Establishment of Babesia bovis in vitro culture using medium free of animal products

Authors: ÁLVAREZ MARTÍNEZA, JESÚS - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); FIGUEROA MILLÁN, JULIO - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); Ueti, Massaro; ROJAS-MARTÍNEZ, CARMEN - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP)

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/16/2021
Publication Date: 6/19/2021
Citation: Álvarez Martíneza, J.A., Figueroa Millán, J.V., Ueti, M.W., Rojas-Martínez, C. 2021. Establishment of Babesia bovis in vitro culture using medium free of animal products. Pathogens. 10(6). Article 770. https://doi.org/10.3390/pathogens10060770.
DOI: https://doi.org/10.3390/pathogens10060770

Interpretive Summary: Bovine babesiosis is widespread in tropical and subtropical regions. The development of new methodologies to reduce the cost of biological material required for diagnostic and vaccines for bovine babesiosis is highly desirable. In this study, we tested several culture media to determine if the media support the growth of B. bovis in vitro condition without supplementation of animal products. We demonstrated the addition of lipid acid mixture in the VP-SF medium improves B. bovis in vitro condition. The impact of the study is the potential of reducing and replacing the use of animals to produce reagents for diagnostic and live vaccines to control bovine babesiosis for endemic areas.

Technical Abstract: 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 medium free-components of animal origin enriched with a 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 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 dilution 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 reagent for diagnostic and live vaccines to control bovine babesiosis.