Limits...
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.

Show MeSH

Related in: MedlinePlus

Influence of the methods of merozoite preparation on erythrocyte invasion efficiency. Parasites from the culture medium were collected by three different methods. The invasion assays were performed in RPMI 1640 + 1 mM CaCl2. The experiment was performed twice with similar results, and one of them is presented here. Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3117698&req=5

Figure 3: Influence of the methods of merozoite preparation on erythrocyte invasion efficiency. Parasites from the culture medium were collected by three different methods. The invasion assays were performed in RPMI 1640 + 1 mM CaCl2. The experiment was performed twice with similar results, and one of them is presented here. Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.

Mentions: Starting with the same culture material, RBC osmotic lysis and RBC electroporation produced 5 times and 29 times more "invasion-competent" parasites respectively than merozoites from the culture supernatant (Figure 3). Similar results were obtained in a second assay (data not shown).


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)

Influence of the methods of merozoite preparation on erythrocyte invasion efficiency. Parasites from the culture medium were collected by three different methods. The invasion assays were performed in RPMI 1640 + 1 mM CaCl2. The experiment was performed twice with similar results, and one of them is presented here. Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Influence of the methods of merozoite preparation on erythrocyte invasion efficiency. Parasites from the culture medium were collected by three different methods. The invasion assays were performed in RPMI 1640 + 1 mM CaCl2. The experiment was performed twice with similar results, and one of them is presented here. Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.
Mentions: Starting with the same culture material, RBC osmotic lysis and RBC electroporation produced 5 times and 29 times more "invasion-competent" parasites respectively than merozoites from the culture supernatant (Figure 3). Similar results were obtained in a second assay (data not shown).

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