RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture

Ishizaki, Takahiro; Sivakumar, Thillaiampalam; Hayashida, Kyoko; Tuvshintulga, Bumduuren; Igarashi, Ikuo; Yokoyama, Naoki

doi:10.1016/j.exppara.2016.03.010

Cited by 7 publications

(6 citation statements)

References 28 publications

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“…The method described by Ishizaki et al (2016) was used to isolate free merozoites liberated from infected RBCs. A five ml sample of an in vitro culture of B. bovis with 30% parasitemia was incubated on ice for 2 h. The culture was then resuspended in five ml of GIT medium.…”

Section: Preparation Of Free Merozoites By Cold Treatmentmentioning

confidence: 99%

“…The RBC invasion-inhibition assay was performed in triplicate in 96-well plates (Nunc, Roskilde, Denmark, Europe) according to the method employed by Ishizaki et al (2016) with some modifications. Briefly, purified free merozoites (obtained from B. bovis in vitro culture) together with uninfected bovine RBCs with a multiplicity of infection (MOI) of 4.2 were added to the GIT medium containing rabbit anti-sBbAMA-1 serum at dilutions of 1:5 and 1:10.…”

Section: In Vitro Invasion-inhibition Assaymentioning

confidence: 99%

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Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Rittipornlertrak

Nambooppha

Muenthaisong

et al. 2021

PeerJ

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Background Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. Results Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. Conclusion Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

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Section: Preparation Of Free Merozoites By Cold Treatmentmentioning

confidence: 99%

Section: In Vitro Invasion-inhibition Assaymentioning

confidence: 99%

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Rittipornlertrak

Nambooppha

Muenthaisong

et al. 2021

PeerJ

Self Cite

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show abstract

“…The method described by Ishizaki et al (2016) [50] was used to isolate free merozoite liberated from the infected RBCs. A 5 ml of an in vitro culture of B. bovis with 30% parasitemia was incubated on ice for 2 h, and then the culture was resuspended in 5 ml of GIT medium.…”

Section: Preparation Of Free Merozoites By Cold Treatmentmentioning

confidence: 99%

“…The RBC invasion-inhibition assay was performed in triplicate in a 96-well plates (Nunc, Roskilde, Denmark) according to Ishizaki et al (2016) [50] with some modi cation. Brie y, puri ed free merozoites (obtained from B. bovis in vitro culture) together with uninfected bovine RBCs with a multiplicity of infection (MOI) of 4.2 were added to GIT medium containing rabbit anti-sBbAMA-1 serum at 1:5 and 1:10 dilutions.…”

Section: In Vitro Invasion-inhibition Assaymentioning

confidence: 99%

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1

Rittipornlertrak

Nambooppha

Muenthaisong

et al. 2020

Preprint

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Background Bovine babesiosis caused by Babesia bovis (B. bovis) affects the cattle industry worldwide with high mobility and mortality. Live-attenuated vaccines are currently used in some of the endemic countries, but their wide use is limited due to various reasons. Although recombinant vaccines have been proposed as an alternative to the live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates for vaccine development against diseases caused by apicomplexan parasite species. In this study, we predicted an epitope from the plasminogen, apple and nematode (PAN) motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Results The anti-sBbAMA-1 serum obtained was evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the predicted BbAMA-1 epitope, which is located on surface-exposed α-helix of PAN motif in domain I at the apex area, elicits antibodies capable of recognizing the native BbAMA-1 in immunoassays. Importantly, as compared to the control groups, the rabbit anti-sBbAMA-1 serum at dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on day 3 and 4 of cultivation and the invasion of merozoites by approximately 60% within 4 h of incubation. Conclusion Our results indicate the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

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“…However, given that the parasite's erythrocytic cycle is quite short (1-2 cycles per day for B. bovis), it is impossible to separate ring from binary forms using these techniques. Synchronous culture could be produced using free merozoites isolated from culture supernatant 9,10 , released by electroporation 11 , or cold treatment 12 . However, all these methods have drawbacks such as the need for large scale culture volume due to their low efficiency or parasite damage in case of using electroporation.…”

Section: Introductionmentioning

confidence: 99%

Isolation of viable Babesia bovis merozoites to study parasite invasion

Hakimi

Asada

Ishizaki

et al. 2021

Preprint

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Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induce alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG) and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no role in invasion. Given the importance of invasion for establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

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RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture

Cited by 7 publications

References 28 publications

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1

Isolation of viable Babesia bovis merozoites to study parasite invasion

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