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

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.

Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin.

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.

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis.

BACKGROUND: Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples. METHODS: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy. RESULTS: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). CONCLUSIONS: Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.

Research on Integrated Management for Cattle Fever Ticks and Bovine Babesiosis in the United States and Mexico: Current Status and Opportunities for Binational Coordination.

Bovine babesiosis is a reportable transboundary animal disease caused by Babesia bovis and Babesiabigemina in the Americas where these apicomplexan protozoa are transmitted by the invasive cattle fever ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus(Boophilus) annulatus. In countries like Mexico where cattle fever ticks remain endemic, bovine babesiosis is detrimental to cattle health and results in a significant economic cost to the livestock industry. These cattle disease vectors continue to threaten the U.S. cattle industry despite their elimination through efforts of the Cattle Fever Tick Eradication Program. Mexico and the U.S. share a common interest in managing cattle fever ticks through their economically important binational cattle trade. Here, we report the outcomes of a meeting where stakeholders from Mexico and the U.S. representing the livestock and pharmaceutical industry, regulatory agencies, and research institutions gathered to discuss research and knowledge gaps requiring attention to advance progressive management strategies for bovine babesiosis and cattle fever ticks. Research recommendations and other actionable activities reflect commitment among meeting participants to seize opportunities for collaborative efforts. Addressing these research gaps is expected to yield scientific knowledge benefitting the interdependent livestock industries of Mexico and the U.S. through its translation into enhanced biosecurity against the economic and animal health impacts of bovine babesiosis and cattle fever ticks.

La vacunación simultánea de bovinos con Lactobacillus casei y la vacuna bivalente contra babesiosis bovina genera una mejor protección contra Babesia bovis y B. bigemina transmitidas por garrapatas en condiciones extremas de campo / The simultaneous vaccination of bovines with Lactobacillus casei and the bivalent vaccine against bovine babesiosis induces a better protection against Babesia bovis and B. bigemina transmitted by ticks in extreme field conditions

The effect of Lactobacillus casei on INIFAP's mixed vaccine against bovine babesiosis (VAC) was assessed in bovines in an endemic babesiosis area. It was previously reported that L. casei increases the efficiency of the Mexican mixed vaccine against bovine babesiosis under controlled conditions. The results of the present study demonstrated the effectiveness of simultaneous vaccination of bovines with L. casei and the mixed vaccine against bovine babesiosis in eliciting a protective immune response under extreme conditions in the field. Twenty Babesia spp free bovines were allocated into three groups: un-vaccinated (Control, n = 9), vaccinated with VAC (n = 5), and vaccinated simultaneously with VAC and Lactobacillus casei (LC-VAC, n = 6). All animals were kept in a tick and Babesia spp free field at Coatepec, Veracruz during 24 days before moving them to Paso del Toro, Veracruz, for a natural exposition to Babesia spp transmitted by Riphicephalus (Boophilus) microplus ticks. Protection against Babesia spp was observed in bovines belonging to VAC and LC-VAC groups, while control animals showed severe clinical babesiosis. Bovines in VAC-LC group showed less clinical signs between days 12-16 after challenge as compared with animals in VAC group. All bovines showed both Babesia spp after challenge. Levels of IgG anti-Babesia in animals from both vaccinated groups, determined by indirect immunofluorescence test, always were higher to Babesia bovis than to B. bigemina after vaccination and challenge. It was demonstrated the efficiency of simultaneous vaccination of bovines with VAC and L. casei, in eliciting a better protective immune response against naturally transmitted Babesia spp under extreme field conditions.

Se evaluó el efecto de Lactobacillus casei en la vacuna mixta contra babesiosis bovina del INIFAP (VAC), en bovinos de un área endémica de babesiosis. Previamente se informó que L. casei incrementa la eficacia de la vacuna mixta mexicana contra babesiosis bovina bajo condiciones controladas. Los resultados aquí expuestos demostraron dicha efectividad para generar una respuesta inmunitaria protectora bajo condiciones extremas en el campo. Veinte bovinos libres de Babesia spp fueron distribuidos al azar en tres grupos: testigo no vacunado (Testigo, n = 9), vacunado con VAC (n = 5), y vacunado simultáneamente con VAC y L. casei (LC-VAC, n = 6). Todos los animales se mantuvieron en un corral libre de garrapatas y Babesia spp en Coatepec, Veracruz durante 24 días antes de transportarlos a Paso del Toro, Veracruz, para una exposición natural a Babesia spp transmitida por garrapatas Riphicephalus (Boophilus). Se observó protección contra Babesia spp en bovinos pertenecientes a los grupos VAC y LC-VAC, mientras que los animales testigo mostraron signos clínicos de babesiosis aguda. Los bovinos del grupo VAC-LC mostraron menos signos clínicos que los del grupo VAC entre los días 12-16. Todos los bovinos mostraron Babesia spp después de la confrontación. Los niveles de IgG anti-Babesia en los animales de los grupos vacunados, determinados por inmunofluorescencia indirecta, siempre fueron más elevados contra Babesia bovis que contra B. bigemina después de la vacunación y de la confrontación. Se demostró la eficacia de la vacunación simultánea con VAC y L. casei en bovinos, para generar una mejor respuesta inmunitaria protectora contra Babesia spp transmitida naturalmente por garrapatas, bajo condiciones extremas de campo.