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The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay.

Sun Y, Moreau E, Chauvin A, Malandrin L - Vet. Res. (2011)

Bottom Line: In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion.The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process.Further investigations are required for their characterisation.

View Article: PubMed Central - HTML - PubMed

Affiliation: ONIRIS, UMR1300, Biologie, Epidémiologie et Analyse de Risque en Santé Animale, Route de Gachet, La Chantrerie, BP 40706, F-44307 Nantes, France. yi.sun@oniris-nantes.fr.

ABSTRACT
Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

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a). Kinetics of erythrocyte invasion. Curves indicate the percentage of erythrocytes invaded with parasites prepared by high-voltage electroporation (squares) or by osmotic lysis (rounds) according to the time of incubation. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. b). Photomicrographs of bovine erythrocytes following an invasion assay with merozoites prepared by electroporation. The photos were taken immediately after contact and at 10 min and 120 min.
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Figure 4: a). Kinetics of erythrocyte invasion. Curves indicate the percentage of erythrocytes invaded with parasites prepared by high-voltage electroporation (squares) or by osmotic lysis (rounds) according to the time of incubation. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. b). Photomicrographs of bovine erythrocytes following an invasion assay with merozoites prepared by electroporation. The photos were taken immediately after contact and at 10 min and 120 min.

Mentions: B. divergens parasites prepared by RBC lysis (osmotic and electroporation) were used to follow the kinetics of invasion by analysing parasitemia after contact with RBC ranging from 0 to 240 min. Although a huge difference in the maximum parasitemia attained in these two assays was observed (which confirmed the choice of electroporation as the better method of parasite preparation), the trends of the two curves were comparable (Figure 4a). B. divergens invasion of RBC occurred within seconds. A contact of 45 s (the time required to mix RBC with parasites, and to perform Percoll separation) was indeed sufficient to attain 50% (RBC osmotic lysis) and 70% (RBC electroporation) of maximum parasitemia. Ten minutes after the initial contact, all "invasion-competent" parasites had penetrated the RBC, since no further increase in parasitemia was observed over time. Microphotographs of Giemsa-stained smears prepared immediately after contact (about 5 s), at 10 min and 120 min after contact and before Percoll separation were taken to show the changes in parasite morphology (Figure 4b). Free parasites, some adhering to the RBC surface, were visible after contact (0, 10 min). Their quantities seemed to diminish with time (120 min). Intra-cellular parasites were visible immediately after the contact with RBC. Their shape changed rapidly, from condensed intra-erythrocytic black particles at the very beginning, to trophozoites with the appearance of cytoplasms (white areas) inside the parasite at ten minutes, which expanded to become clearly visible at 120 min. Parasites with homogeneous size and shape were still visible 120 min after the beginning of the culture.


The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay.

Sun Y, Moreau E, Chauvin A, Malandrin L - Vet. Res. (2011)

a). Kinetics of erythrocyte invasion. Curves indicate the percentage of erythrocytes invaded with parasites prepared by high-voltage electroporation (squares) or by osmotic lysis (rounds) according to the time of incubation. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. b). Photomicrographs of bovine erythrocytes following an invasion assay with merozoites prepared by electroporation. The photos were taken immediately after contact and at 10 min and 120 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117698&req=5

Figure 4: a). Kinetics of erythrocyte invasion. Curves indicate the percentage of erythrocytes invaded with parasites prepared by high-voltage electroporation (squares) or by osmotic lysis (rounds) according to the time of incubation. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. b). Photomicrographs of bovine erythrocytes following an invasion assay with merozoites prepared by electroporation. The photos were taken immediately after contact and at 10 min and 120 min.
Mentions: B. divergens parasites prepared by RBC lysis (osmotic and electroporation) were used to follow the kinetics of invasion by analysing parasitemia after contact with RBC ranging from 0 to 240 min. Although a huge difference in the maximum parasitemia attained in these two assays was observed (which confirmed the choice of electroporation as the better method of parasite preparation), the trends of the two curves were comparable (Figure 4a). B. divergens invasion of RBC occurred within seconds. A contact of 45 s (the time required to mix RBC with parasites, and to perform Percoll separation) was indeed sufficient to attain 50% (RBC osmotic lysis) and 70% (RBC electroporation) of maximum parasitemia. Ten minutes after the initial contact, all "invasion-competent" parasites had penetrated the RBC, since no further increase in parasitemia was observed over time. Microphotographs of Giemsa-stained smears prepared immediately after contact (about 5 s), at 10 min and 120 min after contact and before Percoll separation were taken to show the changes in parasite morphology (Figure 4b). Free parasites, some adhering to the RBC surface, were visible after contact (0, 10 min). Their quantities seemed to diminish with time (120 min). Intra-cellular parasites were visible immediately after the contact with RBC. Their shape changed rapidly, from condensed intra-erythrocytic black particles at the very beginning, to trophozoites with the appearance of cytoplasms (white areas) inside the parasite at ten minutes, which expanded to become clearly visible at 120 min. Parasites with homogeneous size and shape were still visible 120 min after the beginning of the culture.

Bottom Line: In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion.The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process.Further investigations are required for their characterisation.

View Article: PubMed Central - HTML - PubMed

Affiliation: ONIRIS, UMR1300, Biologie, Epidémiologie et Analyse de Risque en Santé Animale, Route de Gachet, La Chantrerie, BP 40706, F-44307 Nantes, France. yi.sun@oniris-nantes.fr.

ABSTRACT
Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

Show MeSH
Related in: MedlinePlus