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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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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.

Palabras clave: Detección, leucocitos, Neospora caninum, PCRABSTRACT. Seropositivity to Neospora caninum was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA) and the DNA was detected by PCR nested with external initiators Np 21-4 and internal Np 9-10 in cow leukocytes from the small-scale milk production system in Amecameca, State of México. A herd with 34 adult females was studied, performing follow-up with blood sampling for ve months. Also, two brain samples from aborted fetuses were analyzed and the sequence of the amplicons in the Genbank was veried. Consistency between techniques was compared using the Kappa test. ELISA seroprevalence was 85.3 %; and DNA of the parasite was detected in 89.4 % of the cows. A brain sample was positive. The similarity of the amplicons ranged from 91 to 96 %, the Kappa index was 0.41. The use of leukocytes increases the likelihood of amplifying N. caninum DNA.

RESUMENLos métodos más comúnmente empleados para el control de las principales enfermedades de los rumiantes causada por protozoarios son la quimioterapia y, en los casos de protozoarios transmitidos por artrópodos, el uso de ixodicidas para el control de los vectores. Sin embargo, la inmunización y el desarrollo de nuevas alternativas que propongan métodos prácticos de vacunación para la prevención de las enfermedades parasitarias, son los grandes avances en materia de inmunogenicidad y bioseguridad a través del uso de la biotecnología moderna. Con estas herramientas ha sido posible desarrollar modelos nuevos de vacunas con características innovadoras que contribuyan al mejoramiento de la eficiencia en la producción ganadera en el mundo.