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

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.

Babesia divergensbuilds a complex population structure composed of specific ratios of infected cells to ensure a prompt response to changing environmental conditions

“…Other methods that isolate free merozoites spontaneously released in B . divergens culture (Montero et al ., ) and electroporation of iRBC (Franssen et al ., ; Sun et al ., ) result in low invasion rates in our hands. By avoiding low temperatures (Cursino‐Santos et al ., ) and multiple centrifugations, and using a novel method to release infectious merozoites to infect RBCs, we were able to obtain highly synchronized parasite populations to exploit for a detailed chronological characterization of the IEC of B .…”

“…Thus, methods based on density differences like Percoll centrifugation or osmotic lysis of mature malaria stages (Lambros and Vanderberg, 1979;Russmann et al, 1982;Wahlgren et al, 1983) have not yielded synchronous cultures. Other methods that isolate free merozoites spontaneously released in B. divergens culture (Montero et al, 2006) and electroporation of iRBC (Franssen et al, 2003;Sun et al, 2011) result in low invasion rates in our hands. By avoiding low temperatures (Cursino-Santos et al, 2014) and multiple centrifugations, and using a novel method to release infectious merozoites to infect RBCs, we were able to obtain highly synchronized parasite populations to exploit for a detailed chronological characterization of the IEC of B. divergens.…”

Evaluation of a Major Surface Antigen of Babesia microti Merozoites as a Vaccine Candidate against Babesia Infection

“…B. microti is a protozoan hemoparasite of the phylum Apicomplexa and is the causative agent of human babesiosis. There are diverse mechanisms through which the host defends against this parasitic attack, however, there have been very limited efforts made to understand the process of invasion of the erythrocytes by B. microti parasites and the many molecules associated with this process (Montero et al, 2009 ; Sun et al, 2011 ). The molecules that are involved in this process of invasion are likely to be important virulence factors (Alzan et al, 2016 ) that can be explored as potential candidates for vaccine development or to develop novel therapies, with the ultimate goal of preventing babesiosis and eradicating Babesia parasites.…”

The Complexity of Piroplasms Life Cycles

“…Parasite orientation and penetration is mediated by proteins secreted from the apical secretory organelles (Dubremetz et al, 1998 ; Soldati et al, 2004 ), and thus is accompanied by apposition of apical organelles with the host cell membrane (Ward and Jack, 1981 ). Similarly with Theileria, Babesia internalizes within a few minutes without parasitophorous vacuole formation (Table 1 ), and thus the parasite lies freely within the host cell cytoplasm (Simpson et al, 1963 ; Rudzinska et al, 1976 ; Potgieter and Els, 1977b ; Kawai et al, 1999a , b ; Guimarães et al, 2003 ; Montero et al, 2009 ; Sun et al, 2011 ; Lobo et al, 2012 ). The invasion process does not differ for Babesia sensu stricto species (Montero et al, 2009 ; Asada et al, 2012 ) and B. microti (Rudzinska, 1976 ; Rudzinska et al, 1976 ).…”