INVESTIGADORES
RUYBAL paula
congresos y reuniones científicas
Título:
Flow Cytometry to Evaluate Anaplasma marginale Parasitemia Using the
Autor/es:
ROSALÍA MORETTA; PAULA RUYBAL; MARIA MESPLET; ROMINA PETRIGH; PABLO NUÑEZ; GONZALO GIL; SILVINA WILCOWSKY; GRACIELA GARBOSSA; MARISA FARBER
Lugar:
Merida, Yucatan , Mexico
Reunión:
Conferencia; 9th Biennial Conference of the Society for Tropical Veterinary Medicine; 2007
Resumen:
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., 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.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.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.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.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.