Research Project: Control Strategies for Bovine Babesiosis

Location: Animal Disease Research

2022 Annual Report

Objectives
This project aims to develop a risk mitigation plan to reduce threats associated with bovine babesiosis for the U.S. livestock industry. To develop bovine babesiosis control strategies, there are significant knowledge gaps regarding Babesia-mammalian-tick host interactions that need to be addressed. These gaps include a lack of understanding of Babesia stage-specific protein expression, limited knowledge regarding drug therapy efficacy and safety, and genetic conservation among geographically distinct Babesia isolates. Additionally, little progress has been made on characterizing the mechanisms regulating a protective immune response to bovine babesiosis, understanding the risk of introducing pathogens into disease-free regions by wildlife, and rapidly changing environmental factors. This plan will address these knowledge gaps by developing diagnostic assays for detecting cattle infected with Babesia spp., discovery and testing of potential Babesia antigen targets expressed by the parasite during its development within cattle or tick vectors, development of efficacious and safe treatment regimens to control babesiosis, characterizing the immune response to Babesia and identification of targets that can be used to develop a vaccine, and developing mathematical models to predict the spread of bovine babesiosis parasites into U.S. herds. This project will address the following objectives. Objective 1: Develop intervention strategies to minimize the impact of bovine babesiosis outbreaks, to include vaccine and therapeutic development to prevent clinical disease or block transmission of bovine babesiosis. Sub-objective 1A: Improve molecular and serologic diagnostic assays for bovine babesiosis. Sub-objective 1B: Evaluate the capacity of bovine antibodies against Babesia gamete and kinete surface antigens to minimize the impact of bovine babesiosis outbreaks. Sub-objective 1C: Assess the efficacy of chemotherapeutic approaches to eliminate Babesia pathogens and disrupt transmission. Objective 2: Characterize the immune response to infection with Babesia bovis, to include identifying targets that can be used as correlates of protection and examining age-specific differences. Sub-objective 2A: Investigate age-specific immune response differences to acute Babesia bovis infection between naïve young calves and adult cattle. Sub-objective 2B: Identify Babesia bovis proteins required to induce an immune response that mitigates disease severity. Objective 3: Develop predictive models of potential Babesia disease spread in the United States to assist in mitigating potential future outbreaks. Sub-objective 3A: Develop prediction models of potential Babesia parasite spread into the United States.

Approach
Objective 1: Develop intervention strategies to minimize the impact of bovine babesiosis outbreaks, to include vaccine and therapeutic development to prevent clinical disease or block transmission of bovine babesiosis. Goals: The research will focus on developing strategies for mitigating or preventing bovine babesiosis outbreaks. Specifically, 1) develop cost-effective diagnostic assays for detecting cattle infected with B. bovis and/or B. bigemina, which are major threats to the U.S. cattle industry, 2) evaluate bovine antibodies against Babesia tick stage-specific parasites for their capacity to prevent transmission and reduce acute disease, and 3) identify efficient drug(s) to clear infection to minimize the impact of bovine babesiosis outbreaks. Objective 2: Characterize the immune response to infection with Babesia bovis, to include identifying targets that can be used as correlates of protection and examining age-specific differences. Goals: The research will focus on a comprehensive characterization of the immune response of calves to infection with B. bovis and defining cytokines/chemokines associated with reduced disease severity. Also, Babesia surface proteins will be identified and utilized to understand immune mechanisms responsible for controlling Babesia infection within the mammalian host. The outcome will provide a strong foundation for future Babesia subunit vaccine development. Objective 3: Develop predictive models of potential Babesia disease spread in the United States to assist in mitigating potential future outbreaks. Goal: The research focus will be to develop and validate models that better predict areas at risk of Babesia parasite introduction into the United States.

Progress Report
In support of Objective 1, significant progress has been made on all sub-objectives. For Sub-objective 1A, molecular and serologic diagnostic assays were improved to detect parasites that cause bovine babesiosis. The results showed that the molecular test is more accurate to identify early infection. The serological test showed more specificity to identify animals exposed to the parasites. For Sub-objective 1B, cattle were vaccinated with a protein found on the parasite during its development within ticks. The results demonstrated that antibodies disrupted parasite development in the ticks and consequently prevented the transmission of parasites to naïve animals. In support of Sub-objective 1C, the efficacy of drugs to kill the parasites was tested in in vitro culture. The results demonstrated drugs can inhibit the growth of parasites. In support of Objective 2, progress has been made in all sub-objectives. For Sub-objective 2A, results demonstrated young bovines vaccinated with live parasites were protected against disease. For adult bovines, the result demonstrated the live parasite vaccine caused mild disease. However, animals completely recovered. In addition, vaccinated adults survived the challenge using a lethal parasite. Immunity induced by live vaccine protected young and adult bovines against lethal parasites. For Sub-objective 2B, parasite proteins were identified and categorized to test as potential vaccine candidates. In support of Objective 3, progress has also been made. For the Sub-objective 3A, models to understand parasite spread was initiated. Data from tick species, and wildlife hosts including white-tailed deer and nilgai, were collected to determine the incidence of pathogens that cause bovine babesiosis. Distribution models for the ticks and wildlife hosts were created. Open-source data for tick and wildlife host occurrence were downloaded. Our collaborator agreed to provide additional data from the Permanent Quarantine Zone in southern Texas, to create more robust predictive models. Also, humidity and temperature data were collected to evaluate their impact on the tick distribution. The models can be used to predict and prevent the spread of bovine babesiosis throughout the United States.

Accomplishments
1. Transfected parasite is able to complete the life cycle within mammalian and tick hosts. Babesia parasites have a complex life cycle during tick and bovine infection. To understand the parasite life cycle, a parasite was transfected to express a marker during its development within tick and mammalian host. ARS scientists in Pullman, Washington, demonstrated that genetically modified parasites were able to infect ticks, transfer to the next tick generation and were transmitted to naïve animals by larval ticks. The development of gene replacement strategy permitted a deeper understanding of the biology of parasite-host-vector triad interactions and facilitated the evaluation of upregulated genes during the parasite's journey through the tick vector. This novel technology will allow introduction of genes encoding proteins lethal to ticks during the development of Babesia within the vector, with the expectation of killing the ticks, which will reduce the use of chemical acaricides by the livestock industry.

2. Potential of gossypol as a drug to control Babesia bigemina. Bovine babesiosis is an acute disease characterized by fever, anemia, and hemoglobinuria, which can be caused by Babesia parasites. Gossypol is a natural compound that already proved to be effective against in vitro cultured parasites. ARS scientists at Pullman, Washington, explored the potential of gossypol as a therapeutic drug against in vitro cultured parasite and calculated the doses of the drug that effectively inhibit the parasite growth. Gossypol efficiently controls the growth of parasite in vitro; however, viability of the host cells might be compromised by the drug. Altogether, the results suggest that gossypol is an effective drug against Babesia parasites, but its toxic effects for the bovine host should be further evaluated in in vivo trials.

3. In vitro adapted and attenuated parasites that are not transmissible by ticks. Bovine babesiosis can be controlled using live vaccines produced by passaging of parasites in calves. These live vaccines are costly and may have the risk of spreading other parasites to the herd. ARS scientists at Pullman, Washington, found phenotypic and genotypic differences among parasites maintained in in vitro cultures for years, with its short-term cultured parasites. The long-term cultured parasites have reduced genetic content, lack the ability to transition to sexual forms, and cannot be transmitted to naïve calves by ticks. These differences might be due to the selection of a culture-adapted parasite, epigenetic changes, or a combination of events. In vitro adapted, live parasites are candidate components of non-transmissible vaccines. Therefore, the livestock industry in endemic areas will have an alternative live vaccine to protect cattle against bovine babesiosis.

Review Publications
Alzan, H.F., Bastos, R.G., Laughery, J.M., Scoles, G.A., Ueti, M.W., Johnson, W.C., Suarez, C.E. 2022. A culture-adapted strain of Babesia bovis has reduced subpopulation complexity and is unable to complete its natural life cycle in ticks. Frontiers in Cellular and Infection Microbiology. 12. Article 827347. https://doi.org/10.3389/fcimb.2022.827347.
Sears, K.P., Knowles, D.P., Fry, L.M. 2022. Clinical progression of Theileria haneyi in splenectomized horses reveals decreased virulence compared to Theileria equi. Pathogens. 11(2). Article 254. https://doi.org/10.3390/pathogens11020254.
Ozubek, S., Alzan, H.F., Bastos, R.G., Laughery, J.M., Suarez, C.E. 2022. Identification of CCp5 and FNPA as novel non-canonical members of the CCp protein family in Babesia bovis. Frontiers in Veterinary Science. 9. Article 833183. https://doi.org/10.3389/fvets.2022.833183.
Gallenti, R., Hussein, H.E., Alzan, H.F., Suarez, C.E., Ueti, M.W., Asurmendi, S., Benitez, D., Araujo, F.R., Rolls, P., Sibeko-Matjila, K., Schnittger, L., Florin-Christensen, M. 2022. Unraveling the complexity of the rhomboid serine protease 4 family of Babesia bovis by bioinformatics and experimental studies. Pathogens. 11(3) Article 344. https://doi.org/10.3390/pathogens11030344.
He, L., Bastos, R.G., Yu, L., Laughery, J.M., Suarez, C.E. 2022. Lactate dehydrogenase as a potential therapeutic drug target to control Babesia bigemina. Frontiers in Cellular and Infection Microbiology. 12. Article 870852. https://doi.org/10.3389/fcimb.2022.870852.
Taus, N.S., Cywes-Bently, C., Johnson, W.C., Pier, G.B., Fry, L.M., Mousel, M.R., Ueti, M.W. 2021. Immunization against a conserved surface polysaccharide stimulates bovine antibodies with opsonic killing activity but does not protect against Babesia bovis challenge. Pathogens. 10(12). Article 1598. https://doi.org/10.3390/pathogens10121598.
Paoletta, M.S., Laughery, J.M., Arias, L.S.L., Ortiz, J.M.J., Montenegro, V.N., Petrigh, R., Ueti, M.W., Suarez, C.E., Farber, M.D., Wilkowsky, S.E. 2021. The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell. International Journal for Parasitology. 51(8):643-658. https://doi.org/10.1016/j.ijpara.2020.12.010.
Scoles, G.A., Lohmeyer, K.H., Ueti, M.W., Bonilla, D., Lahmers, K.K., Piccione, J., Rogovskyy, A.S. 2021. Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens. Ticks and Tick Borne Diseases. 12(5). Article 101708. https://doi.org/10.1016/j.ttbdis.2021.101708.
Hussein, H.E., Johnson, W.C., Taus, N.S., Capelli-Peixoto, J., Suarez, C.E., Mousel, M.R., Ueti, M.W. 2021. Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages. Parasites & Vectors. 14. Article 395. https://doi.org/10.1186/s13071-021-04902-3.
Vimonish, R., Dinkel, K.D., Fry, L.M., Johnson, W.C., Capelli-Peixoto, J., Bastos, R.G., Scoles, G.A., Knowles, D.P., Madder, M., Chaka, G., Ueti, M.W. 2021. Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system. Parasites & Vectors. 14. Article 616. https://doi.org/10.1186/s13071-021-05120-7.
Salinas-Estrella, E., Ueti, M.W., Lobanov, V.A., Castillo-Payró, E., Lizcano-Mata, A., Badilla, C., Martínez-Ibáñez, F., Mosqueda, J. 2022. Serological and molecular detection of Babesia caballi and Theileria equi in Mexico: A prospective study. PLoS ONE. 17(3). Article e0264998. https://doi.org/10.1371/journal.pone.0264998.
He, L., Bastos, R.G., Sun, Y., Hua, G., Guan, G., Zhao, J., Suarez, C.E. 2021. Babesiosis as a potential threat for bovine production in China. Parasites & Vectors. 14. Article 460. https://doi.org/10.1186/s13071-021-04948-3.
Onzere, C.K., Fry, L.M., Bishop, R.P., Da Silva, M., Madsen-Bouterse, S.A., Bastos, R.G., Knowles, D.P., Suarez, C.E. 2022. Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development. International Journal for Parasitology. 52(6):385-397. https://doi.org/10.1016/j.ijpara.2022.01.004.
Hidalgo-Ruiz, M., Mejia-López, S., Perez-Serrano, R.M., Zaldívar-Lelo de Larrea, R., Ganzinelli, S., Florin-Christensen, M., Suarez, C.E., Hernández-Ortiz, R., Mercado-Uriostegui, M.A., Rodríguez-Torres, A., Carvajal-Gamez, B.I., Camacho-Nuez, M., Wilkowsky, S.E., Mosqueda, J. 2022. Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle. Vaccine. 40(8):1108-1115. https://doi.org/10.1016/j.vaccine.2022.01.023.
Basto, R.G., Thekkiniath, J., Mamoun, C.B., Fuller, L., Molestina, R.E., Florin-Christensen, M., Schnittger, L., Alzan, H.F., Suarez, C.E. 2021. Babesia microti immunoreactive rhoptry-associated protein-1 paralogs are ancestral members of the piroplasmid-confined RAP-1 family. Pathogens. 10(11). Article 1384. https://doi.org/10.3390/pathogens10111384.
Florin-Christensen, M., Wieser, S.N., Suarez, C.E., Schnittger, L. 2021. In silico survey and characterization of Babesia microti functional and non-functional proteases. Pathogens. 10(11). Article 1457. https://doi.org/10.3390/pathogens10111457.
Bastos, R.G., Alzan, H.F., Rathinasamy, V.A., Cooke, B.M., Dellagostin, O.A., Barletta, R.G., Suarez, C.E. 2022. Harnessing Mycobacterium bovis BCG trained immunity to control human and bovine babesiosis. Vaccines. 10(1). Article 123. https://doi.org/10.3390/vaccines10010123.
Elnaggar, M.M., Knowles, D.P., Davis, W., Fry, L.M. 2021. Flow cytometric analysis of the cytotoxic T-cell recall response to Theileria parva in cattle following vaccination by the infection and treatment method. Veterinary Sciences. 8(6). Article 114. https://doi.org/10.3390/vetsci8060114.
Davis, W.C., Abdellrazeq, G.S., Mahmoud, A.H., Park, K., Elnaggar, M.M., Donofrio, G., Hulubei, V., Fry, L.M. 2021. Advances in understanding of the immune response to mycobacterial pathogens and vaccines through use of cattle and Mycobacterium avium subsp. paratuberculosis as a prototypic mycobacterial pathogen. Vaccines. 9(10). Article 1085. https://doi.org/10.3390/vaccines9101085.
Scoles, G.A., Hussein, H.E., Olds, C.L., Mason, K.L., Davis, S.K. 2022. Vaccination of cattle with synthetic peptides corresponding to predicted extracellular domains of Rhipicephalus (Boophilus) microplus Aquaporin-2 reduces the number of ticks feeding to repletion. Parasites & Vectors. https://doi.org/10.1186/s13071-022-05166-1.
Johnson, W.C., Hussein, H.E., Capelli-Peixoto, J., Laughery, J.M., Taus, N.S., Suarez, C.E., Ueti, M.W. 2022. A transfected Babesia bovis parasite line expressing eGFP is able to complete the full life cycle of the parasite in mammalian and tick hosts. Pathogens. 11(6). Article 623. https://doi.org/10.3390/pathogens11060623.