Establishment of Babesia bovis In Vitro Culture Using Medium Free of Animal Products

Babesia bovis, an etiological agent of bovine babesiosis, causes a significant burden to the cattle industry worldwide. The most efficient method to mitigate bovine babesiosis is a live vaccine produced by serial passage in splenectomized cattle. However, there are several concerns regarding live vaccine production, including variation between batches and the use of many animals. In this study, we report a B. bovis-SF strain continuously cultured in a medium free of components of animal origin enriched with a chemically defined lipid mixture (CD lipid mixture) and the use of a perfusion bioreactor to harvest a large amount of B. bovis. Six culture media were compared, including VP-SFM, CD-CHO, CD-Hydrolyzed, CD-CHO, SFM, and ADMEM/F12. We found that the VP-SFM medium performed the best for B. bovis growth, with a maximum percentage of parasitized erythrocytes (PPE) of 8.6%. The effect of six dilutions of a commercial mixture of CD lipids added to VP-SFM showed that the CD lipid mixture at a dilution of 1:100 had the best B. bovis growth curve, with a maximum PPE of 13.9%. Propagation of the in vitro B. bovis culture was scaled up in a perfusion bioreactor using VP-SFM with a CD lipid mixture, and the PPE reached over 32%. The continuous in vitro B. bovis culture in a medium free of animal origin components could potentially reduce and replace the use of animals to produce a reagent for diagnostics and live vaccines to control bovine babesiosis.

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Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin

Pathogens ◽

10.3390/pathogens9050343 ◽

2020 ◽

Vol 9 (5) ◽

pp. 343

Author(s):

Jesús A. Álvarez Martínez ◽

Julio V. Figueroa Millán ◽

Massaro W. Ueti ◽

Carmen Rojas-Martínez

Keyword(s):

In Vitro Culture ◽

Culture Media ◽

Culture Method ◽

Animal Origin ◽

Acid Mixture ◽

Babesia Bigemina ◽

Lipid Mixture ◽

Proliferation In Vitro ◽

Animal Component

In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes.

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

10.21203/rs.3.rs-58018/v1 ◽

2020 ◽

Author(s):

Carlos Suarez ◽

Marta G. Silva ◽

Reginaldo G. Bastos ◽

J. Stone Doggett ◽

Michael K. Riscoe ◽

...

Keyword(s):

Mononuclear Cells ◽

Babesia Bovis ◽

Host Cells ◽

Bovine Babesiosis ◽

Theileria Equi ◽

Peripheral Blood Mononuclear ◽

Equine Piroplasmosis ◽

Apicomplexan Parasites ◽

And Control

Abstract Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of actively replicating horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by ELISA. Results: We calculated IC50 ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 hr among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQs-300 and ELQ-316 at IC100, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 hours. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions: ELQ-300 and ELQ-316 have a significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

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Diagnostic Tools for the Identification of Babesia sp. in Persistently Infected Cattle

Pathogens ◽

10.3390/pathogens8030143 ◽

2019 ◽

Vol 8 (3) ◽

pp. 143 ◽

Cited By ~ 7

Author(s):

J. Antonio Alvarez ◽

Carmen Rojas ◽

Julio V. Figueroa

Keyword(s):

Clinical Signs ◽

International Health ◽

Babesia Bovis ◽

Diagnostic Tools ◽

Infected Cattle ◽

Babesia Bigemina ◽

Bovine Babesiosis ◽

Culture Methods ◽

Persistently Infected

Bovine babesiosis is a tick-borne disease of cattle caused by the protozoan parasites of the genus Babesia. Babesia bovis, Babesia bigemina and Babesia divergens are considered by International health authorities (OIE) as the principal species of Babesia that cause bovine babesiosis. Animals that recover from a babesial primo infection may remain as persistent carriers with no clinical signs of disease and can be the source of infection for ticks that are able to acquire Babesia parasites from infected cattle and to transmit Babesia parasites to susceptible cattle. Several procedures that have been developed for parasite detection and diagnosis of this infectious carrier state constitute the basis for this review: A brief description of the direct microscopic detection of Babesia-infected erytrocytes; PCR-based diagnostic assays, which are very sensitive particularly in detecting Babesia in carrier cattle; in-vitro culture methods, used to demonstrate presence of carrier infections of Babesia sp.; animal inoculation, particularly for B. divergens isolation are discussed. Alternatively, persistently infected animals can be tested for specific antibabesial antibodies by using indirect serological assays. Serological procedures are not necessarily consistent in identifying persistently infected animals and have the disadvantage of presenting with cross reactions between antibodies to Babesia sp.

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

10.21203/rs.3.rs-58018/v3 ◽

2020 ◽

Author(s):

Marta G. Silva ◽

Reginaldo G. Bastos ◽

J. Stone Doggett ◽

Michael K. Riscoe ◽

Sovitj Pou ◽

...

Keyword(s):

Mononuclear Cells ◽

Colorimetric Assay ◽

Babesia Bovis ◽

Host Cells ◽

Bovine Babesiosis ◽

Theileria Equi ◽

Peripheral Blood Mononuclear ◽

Equine Piroplasmosis ◽

And Control

Abstract Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay.Results: We calculated IC50 ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 h among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQ-300 and ELQ-316 at IC100, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions: ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

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Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

Parasitology International ◽

10.1016/j.parint.2017.11.004 ◽

2018 ◽

Vol 67 (2) ◽

pp. 190-195 ◽

Cited By ~ 6

Author(s):

Carmen Rojas-Martínez ◽

Roger Iván Rodríguez-Vivas ◽

Julio Vicente Figueroa Millán ◽

Carlos Ramón Bautista-Garfias ◽

Roberto Omar Castañeda-Arriola ◽

...

Keyword(s):

Babesia Bovis ◽

Serum Free Medium ◽

Free Medium ◽

Babesia Bigemina ◽

Bovine Babesiosis ◽

Serum Free

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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

PeerJ ◽

10.7717/peerj.11765 ◽

2021 ◽

Vol 9 ◽

pp. e11765

Author(s):

Amarin Rittipornlertrak ◽

Boondarika Nambooppha ◽

Anucha Muenthaisong ◽

Veerasak Punyapornwithaya ◽

Saruda Tiwananthagorn ◽

...

Keyword(s):

Vaccine Development ◽

Apical Membrane ◽

Rabbit Antiserum ◽

Babesia Bovis ◽

Inhibitory Effects ◽

Bovine Babesiosis ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen ◽

Domain I

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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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

10.21203/rs.3.rs-58018/v4 ◽

2020 ◽

Author(s):

Marta G. Silva ◽

Reginaldo G. Bastos ◽

J. Stone Doggett ◽

Michael K. Riscoe ◽

Sovitj Pou ◽

...

Keyword(s):

Mononuclear Cells ◽

Colorimetric Assay ◽

Babesia Bovis ◽

Host Cells ◽

Bovine Babesiosis ◽

Theileria Equi ◽

Peripheral Blood Mononuclear ◽

Equine Piroplasmosis ◽

And Control

Abstract Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay.Results: We calculated IC50 ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 h among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQ-300 and ELQ-316 at IC100, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions: ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

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Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651984000200005 ◽

1984 ◽

Vol 26 (2) ◽

pp. 97-104 ◽

Cited By ~ 1

Author(s):

Edda de Rizzo ◽

Celidéia Aparecida Coppi Vaz ◽

Inácio França Mendes ◽

Anatércia Ferreira Bonfim Yano

Keyword(s):

Bovine Serum ◽

Culture Media ◽

Fetal Bovine Serum ◽

Calf Serum ◽

Animal Origin ◽

Electrophoretic Study ◽

Fetal Bovine ◽

Growth Promoting ◽

Antiproteolytic Activity

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

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Recent Advances in Molecular Genetic Tools for Babesia

Veterinary Sciences ◽

10.3390/vetsci8100222 ◽

2021 ◽

Vol 8 (10) ◽

pp. 222

Author(s):

Hassan Hakimi ◽

Masahito Asada ◽

Shin-ichiro Kawazu

Keyword(s):

Drug Targets ◽

Molecular Genetic ◽

Essential Genes ◽

Babesia Bovis ◽

Bovine Babesiosis ◽

Genetic Tools ◽

Genome Wide ◽

Glms Ribozyme ◽

Novel Drug

Development of in vitro culture and completion of genome sequencing of several Babesia parasites promoted the efforts to establish transfection systems for these parasites to dissect the gene functions. It has been more than a decade since the establishment of first transfection for Babesia bovis, the causative agent of bovine babesiosis. However, the number of genes that were targeted by genetic tools in Babesia parasites is limited. This is partially due to the low efficiencies of these methods. The recent adaptation of CRISPR/Cas9 for genome editing of Babesia bovis can accelerate the efforts for dissecting this parasite’s genome and extend the knowledge on biological aspects of erythrocytic and tick stages of Babesia. Additionally, glmS ribozyme as a conditional knockdown system is available that could be used for the characterization of essential genes. The development of high throughput genetic tools is needed to dissect the function of multigene families, targeting several genes in a specific pathway, and finally genome-wide identification of essential genes to find novel drug targets. In this review, we summarized the current tools that are available for Babesia and the genes that are being targeted by these tools. This may draw a perspective for the future development of genetic tools and pave the way for the identification of novel drugs or vaccine targets.

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

10.21203/rs.3.rs-58018/v2 ◽

2020 ◽

Author(s):

Marta G. Silva ◽

Reginaldo G. Bastos ◽

J. Stone Doggett ◽

Michael K. Riscoe ◽

Sovitj Pou ◽

...

Keyword(s):

Mononuclear Cells ◽

Colorimetric Assay ◽

Babesia Bovis ◽

Host Cells ◽

Bovine Babesiosis ◽

Theileria Equi ◽

Peripheral Blood Mononuclear ◽

Equine Piroplasmosis ◽

And Control

Abstract Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay.Results: We calculated IC50 ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 h among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQ-300 and ELQ-316 at IC100, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions: ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

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