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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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Inhibition of invasion by sera directed against B. divergens. Invasion was performed with antisera of calf C139 and sheep S-M-SC (black bars) and compared to the negative sera of calf and sheep (white bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between antisera and negative sera are indicated with stars.
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Figure 7: Inhibition of invasion by sera directed against B. divergens. Invasion was performed with antisera of calf C139 and sheep S-M-SC (black bars) and compared to the negative sera of calf and sheep (white bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between antisera and negative sera are indicated with stars.

Mentions: The invasion assay was used to test the inhibitory effect of two selected sera on invasion (Figure 7). For both sera, an inhibition of invasion was observed at each dilution tested (up to 1/8) and the effect was dose-dependent. Invasion was reduced by up to 80% in the case of calf antiserum. Sheep negative serum also had an inhibitory effect on invasion, an effect that had already been noted on growth of B. divergens [36]. It could therefore be postulated that the proteins detected by Western blotting with these sera had a role in the invasion process.


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)

Inhibition of invasion by sera directed against B. divergens. Invasion was performed with antisera of calf C139 and sheep S-M-SC (black bars) and compared to the negative sera of calf and sheep (white bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between antisera and negative sera are indicated with stars.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Inhibition of invasion by sera directed against B. divergens. Invasion was performed with antisera of calf C139 and sheep S-M-SC (black bars) and compared to the negative sera of calf and sheep (white bars). Each figure represents the average value of the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between antisera and negative sera are indicated with stars.
Mentions: The invasion assay was used to test the inhibitory effect of two selected sera on invasion (Figure 7). For both sera, an inhibition of invasion was observed at each dilution tested (up to 1/8) and the effect was dose-dependent. Invasion was reduced by up to 80% in the case of calf antiserum. Sheep negative serum also had an inhibitory effect on invasion, an effect that had already been noted on growth of B. divergens [36]. It could therefore be postulated that the proteins detected by Western blotting with these sera had a role in the invasion process.

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