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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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Improvement of invasion efficiency evaluation. Merozoites collected from culture supernatant were used to perform the invasion assay in RPMI 1640. Parasitemia was counted before (white bars), after Percoll separation (grey bars) and after 5 h incubation in culture medium (black bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.
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Figure 1: Improvement of invasion efficiency evaluation. Merozoites collected from culture supernatant were used to perform the invasion assay in RPMI 1640. Parasitemia was counted before (white bars), after Percoll separation (grey bars) and after 5 h incubation in culture medium (black bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.

Mentions: Parasitemia was measured before and after Percoll separation, and after 5 h incubation in culture medium to determine the efficiency of Percoll separation in eliminating parasites that did not invade erythrocytes during the invasion test and to control the non-toxicity of Percoll on intra-erythrocytic parasites. These measurements were performed 5, 10 and 120 min after the initial contact between RBC and merozoites. Regardless of the time of contact, a significant decrease in parasitemia from 14 fold (5 min) to 4 fold (120 min), was obtained after Percoll separation (Figure 1). The observed decrease in parasitemia according to the time of contact before Percoll separation was not correlated with a corresponding increase of invading parasites (after Percoll). No significant differences in parasitemia were observed before or after additional incubation in culture medium at any of the three contact times (Figure 1). That is to say that Percoll is compatible with the subsequent in vitro growth of B. divergens.


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)

Improvement of invasion efficiency evaluation. Merozoites collected from culture supernatant were used to perform the invasion assay in RPMI 1640. Parasitemia was counted before (white bars), after Percoll separation (grey bars) and after 5 h incubation in culture medium (black bars). 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 1: Improvement of invasion efficiency evaluation. Merozoites collected from culture supernatant were used to perform the invasion assay in RPMI 1640. Parasitemia was counted before (white bars), after Percoll separation (grey bars) and after 5 h incubation in culture medium (black bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation.
Mentions: Parasitemia was measured before and after Percoll separation, and after 5 h incubation in culture medium to determine the efficiency of Percoll separation in eliminating parasites that did not invade erythrocytes during the invasion test and to control the non-toxicity of Percoll on intra-erythrocytic parasites. These measurements were performed 5, 10 and 120 min after the initial contact between RBC and merozoites. Regardless of the time of contact, a significant decrease in parasitemia from 14 fold (5 min) to 4 fold (120 min), was obtained after Percoll separation (Figure 1). The observed decrease in parasitemia according to the time of contact before Percoll separation was not correlated with a corresponding increase of invading parasites (after Percoll). No significant differences in parasitemia were observed before or after additional incubation in culture medium at any of the three contact times (Figure 1). That is to say that Percoll is compatible with the subsequent in vitro growth of B. divergens.

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