2010 Annual Report
1a.Objectives (from AD-416)
The objectives of this research are: (1) the testing of the hypothesis that tick antigens capable of inducing anti-tick immunity can be delivered through a transfected parasite; (2) discovery of new tick antigen vaccine targets; (3) an understanding of the vector competence of certain U. S. ticks for transmission of equine babesiosis, and (4) the determination if certain anti-babesial drugs are capable of clearing horses of persistent babesial infection.
1b.Approach (from AD-416)
The above objectives will be approached through the use of transfection to create replication competent parasites containing tick antigens known to induce anti-tick immunity. In parallel with this approach new tick antigens will be discovered and characterized through genetic approaches including suppressive subtractive hybridization and Serial Analysis of Gene Expression. New antigens will first be tested by subunit immunization for their ability to induce anti-tick immunity. Tick vectors present in the U. S. and known to feed on horses will be tested for their ability to transmit B. equi and/or B. caballi and finally selected chemotherapeutics will be tested for their ability to clear persistent B. equi and/or B. caballi infections. Clearance will be defined as the lack of detectable anti-parasite antibody, the lack of PCR detectable genetic elements of B. equi and/or B. caballi and finally by testing for the ability of know competent ticks to acquire infection from treated horses. Formerly 5348-32000-020-00D (12/06).
This research addresses critical gaps in our knowledge of the transmission, pathogenesis and treatment of tick-borne Babesia parasites of cattle and horses. Estimates of the impact of tick-borne pathogens indicate economic losses exceeding 50 million dollars per year in the U. S. alone. Global economic impact, especially in developing countries is much greater. The emergence of acaricide resistant Rhipicephalus (Boophilus) ticks and their movement northward, in the U. S. is a primary concern. Should the population level of these ticks reach threshold levels and babesiosis reemerge in U. S. cattle, the economic consequences would be dire. The emergence of the foreign animal disease, equine babesiosis in the U. S. is a striking reminder of the vigilance required to remain free of the class of infectious diseases that include persistence in the individual host in their pathogenesis.
Concerning the emergence of equine babesiosis in the U. S. responses by ARS scientists located at Pullman, WA include: partnering with APHIS to provide research leadership in support of control and eventual eradication. Progress resulting from this support includes identification of a new tick vector for Babesia equi in the U. S. This vector is Amblyomma cajennense. Also, a model for demonstrating the complete removal of the parasitic causes of equine babesiosis was published and is being applied to horses found infected in the current U. S. outbreak. A study testing the efficacy of ponazuril (with a commercial partner) for control of B. equi was completed. Genomic sequencing and annotation of the B. equi genome was completed.
A critical need for control of tick borne diseases in livestock is a long acting anti-parasite and/or tick vaccine. Progress toward this goal was achieved by demonstrating that an attenuated stably transfected (a foreign gene was placed into the attenuated parasite and shown to express its protein) bovine babesia parasite is capable of establishing persistence in immunized cattle. The concept is vaccine development which will provide the economic advantage of one administration.
As part of our ongoing collaboration with the International livestock Research Institute (ILRI) in Nairobi, Kenya as well as our own research in-house we are identifying new anti-tick and/or transmission blocking vaccines for use in either the transfection based approach or in a traditional subunit vaccine approach. We have initiated experiments using RNA interference (RNAi) for targeted silencing of tick genes as a tool for studying these as potential vaccine targets. As part of this work we hosted a visiting scientist from ILRI and then made a reciprocal visit to ILRI To test candidate tick antigens using RNAi and to transfer the RNAi technology to ILRI.
We have received extramural funding to augment appropriated funds to take a population genetics based approach to studying the problem of deer as alternate hosts for exotic Rhipicephalus (Boophilus) ticks in southern Texas and to determine what role deer might play as alternate reservoir hosts for the parasite.
A model for showing horses infected with Babesia caballi are free of transmission risk. The reemergence of equine babesiosis in the U. S. represents a significant economic issue for the industry and regulatory agencies. ARS scientists at Pullman, WA and their collaborators at Washington State University and the University of Idaho developed a therapeutic model to show that horses infected with Babesia caballi can be completely cleared of infection and transmission risk. The impact of this outcome for the equine industry is clear and the reemergence of equine babesiosis in the U. S. is an important reminder of the necessity for continued research into bovine babesisois.
Using Targeted Gene Silencing to study tick genes involved in vector/parasite interactions. The cattle tick Rhipicephalus (Boophilus) microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of the a putative immunophilin gene (Imnp), a putative Kunitz-type serine protease inhibitor (Spi) or a putative lipocalin (Lpc) decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. Data from silencing the Imnp gene also suggests that it is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny. The impact of this work is that we now have a method for assessment of the function of tick genes that are being considered as potential anti-tick or transmission blocking vaccine targets.
Critical progress obtained toward development of a long acting anti-babesial and anti-tick vaccine. Babesiosis is an economically devastating disease of cattle. No vaccines against Babesia are currently available in the US and the emergence of resistance to control measures in ticks which transmit babesiosis, requires development of novel control methods. ARS scientists at Pullman, WA and their collaborators at Washington State University tested whether attenuated transfected babesial parasites expressing foreign antigens were able to establish acute and chronic infection in experimentally infected cattle. In addition ARS scientists at Pullman, WA and collaborators designed a system that allows investigating occurrence of Babesia superinfections in cattle using a novel tandem repeat-based multilocus typing system for distinguishing geographic isolates. The impact of this research is that it will allow testing of the hypothesis that transfected parasites are able to superinfect horses and/or cattle with preexisting babesial infections.
Leading research support of APHIS to control emergence of the foreign animal disease, equine babesiosis in the U. S. Beginning in 2009 and continuing into 2010 there has been a large outbreak of equine piroplasmosis (EP) in southern Texas. The U.S. has been considered free of this disease by the World Organization for Animal Health (OIE) for 30 years. Greater than 300 infected horses have been identified and fourteen additional states are involved. ARS scientists in Pullman, WA in collaboration with colleagues at Washington State University, University of Idaho and Texas A & M, Kingville showed for the first time that the Cayenne tick, Amblyomma cajennense, the predominant tick species found on horses at the center of the outbreak were capable of transmitting B. equine.
Bastos, R., Ueti, M.W., Guerrero, F., Knowles Jr, D.P., Scoles, G.A. 2009. Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus) microplus increases the infection rate of Babesia bovis in larval progeny. BioMed Central (BMC) Vectors and Parasites. 2(1):57.
Laughery, J.M., Lau, A.O., White, S.N., Howell, J.M., Suarez, C.E. 2009. Babesia bovis: Transcriptional analysis of rRNA gene unit expression. Experimental Parasitology. 123(1):45-50.
Silva, M.G., Ueti, M., Norimine, J., Florin-Christensen, M., Bastos, R., Goff, W.L., Brown, W.C., Oliva, A., Suarez, C.E. 2010. Babesia bovis expresses a neutralization-sensitive antigen that contains a microneme adhesive repeat (MAR) domain. Parasitology International. Available: doi:10.1016/j.parint.2010.03.004
Schwint, O.N., Ueti, M.W., Palmer, G.H., Kappmeyer, L.S., Hines, M.T., Cordes, R.T., Knowles Jr, D.P., Scoles, G.A. 2009. Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential. Antimicrobial Agents and Chemotherapy. 53(10):4327-4332.
Perez De Leon, A.A., Strickman, D.A., Knowles Jr, D.P., Fish, D., Thacker, E.L., De La Fuente, J., Krause, P.J., Wikel, S.K., Miller, R., Wagner, G.G., Almazan, C., Hillman, R., Messenger, M.T., Ugstad, P.O., Duhaime, R.A., Teel, P.D., Ortega-Santos, A., Hewitt, D.G., Bowers, E.J., Bent, S.J., Cochran, M.H., Mcelwain, T.F., Scoles, G.A., Suarez, C.E., Davey, R.B., Freeman, J.M., Lohmeyer, K.H., Li, A.Y., Guerrero, F., Kammlah, D.M., Phillips, P.L., Pound, J.M. 2010. One Health approach to identify research needs in bovine and human babesioses: Workshop report. Parasites & Vectors. 3:Article 36.
Dominguez, M., Echaide, I., Torioni De Echaide, S., Mosqueda, J., Cetra, B., Suarez, C.E., Florin-Christensen, M. 2010. In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive. Veterinary Parasitology. 167(2-4):216-226.
Perez-Llaneza, A., Caballero, M., Baravalle, E., Mesplet, M., Mosqueda, J., Suarez, C.E., Echaide, I., Katzer, F., Pacheco, G.M., Florin-Christensen, M., Schnittger, L. 2010. Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates. Veterinary Parasitology. 167(2-4):196-204.
Suarez, C.E., Mcelwain, T.F. 2010. Transfection systems for Babesia bovis: a review of methods for the transient and stable expression of exogenous genes. Veterinary Parasitology. 167(2-4):205-215.
Kerber, C.E., Labruna, M.B., Ferreira, F., De Waal, D.T., Knowles Jr, D.P., Gennari, S.M. 2009. Prevalence of equine Piroplasmosis and its association with tick infestation in the State of Sao Paulo, Brazil. Brazilian Journal of Veterinary Parasitology. 18(4):1-8.
Knowles Jr, D.P. 2010. Understanding Piroplasmosis. Equine Disease Quarterly. 19(3):4-5.