{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T04:30:10Z","timestamp":1778905810971,"version":"3.51.4"},"reference-count":31,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,5,27]],"date-time":"2022-05-27T00:00:00Z","timestamp":1653609600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Agricultural Research Service Project","award":["#2090-32000-040-00D"],"award-info":[{"award-number":["#2090-32000-040-00D"]}]},{"name":"Agricultural Research Service Project","award":["# 2016-67015-24968"],"award-info":[{"award-number":["# 2016-67015-24968"]}]},{"name":"National Institute of Food and Agriculture-Agriculture and Food Research Initiative Competitive Grant Project","award":["#2090-32000-040-00D"],"award-info":[{"award-number":["#2090-32000-040-00D"]}]},{"name":"National Institute of Food and Agriculture-Agriculture and Food Research Initiative Competitive Grant Project","award":["# 2016-67015-24968"],"award-info":[{"award-number":["# 2016-67015-24968"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia, including B. bovis. This protozoan parasite has a complex life cycle involving dynamic changes to its transcriptome during the transition between the invertebrate and vertebrate hosts. Studying the role of genes upregulated by tick stage parasites has been hindered by the lack of appropriate tools to study parasite gene products in the invertebrate host. Herein, we present tfBbo5480, a transfected B. bovis cell line, constitutively expressing enhanced green fluorescent protein (eGFP) created by a whole gene replacement transfection strategy, that was capable of completing the parasite\u2019s entire life cycle in both the vertebrate and invertebrate hosts. tfBbo5480 was demonstrated to respond to in vitro sexual stage induction and upon acquisition by the female tick vector, Rhipicephalus microplus, the tick specific kinete stage of tfBbo5480 was detected in tick hemolymph. Larvae from tfBbo5480 exposed R. microplus female ticks successfully transmitted the transfected parasite to a na\u00efve calf. The development of the whole gene replacement strategy will permit a deeper understanding of the biology of parasite-host-vector triad interactions and facilitate the evaluation of upregulated genes during the parasite\u2019s journey through the tick vector leading to new intervention strategies for the control of bovine babesiosis.<\/jats:p>","DOI":"10.3390\/pathogens11060623","type":"journal-article","created":{"date-parts":[[2022,5,29]],"date-time":"2022-05-29T23:43:03Z","timestamp":1653867783000},"page":"623","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["A Transfected Babesia bovis Parasite Line Expressing eGFP Is Able to Complete the Full Life Cycle of the Parasite in Mammalian and Tick Hosts"],"prefix":"10.3390","volume":"11","author":[{"given":"Wendell C.","family":"Johnson","sequence":"first","affiliation":[{"name":"Animal Diseases Research Unit, USDA-ARS, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1912-2377","authenticated-orcid":false,"given":"Hala E.","family":"Hussein","sequence":"additional","affiliation":[{"name":"Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA"},{"name":"Department of Entomology, Faculty of Science, Cairo University, Giza 12613, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8571-5870","authenticated-orcid":false,"given":"Janaina","family":"Capelli-Peixoto","sequence":"additional","affiliation":[{"name":"Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jacob M.","family":"Laughery","sequence":"additional","affiliation":[{"name":"Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5928-6232","authenticated-orcid":false,"given":"Naomi S.","family":"Taus","sequence":"additional","affiliation":[{"name":"Animal Diseases Research Unit, USDA-ARS, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos E.","family":"Suarez","sequence":"additional","affiliation":[{"name":"Animal Diseases Research Unit, USDA-ARS, Pullman, WA 99164, USA"},{"name":"Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Massaro W.","family":"Ueti","sequence":"additional","affiliation":[{"name":"Animal Diseases Research Unit, USDA-ARS, Pullman, WA 99164, USA"},{"name":"Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA"},{"name":"Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1007\/s11538-013-9912-8","article-title":"A bovine babesiosis model with dispersion","volume":"76","author":"Friedman","year":"2014","journal-title":"Bull. Math. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"104747","DOI":"10.1016\/j.prevetmed.2019.104747","article-title":"Risks of cattle babesiosis (Babesia bovis) outbreaks in a semi-arid region of Argentina","volume":"170","author":"Morel","year":"2019","journal-title":"Prev. Vet. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"S247","DOI":"10.1017\/S0031182004005190","article-title":"Babesiosis of cattle","volume":"129","author":"Bock","year":"2004","journal-title":"Parasitology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.vetpar.2011.05.032","article-title":"Emerging perspectives in the research of bovine babesiosis and anaplasmosis","volume":"180","author":"Suarez","year":"2011","journal-title":"Vet. Parasitol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"248","DOI":"10.3389\/fcimb.2018.00248","article-title":"The Complexity of piroplasms life cycles","volume":"8","author":"Jalovecka","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1021\/acs.jproteome.6b00947","article-title":"Analysis of stage-specific protein expression during Babesia bovis development within female Rhipicephalus\u00a0microplus","volume":"16","author":"Johnson","year":"2017","journal-title":"J. Proteome Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3155","DOI":"10.1128\/JCM.00766-07","article-title":"Persistently infected calves as reservoirs for acquisition and transovarial transmission of Babesia bovis by Rhipicephalus (Boophilus)\u00a0microplus","volume":"45","author":"Howell","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1218","DOI":"10.1126\/science.7355284","article-title":"Babesia bovis: Continuous cultivation in a microaerophilous stationary phase culture","volume":"207","author":"Levy","year":"1980","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2363","DOI":"10.1128\/iai.56.9.2363-2368.1988","article-title":"Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies","volume":"56","author":"Goff","year":"1988","journal-title":"Infect. Immun."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Rizk, M., El-Sayed, S.A., Terkawi, M.A., Youssef, M.A., Said, E.S.E.S.E., Elsayed, G., El-Khodery, S., El-Ashker, M., ElSify, A., and Omar, M. (2015). Optimization of a fluorescence-based assay for large-scale drug screening against Babesia and Theileria parasites. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0125276"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.ijpddr.2018.04.004","article-title":"Assessment of Draxxin\u00ae (tulathromycin) as an inhibitor of in vitro growth of Babesia bovis, Babesia bigemina and Theileria\u00a0equi","volume":"8","author":"Silva","year":"2018","journal-title":"Int. J. Parasitol. Drugs Drug Resist."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e00109-19","DOI":"10.1128\/mSphere.00109-19","article-title":"Genome Editing of Babesia bovis Using the CRISPR\/Cas9 System","volume":"4","author":"Hakimi","year":"2019","journal-title":"mSphere"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/j.ijpara.2008.08.006","article-title":"Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites","volume":"39","author":"Suarez","year":"2009","journal-title":"Int. J. Parasitol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Asada, M., Yahata, K., Hakimi, H., Yokoyama, N., Igarashi, I., Kaneko, O., Suarez, C.E., and Kawazu, S.-I. (2015). Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0125993"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Oldiges, D.P., Laughery, J.M., Tagliari, N.J., Leite Filho, R.V., Davis, W.C., da Silva Vaz, I., Termignoni, C., Knowles, D.P., and Suarez, C.E. (2016). Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0005152"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.vetpar.2018.03.016","article-title":"The application of the HyPer fluorescent sensor in the real-time detection of H2O2 in Babesia bovis merozoites in vitro","volume":"255","author":"Asada","year":"2018","journal-title":"Vet. Parasitol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Talman, A.M., Blagborough, A.M., and Sinden, R.E. (2010). A Plasmodium falciparum strain expressing GFP throughout the parasite\u2019s life-cycle. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0009156"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Hussein, H.E., Bastos, R.G., Schneider, D.A., Johnson, W.C., Adham, F.K., Davis, W.C., Laughery, J.M., Herndon, D.R., Alzan, H.F., and Ueti, M.W. (2017). The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction. PLoS Negl. Trop. Dis., 11.","DOI":"10.1371\/journal.pntd.0005965"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Alzan, H.F., Lau, A.O., Knowles, D.P., Herndon, D.R., Ueti, M.W., Scoles, G.A., Kappmeyer, L., and Suarez, C.E. (2016). Expression of 6-Cys Gene Superfamily Defines Babesia bovis sexual stage development within Rhipicephalus\u00a0microplus. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0163791"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1186\/s13071-018-3052-9","article-title":"Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages","volume":"11","author":"Bohaliga","year":"2018","journal-title":"Parasites Vectors"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.ijpara.2020.08.006","article-title":"Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation","volume":"51","author":"Ueti","year":"2021","journal-title":"Int. J. Parasitol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1038\/s41579-019-0306-2","article-title":"Plasmodium asexual growth and sexual development in the haematopoietic niche of the host","volume":"18","author":"Venugopal","year":"2020","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Hakimi, H., Asada, M., and Kawazu, S.I. (2021). Recent advances in molecular genetic tools for\u00a0Babesia. Vet. Sci., 8.","DOI":"10.3390\/vetsci8100222"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Alzan, H.F., Knowles, D.P., and Suarez, C.E. (2016). Comparative bioinformatics analysis of transcription factor genes indicates conservation of key regulatory domains among Babesia bovis, Babesia microti, and Theileria\u00a0equi. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004983"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1186\/s13071-021-04902-3","article-title":"Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages","volume":"14","author":"Hussein","year":"2021","journal-title":"Parasites Vectors"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1146\/annurev.mi.44.100190.002241","article-title":"Sexual differentiation in malaria parasites","volume":"44","author":"Alano","year":"1990","journal-title":"Ann. Rev. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"827347","DOI":"10.3389\/fcimb.2022.827347","article-title":"A Culture-adapted strain of Babesia bovis has reduced subpopulation complexity and is unable to complete its natural life cycle in ticks","volume":"12","author":"Alzan","year":"2022","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.ttbdis.2014.11.007","article-title":"Co-transmission of the non-transmissible South African Babesia bovis S24 vaccine strain during mixed infection with a field isolate","volume":"6","author":"Combrink","year":"2015","journal-title":"Ticks Tick Borne Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1128\/JCM.01757-06","article-title":"Transovarial transmission efficiency of Babesia bovis tick stages acquired by Rhipicephalus (Boophilus) microplus during acute infection","volume":"45","author":"Howell","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1186\/s13071-019-3531-7","article-title":"Identification of proteins expressed by Babesia bigemina kinetes","volume":"12","author":"Bohaliga","year":"2019","journal-title":"Parasites Vectors"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/0304-4017(93)90008-B","article-title":"Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood","volume":"50","author":"Figueroa","year":"1993","journal-title":"Vet. Parasitol."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/6\/623\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:20:00Z","timestamp":1760138400000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/6\/623"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,27]]},"references-count":31,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["pathogens11060623"],"URL":"https:\/\/doi.org\/10.3390\/pathogens11060623","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,27]]}}}