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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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Influence of medium composition on erythrocyte invasion efficiency. The invasion assays were performed in 3 basic media (white bars), and compared to the complete medium (gray bar). CaCl2 (striped bars) or MgCl2 (black bars) were added at a concentration of 1 mM into the basic media separately. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between complete medium and basic media are indicated with stars, while significant differences due to addition of calcium of magnesium in each basic medium are indicated with crosses.
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Figure 2: Influence of medium composition on erythrocyte invasion efficiency. The invasion assays were performed in 3 basic media (white bars), and compared to the complete medium (gray bar). CaCl2 (striped bars) or MgCl2 (black bars) were added at a concentration of 1 mM into the basic media separately. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between complete medium and basic media are indicated with stars, while significant differences due to addition of calcium of magnesium in each basic medium are indicated with crosses.

Mentions: In order to choose a suitable synthetic medium for performing the invasion test, we compared the invasion efficiency in culture medium (RPMI 1640 + 20% FCS) with the invasion efficiencies obtained using three synthetic media (PBS, Tris-Sucrose and RPMI 1640). Calcium and magnesium were added to each synthetic medium to find out if they could improve invasion efficiency. Free parasites were obtained by RBC osmotic lysis, and a 30 min incubation time was used. Among the three synthetic media tested, the same invasion efficiency as the culture medium was only obtained with RPMI 1640 (Figure 2). Parasitemia was significantly lower than the control when PBS or Tris-Sucrose was used. Adding calcium or magnesium had no significant effect except for RPMI 1640 supplemented with calcium. Therefore RPMI 1640 + 1 mM CaCl2 was selected for the in vitro invasion assays.


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 medium composition on erythrocyte invasion efficiency. The invasion assays were performed in 3 basic media (white bars), and compared to the complete medium (gray bar). CaCl2 (striped bars) or MgCl2 (black bars) were added at a concentration of 1 mM into the basic media separately. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between complete medium and basic media are indicated with stars, while significant differences due to addition of calcium of magnesium in each basic medium are indicated with crosses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Influence of medium composition on erythrocyte invasion efficiency. The invasion assays were performed in 3 basic media (white bars), and compared to the complete medium (gray bar). CaCl2 (striped bars) or MgCl2 (black bars) were added at a concentration of 1 mM into the basic media separately. Each figure represents the average value of parasitemia from the triplicate assays and error bars indicate the standard deviation. Significant differences (P < 0.05) between complete medium and basic media are indicated with stars, while significant differences due to addition of calcium of magnesium in each basic medium are indicated with crosses.
Mentions: In order to choose a suitable synthetic medium for performing the invasion test, we compared the invasion efficiency in culture medium (RPMI 1640 + 20% FCS) with the invasion efficiencies obtained using three synthetic media (PBS, Tris-Sucrose and RPMI 1640). Calcium and magnesium were added to each synthetic medium to find out if they could improve invasion efficiency. Free parasites were obtained by RBC osmotic lysis, and a 30 min incubation time was used. Among the three synthetic media tested, the same invasion efficiency as the culture medium was only obtained with RPMI 1640 (Figure 2). Parasitemia was significantly lower than the control when PBS or Tris-Sucrose was used. Adding calcium or magnesium had no significant effect except for RPMI 1640 supplemented with calcium. Therefore RPMI 1640 + 1 mM CaCl2 was selected for the in vitro invasion assays.

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