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

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 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.

Establishment of Continuous In Vitro Culture of Babesia gibsoni by Using VP-SFM Medium with Low-Concentration Serum

“…Previous modifications to the culture medium of Babesia in vitro were made by reducing or removing the serum component and replacing it with other components, such as a mixture of lipids or bovine serum albumin rich in lipids called AlbuMAX I and II ( 7 , 11 , 12 ). The VP-SFM medium allowed the continuous in vitro proliferation of Toxoplasma gondii , B. bovis , and Babesia bigemina ( 13 – 15 ). Nevertheless, VP-SFM and AlbuMAX have never been evaluated for the in vitro culture of B. gibsoni .…”

Sudden Death of Cattle Caused by Babesia bovis Transmitted by Rhipicephalus microplus (Acari: Ixodidae) Ticks

“…At present, live attenuated vaccines have achieved good efficiency in disease prevention and animal immune protection and have been widely used in Australia, Argentina, Brazil, Uruguay and Israel [4]. In addition, the technology of in vitro culture of B. bovis is convenient for biochemical, immunological and antigen source research [5]. In several studies conducted in Mexico, an attenuated vaccine of B. bovis in vitro produced good protection for host animals, but the disadvantage of this method is that it requires a large-scale culture of B. bovis.…”