms. Flow citometry using the vital dye hidroethidine (HE) has been applied for the detection of viable parasites in a short-term cell culture of A. marginale to test drug efficacy in the treatment of anaplasmosis. On the other hand, SYTO16, a recently introduced dye that can penetrate intact live cells and bind strongly to nucleic acids, has been used to evaluate Theileria sergenti parasitemia. In that report, the authors demonstrated that SYTO16 could better differentiate between infected and non-infected erythrocytes in comparison to HE. Considering this advantage we decided to test SYTO16 to follow up the percentage of infected erythrocytes along an experimental infection with A. marginale. BD FACSCalibur Flow Cytometer was used and the samples were prepared as follows. Briefly, 3 ul of blood collected with citrate buffer were resuspended in 1 ml of PBS, SYTO16 was added to a final concentration of 250 mM and incubated at 37ºC for 30 min. Afterwards, cells were washed two times with 2ml of PBS 1X and finally resuspended in BDFACS Flow buffer. To determine sensitivity and linearity of the staining method, blood samples coming from infected cattle with high parasitemia (acute phase) were diluted with non-infected erythrocytes and the percentage of SYTO16-positive cells was checked against the expected percentage of parasitized erythrocytes (%PPE) at each dilution. A. marginale infected erythrocytes were accurately detected in samples with parasitemias ranging from 1 to 25 % PPE. Moreover, we used this methodology to follow up the parasitemia along the time course of an experimental infection during the prepatent and acute phases. Complementary studies with molecular tools such as PCR will be used to validate this method.