# Improvement of Cryopreservation and Production of Attenuated Babesia Parasites to Prevent Bovine Babesiosis

## TL;DR

This study shows that using 20% PVP-40 to cryopreserve and vaccinate with weakened Babesia parasites effectively protects cattle from severe babesiosis.

## Contribution

The study introduces an improved cryopreservation method and demonstrates effective vaccine protection using attenuated Babesia parasites.

## Key findings

- Cryopreservation with 20% PVP-40 improved recovery and proliferation of Babesia parasites in blood culture.
- Vaccinated animals showed mild disease and were protected from severe babesiosis compared to the control group.
- Parasites were detected in vaccinated animals for only one day, while the control group required treatment for severe symptoms.

## 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. We also assessed a reduced dose of attenuated Babesia strains to determine its efficacy in preventing clinical disease. A microaerophilic stationary phase blood culture system was used to recover Babesia parasites that were cryopreserved in solutions with various PVP-40 concentrations and Babesia parasites in 20% PVP-40 were used to vaccinate naïve cattle. The animals were vaccinated intramuscularly with frozen parasites cryopreserved in 20% PVP-40, with a dose of either 1 × 108 or 1 × 107 erythrocytes infected with both attenuated B. bigemina and B. bovis produced from blood cultures. The control group received uninfected erythrocytes. During the vaccination, clinical parameters such as rectal temperature and hematocrit levels were unaffected. The animals were relocated to a farm in a Babesia hyperendemic area to test the efficacy of these live vaccines in controlling disease onset. Some vaccinated animals showed mild disease. In the vaccinated groups, parasites were detected in blood smears for only one day during the challenge. In contrast, the control group experienced fever for three consecutive days, a decline in hematocrit levels, and significant health deterioration. In this group, parasites were detected in smears for four consecutive days. All the animals in the control group required treatment to manage their high parasitemia and prevent mortality. In this study, we demonstrated that increasing the concentration of PVP-40 to cryopreserve parasites improved the recovery and proliferation of Babesia spp. in blood culture, and we also showed that when animals were vaccinated with cryopreserved, in vitro cultured, attenuated Babesia parasites in 20% PVP-40, they were effectively protected from severe clinical babesiosis.