Improvement of cryopreservation and production of attenuated Babesia parasites to prevent bovine babesiosis

Authors: ROJAS-MARTÍNEZ, CARMEN - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); LIRA-AMAYA, JOSÉ - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); Ueti, Massaro; CASTAÑEDA-ARRIOLA, ROBERTO - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); FIGUEROA MILLÁN, JULIO - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP); ÁLVAREZ MARTÍNEZ, JESÚS - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP)

Submitted to: Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/16/2025
Publication Date: 5/20/2025
Citation: Rojas-Martínez, C., Lira-Amaya, J.J., Ueti, M.W., Castañeda-Arriola, R.0., Figueroa Millán, J.V., Álvarez Martínez, J.A. 2025. Improvement of cryopreservation and production of attenuated Babesia parasites to prevent bovine babesiosis. Pathogens and Disease. 14(5). Article 498. https://doi.org/10.3390/pathogens14050498.
DOI: https://doi.org/10.3390/pathogens14050498

Interpretive Summary: In this study, we evaluated the effects of various concentrations of PVP-40 on the in vitro cryopreservation and recovery of Babesia bovis and Babesia bigemina. We demonstrated that 20% PVP-40 improved the recovery of frozen parasites in blood culture. We also showed that a reduced dose of attenuated Babesia parasites prevented clinical disease in cattle exposed to ticks from an area hyperendemic for Babesia spp. The animals were vaccinated intramuscularly with frozen parasites produced in blood cultures, and the clinical parameters were unaffected during vaccination. The control group received uninfected erythrocytes. During a challenge on a tick-infested ranch, some vaccinated animals did show mild fever (>40 °C). We detected the parasitemia these animals only one day. In contrast, the control group experienced severe disease. In this group, we detected parasites for four consecutive days. The vaccinated animals had a 100% protection rate against severe clinical babesiosis, and all animals in the control group required treatment to prevent mortality. In conclusion, the cryopreserved in vitro cultured attenuated Babesia parasites in 20% PVP-40 effectively protected the animals against severe clinical babesiosis during field challenges.

Technical Abstract: This study evaluated the effects of various concentrations of PVP-40 on the in vitro cryopreservation and recovery of Babesia bovis and Babesia bigemina. The optimal concentration identified for cryopreservation was 20% PVP-40. Additionally, we assessed a reduced dose of attenuated Babesia parasites to determine its efficacy in preventing clinical disease in cattle relocated to a farm in an area hyperendemic for Babesia spp. The animals were vaccinated intramuscularly with frozen parasites cryopreserved in 20% PVP-40 with a dose of either 1 x 108 or 1 x 107 infected erythrocytes from each attenuated B. bigemina and B. bovis produced in blood cultures. The control group received uninfected erythrocytes. During the vaccination period, clinical parameters such as rectal temperature and hematocrit levels were unaffected. However, when challenged on a tick-infested ranch, some vaccinated animals did show mild fever (>40 °C). Parasites were only detected in blood smears on one day. In contrast, the control group experienced fever for three consecutive days, a decline in hematocrit levels, and significant health deterioration among the animals. In this group, parasites were detected in smears for four consecutive days. As a result, the vaccinated animals had a 100% protection rate against severe clinical babesiosis for both doses. In contrast, all animals in the control group required treatment to manage high parasitemia and prevent mortality. The cryopreserved in vitro cultured attenuated Babesia parasites in 20% PVP-40 effectively protected the animals against severe clinical babesiosis during field challenges.