{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T12:58:12Z","timestamp":1784552292600,"version":"3.55.0"},"reference-count":58,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2023,6,9]],"date-time":"2023-06-09T00:00:00Z","timestamp":1686268800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA\u2192AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.<\/jats:p>","DOI":"10.3390\/life13061354","type":"journal-article","created":{"date-parts":[[2023,6,9]],"date-time":"2023-06-09T01:32:34Z","timestamp":1686274354000},"page":"1354","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Detection and Molecular Characterization of Canine Babesiosis Causative Agent Babesia canis in Naturally Infected Dogs in the Dobrogea Area (Southeastern Romania)"],"prefix":"10.3390","volume":"13","author":[{"given":"Mariana","family":"Ionita","sequence":"first","affiliation":[{"name":"Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 11464 Bucharest, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laurentiu","family":"Leica","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 11464 Bucharest, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6622-9997","authenticated-orcid":false,"given":"Marion","family":"Wassermann","sequence":"additional","affiliation":[{"name":"Parasitology Unit, Institute of Biology, University of Hohenheim, 70599 Stuttgart, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emanuel","family":"Mitrea","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 11464 Bucharest, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabela Madalina","family":"Nicorescu","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 11464 Bucharest, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0567-9441","authenticated-orcid":false,"given":"Ioan Liviu","family":"Mitrea","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 11464 Bucharest, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1016\/S0195-5616(03)00039-1","article-title":"Canine babesiosis","volume":"33","author":"Boozer","year":"2003","journal-title":"Vet. Clin. N. Am. Small Anim. Pract."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1186\/s13071-016-1596-0","article-title":"A review of canine babesiosis: The European perspective","volume":"9","author":"Sainz","year":"2016","journal-title":"Parasites Vectors"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"S4","DOI":"10.1186\/1756-3305-2-S1-S4","article-title":"Canine babesiosis: From molecular taxonomy to control","volume":"2","author":"Irwin","year":"2009","journal-title":"Parasites Vectors"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.vetpar.2011.04.023","article-title":"Babesiosis in dogs and cats\u2013expanding parasitological and clinical spectra","volume":"181","author":"Baneth","year":"2011","journal-title":"Vet. Parasitol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1788","DOI":"10.1016\/j.meegid.2012.07.004","article-title":"Babesia: A world emerging","volume":"12","author":"Schnittger","year":"2012","journal-title":"Infect. Genet. Evol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1051\/parasite\/2014015","article-title":"Questionnaire-based survey on the distribution and incidence of canine babesiosis in countries of Western Europe","volume":"21","author":"Halos","year":"2014","journal-title":"Parasite"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.vetpar.2005.04.020","article-title":"Autochthonous canine babesiosis in The Netherlands","volume":"131","author":"Matjila","year":"2005","journal-title":"Vet. Parasitol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/j.vetpar.2010.03.024","article-title":"First case of babesiosis caused by Babesia canis canis in a dog from Norway","volume":"171","author":"Storli","year":"2010","journal-title":"Vet. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1186\/s13071-014-0477-7","article-title":"The first German map of georeferenced ixodid tick locations","volume":"7","author":"Rubel","year":"2014","journal-title":"Parasites Vectors"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1080\/01652176.1989.9694194","article-title":"Three groups of Babesia canis distinguished and a proposal for nomenclature","volume":"11","author":"Uilenberg","year":"1989","journal-title":"Vet. Q."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1017\/S0031182097001686","article-title":"Different Babesia canis isolates, different diseases","volume":"115","author":"Schetters","year":"1997","journal-title":"Parasitology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/S0304-4017(03)00130-4","article-title":"Humoral immunity and reinfection resistance in dogs experimentally inoculated with Babesia canis and either treated or untreated with imidocarb dipropionate","volume":"114","author":"Brandao","year":"2003","journal-title":"Vet. Parasitol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1186\/1756-3305-4-51","article-title":"Why is Southern African canine babesiosis so virulent? An evolutionary perspective","volume":"4","author":"Penzhorn","year":"2011","journal-title":"Parasites Vectors"},{"key":"ref_14","first-page":"134","article-title":"The pathophysiology of canine babesiosis: New approaches to an old puzzle","volume":"65","author":"Jacobson","year":"1994","journal-title":"J. S. Afr. Vet. Assoc."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1080\/00034983.1998.11813308","article-title":"Canine babesiosis in South Africa: More than one disease. Does this serve as a model for falciparum malaria?","volume":"92","author":"Reyers","year":"1998","journal-title":"Ann. Trop. Med. Parasitol."},{"key":"ref_16","first-page":"119","article-title":"Canine babesiosis: A perspective on clinical complications, biomarkers, and treatment","volume":"6","author":"Lobetti","year":"2015","journal-title":"Vet. Med. Res. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/S1471-4922(00)01856-0","article-title":"Tick-borne infectious diseases of dogs","volume":"17","author":"Shaw","year":"2001","journal-title":"Trends Parasitol."},{"key":"ref_18","first-page":"216","article-title":"The subspecies specificity of Babesia canis","volume":"109","author":"Hauschild","year":"1996","journal-title":"Berl. Munch. Tierarztl. Wochenschr."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1007\/s004360050445","article-title":"Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs","volume":"84","author":"Zahler","year":"1998","journal-title":"Parasitol. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1659","DOI":"10.1007\/s00436-011-2683-y","article-title":"Canine babesiosis in Romania due to Babesia canis and Babesia vogeli: A molecular approach","volume":"110","author":"Ionita","year":"2012","journal-title":"Parasitol. Res."},{"key":"ref_21","first-page":"27","article-title":"Retrospective study on prevalence of canine vector borne diseases in Bucharest area","volume":"26","author":"Anghel","year":"2016","journal-title":"Rev. Rom. Med. Vet."},{"key":"ref_22","first-page":"45","article-title":"Clinico-pathological findings in vector-borne pathogen co-infections in dogs, from Bucharest area","volume":"63","author":"Anghel","year":"2017","journal-title":"Sci. Works Ser. C Vet. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1645\/18-137","article-title":"Clinical occurrence of canine babesiosis in the coastal area of the Black Sea (Dobrogea) in southeastern Romania and associated epidemiological implications","volume":"105","author":"Leica","year":"2019","journal-title":"J. Parasitol."},{"key":"ref_24","unstructured":"Day, M., Mackin, A., and Littlewood, J. (2000). Manual of Canine and Feline Haematology and Transfusion Medicine, British Small Animal Veterinary Association."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.vetpar.2006.01.047","article-title":"The south african form of severe and complicated canine babesiosis: Clinical advances 1994\u20132004","volume":"138","author":"Jacobson","year":"2006","journal-title":"Vet. Parasitol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular evolutionary genetics analysis across computing platforms","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1645\/0022-3395(2000)086[0228:QPISOH]2.0.CO;2","article-title":"Quantifying parasites in samples of hosts","volume":"86","author":"Rozsa","year":"2000","journal-title":"J. Parasitol."},{"key":"ref_28","first-page":"65","article-title":"Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland","volume":"13","author":"Casati","year":"2006","journal-title":"Ann. Agric. Environ. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/S0304-4017(02)00112-7","article-title":"Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs","volume":"106","author":"Caccio","year":"2002","journal-title":"Vet. Parasitol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1051\/vetres:2004018","article-title":"Canine babesiosis in Slovenia: Molecular evidence of Babesia canis canis and Babesia canis vogeli","volume":"35","author":"Duh","year":"2004","journal-title":"Vet. Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.vetpar.2004.10.016","article-title":"Babesia canis canis in dogs from Hungary: Detection by PCR and sequencing","volume":"127","author":"Hell","year":"2005","journal-title":"Vet. Parasitol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1089\/vbz.2005.5.285","article-title":"Babesia DNA detection in canine blood and Dermacentor reticulatus ticks in southwestern Siberia, Russia","volume":"5","author":"Rar","year":"2005","journal-title":"Vector Borne Zoonotic Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.vetpar.2007.12.024","article-title":"Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland","volume":"152","author":"Adaszek","year":"2008","journal-title":"Vet. Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1016\/j.ijpara.2008.12.005","article-title":"Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia","volume":"39","author":"Beck","year":"2009","journal-title":"Int. J. Parasitol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/s13028-016-0189-4","article-title":"Fatal Babesia canis canis infection in a splenectomized Estonian dog","volume":"58","author":"Tiskina","year":"2016","journal-title":"Acta Vet. Scand."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1017\/S0031182017001536","article-title":"Occurrence and diversity of arthropod-transmitted pathogens in red foxes (Vulpes vulpes) in western Austria, and possible vertical (transplacental) transmission of Hepatozoon canis","volume":"145","author":"Hodzic","year":"2018","journal-title":"Parasitology"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"D\u2019Amico, G., Ionic\u0103, A.M., Gy\u00f6rke, A., and Dumitrache, M.O. (2022). Epidemiological Survey of the Main Tick-Borne Pathogens Infecting Dogs from the Republic of Moldova. Pathogens, 11.","DOI":"10.3390\/pathogens11111267"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1111\/j.1550-7408.1999.tb05128.x","article-title":"Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: Differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes","volume":"46","author":"Carret","year":"1999","journal-title":"J. Eukaryot. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1016\/j.vetpar.2014.09.001","article-title":"Detection and molecular characterization of canine babesiosis causative agent Babesia canis in the naturally infected dog in Lithuania","volume":"205","author":"Paulauskas","year":"2014","journal-title":"Vet. Parasitol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1186\/s13071-015-1099-4","article-title":"Pathogens vectored by the tick, Dermacentor reticulatus, in endemic regions and zones of expansion in Poland","volume":"8","author":"Mierzejewska","year":"2015","journal-title":"Parasites Vectors"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"101459","DOI":"10.1016\/j.ttbdis.2020.101459","article-title":"Babesiosis in Latvian domestic dogs, 2016\u20132019","volume":"11","author":"Seleznova","year":"2020","journal-title":"Ticks Tick. Borne Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.vetpar.2004.07.028","article-title":"First molecular detection of Babesia vogeli in dogs from Brazil","volume":"127","author":"Passos","year":"2005","journal-title":"Vet. Parasitol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"101986","DOI":"10.1016\/j.ttbdis.2022.101986","article-title":"Pathogens in ticks collected in Israel: II. Bacteria and protozoa found in Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus","volume":"13","author":"Mumcuoglu","year":"2022","journal-title":"Ticks Tick. Borne Dis."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1016\/j.ttbdis.2013.06.006","article-title":"Survey of babesiosis in symptomatic dogs from Romania: Occurrence of Babesia gibsoni associated with breed","volume":"4","author":"Imre","year":"2013","journal-title":"Ticks Tick. Dis."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"669","DOI":"10.2478\/s11686-020-00199-x","article-title":"Epidemiological study of canine babesiosis and hepatozoonosis in the South of Romania","volume":"65","author":"Cimpan","year":"2020","journal-title":"Acta Parasitol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Ciuc\u0103, L., Martinescu, G., Miron, L.D., Roman, C., Acatrinei, D., Cringoli, G., Rinaldi, L., and Maurelli, M.P. (2021). Occurrence of Babesia species and co-infection with Hepatozoon canis in symptomatic dogs and in their ticks in Eastern Romania. Pathogens, 10.","DOI":"10.3390\/pathogens10101339"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1515\/jvetres-2016-0063","article-title":"Occurrence of different strains of Babesia canis in dogs in eastern Poland","volume":"60","author":"Bartnicki","year":"2016","journal-title":"J. Vet. Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.ttbdis.2013.01.007","article-title":"Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands","volume":"4","author":"Schaarschmidt","year":"2013","journal-title":"Ticks Tick. Borne Dis."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Radzijevskaja, J., Mardosait\u0117-Busaitien\u0117, D., Aleksandravi\u010dien\u0117, A., Karvelien\u0117, B., Razg\u016bnait\u0117, M., Stadalien\u0117, I., and Paulauskas, A. (2022). Genetic Diversity of Babesia canis Strains in Dogs in Lithuania. Microorganisms, 10.","DOI":"10.3390\/microorganisms10071446"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"3641","DOI":"10.1093\/nar\/19.13.3641","article-title":"Sequence microheterogeneity of the three small subunit ribosomal RNA genes of Babesia bigemina: Expression in erythrocyte culture","volume":"19","author":"Reddy","year":"1991","journal-title":"Nucleic Acids Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1016\/0020-7519(92)90139-C","article-title":"Babesia bovis, Babesia bigemina, Babesia canis, Babesia microti and Babesia rodhaini: Comparison of ribosomal RNA gene organization","volume":"22","author":"Dalrymple","year":"1992","journal-title":"Int. J. Parasitol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"4172","DOI":"10.1128\/JCM.41.9.4172-4177.2003","article-title":"Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples","volume":"41","author":"Birkenheuer","year":"2003","journal-title":"J. Clin. Microbiol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"59","DOI":"10.4102\/ojvr.v76i1.66","article-title":"Canine babesiosis","volume":"76","author":"Schoeman","year":"2009","journal-title":"Onderstepoort J. Vet. Res."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.vetpar.2008.07.024","article-title":"Babesia canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease","volume":"157","author":"Trotta","year":"2008","journal-title":"Vet. Parasitol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1294","DOI":"10.14202\/vetworld.2020.1294-1302","article-title":"Clinicopathological and molecular profiles of Babesia vogeli infection and Ehrlichia canis coinfection","volume":"13","author":"Rawangchue","year":"2020","journal-title":"Vet. World"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1186\/1756-3305-6-97","article-title":"Are vector-borne pathogen co-infections complicating the clinical presentation in dogs?","volume":"6","author":"Otranto","year":"2013","journal-title":"Parasites Vectors"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.ttbdis.2015.10.006","article-title":"Molecular detection of Rickettsia conorii and other zoonotic spotted fever group rickettsiae in ticks, Romania","volume":"7","author":"Ionita","year":"2016","journal-title":"Ticks Tick. Borne Dis."},{"key":"ref_58","unstructured":"Ionita, M., Mitrea, I.L., Buzatu, M.C., and Dascalu, L. (2010, January 15\u201320). Seasonal dynamics of tick (Acari: Ixodidae) populations in different areas of Romania and the associated risks of tick-borne diseases. Proceedings of the XIIth International Congress of Parasitology (ICOPA), Melbourne, VIC, Australia."}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/13\/6\/1354\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:51:18Z","timestamp":1760125878000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/13\/6\/1354"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,9]]},"references-count":58,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2023,6]]}},"alternative-id":["life13061354"],"URL":"https:\/\/doi.org\/10.3390\/life13061354","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,6,9]]}}}