{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T09:40:59Z","timestamp":1789724459674,"version":"4.0.1"},"reference-count":39,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2016,9,13]],"date-time":"2016-09-13T00:00:00Z","timestamp":1473724800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation) showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.<\/jats:p>","DOI":"10.3390\/vetsci3030023","type":"journal-article","created":{"date-parts":[[2016,9,13]],"date-time":"2016-09-13T10:10:00Z","timestamp":1473761400000},"page":"23","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["An Evaluation of Quantitative PCR Assays (TaqMan\u00ae and SYBR Green) for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates"],"prefix":"10.3390","volume":"3","author":[{"given":"Bing","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Agriculture & Fisheries, Agri-Science Queensland, Animal Science, Dutton Park QLD 4102, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Sambono","sequence":"additional","affiliation":[{"name":"Department of Agriculture & Fisheries, Biosecurity Queensland, Tick Fever Centre, Wacol QLD 4076, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jess","family":"Morgan","sequence":"additional","affiliation":[{"name":"Department of Agriculture & Fisheries, Agri-Science Queensland, Animal Science, Dutton Park QLD 4102, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bronwyn","family":"Venus","sequence":"additional","affiliation":[{"name":"Queensland Alliance for Agriculture & Food Innovation, University of Queensland, Centre for Animal Science, St Lucia QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Rolls","sequence":"additional","affiliation":[{"name":"Department of Agriculture & Fisheries, Biosecurity Queensland, Tick Fever Centre, Wacol QLD 4076, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ala","family":"Lew-Tabor","sequence":"additional","affiliation":[{"name":"Queensland Alliance for Agriculture & Food Innovation, University of Queensland, Centre for Animal Science, St Lucia QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2016,9,13]]},"reference":[{"key":"ref_1","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_2","unstructured":"Sackett, D., Holmes, P., Abbott, K., Jephcott, S., and Barber, M. (2006). Assessing the Economic Cost of Endemic Disease on the Profitability of Australian Beef Cattle and Sheep Producers, Meat & Livestock Australia Limited."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/j.ttbdis.2015.12.012","article-title":"A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases","volume":"7","year":"2016","journal-title":"Ticks Tick Borne Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1373","DOI":"10.1128\/jcm.30.6.1374-1379.1992","article-title":"Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA","volume":"30","author":"Fahrimal","year":"1992","journal-title":"J. Clin. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/j.ijpara.2003.12.012","article-title":"Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda","volume":"34","author":"Oura","year":"2004","journal-title":"Int. J. Parasitol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"832","DOI":"10.1111\/j.1751-0813.2001.tb10931.x","article-title":"Immunity following use of the Australian tick fever vaccine: A review of the evidence","volume":"79","author":"Bock","year":"2001","journal-title":"Aust. Vet. J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/S0304-4017(97)00033-2","article-title":"Sustainable tick and tickborne disease control in livestock improvement in developing countries","volume":"71","year":"1997","journal-title":"Vet. Parasitol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/S0304-4017(01)00464-2","article-title":"Bovine piroplasms in Minorca (Balearic Islands, Spain): A comparison of PCR-based and light microscopy detection","volume":"99","author":"Almeria","year":"2001","journal-title":"Vet. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2748","DOI":"10.1128\/jcm.34.11.2748-2755.1996","article-title":"Monitoring Babesia bovis infections in cattle by using PCR-based tests","volume":"34","author":"Calder","year":"1996","journal-title":"J. Clin. Microbiol."},{"key":"ref_10","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."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.vetpar.2005.02.014","article-title":"Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences","volume":"133","author":"Luo","year":"2005","journal-title":"Vet. Parasitol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.ijpara.2004.09.002","article-title":"PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle","volume":"35","author":"Oliveira","year":"2005","journal-title":"Int. J. Parasitol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1016\/S0304-4017(97)00025-3","article-title":"PCR methods for the discrimination of Babesia bovis isolates","volume":"71","author":"Lew","year":"1997","journal-title":"Vet. Parasitol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.vetpar.2006.01.011","article-title":"Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis","volume":"137","author":"Francino","year":"2006","journal-title":"Vet. Parasitol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1128\/AAC.50.4.1195-1201.2006","article-title":"Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation","volume":"50","author":"Torrado","year":"2006","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.vetpar.2007.03.031","article-title":"A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina","volume":"147","author":"Buling","year":"2007","journal-title":"Vet. Parasitol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"837","DOI":"10.4269\/ajtmh.2007.77.837","article-title":"Development of TaqMan-based real-time PCR assays for diagnostic detection of Babesia bovis and Babesia bigemina","volume":"77","author":"Kim","year":"2007","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1111\/j.1751-0813.1997.tb14197.x","article-title":"Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination","volume":"75","author":"Lew","year":"1997","journal-title":"Aust. Vet. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1111\/j.1751-0813.1995.tb03558.x","article-title":"Studies on failure of T strain live Babesia bovis vaccine","volume":"72","author":"Bock","year":"1995","journal-title":"Aust. Vet. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.vetpar.2013.10.008","article-title":"Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa","volume":"199","author":"Combrink","year":"2014","journal-title":"Vet. Parasitol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.vetpar.2014.12.033","article-title":"Differentiation between Israeli B. bovis vaccine strain and field isolates","volume":"208","author":"Molad","year":"2015","journal-title":"Vet. Parasitol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.vetpar.2009.09.021","article-title":"Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates","volume":"167","author":"Caballero","year":"2010","journal-title":"Vet. Parasitol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1006\/mcpr.1998.0223","article-title":"Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis","volume":"13","author":"Salem","year":"1999","journal-title":"Mol. Cell. Probes"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1038\/72708","article-title":"An economic method for the fluorescent labeling of PCR fragments","volume":"18","author":"Schuelke","year":"2000","journal-title":"Nat. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1002\/elps.201300286","article-title":"A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria","volume":"35","author":"Godwin","year":"2014","journal-title":"Electrophoresis"},{"key":"ref_26","first-page":"601","article-title":"Bovine Babesiosis","volume":"Volume 1","author":"Rolls","year":"2012","journal-title":"OIE Manual of Diagnostic Tests and Vaccine for Terrestrial Animals"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1128\/JB.01330-09","article-title":"Complete genome sequence of Anaplasma marginale subsp","volume":"192","author":"Herndon","year":"2010","journal-title":"centrale. J. Bacteriol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2576","DOI":"10.1128\/jcm.30.10.2576-2582.1992","article-title":"Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification","volume":"30","author":"Figueroa","year":"1992","journal-title":"J. Clin. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.4315\/0362-028X-72.5.1055","article-title":"Real-time PCR detection and identification of prohibited mammalian and avian material in animal feeds","volume":"72","author":"Cawthraw","year":"2009","journal-title":"J. Food Prot."},{"key":"ref_30","unstructured":"Theiler, A. (1911). Further Investigation into Anaplasmosis of South African Cattle."},{"key":"ref_31","first-page":"363","article-title":"Epidemiology and control of anaplasmosis in Australia","volume":"50","author":"Rogers","year":"1979","journal-title":"J. S. Afr. Vet. Assoc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.vetpar.2010.10.035","article-title":"Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis","volume":"176","author":"Ramos","year":"2011","journal-title":"Vet. Parasitol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.pt.2005.05.012","article-title":"Emerging technologies for the detection and genetic characterization of protozoan parasites","volume":"21","author":"Monis","year":"2005","journal-title":"Trends Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.vetpar.2015.12.030","article-title":"Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field","volume":"217","author":"Mira","year":"2016","journal-title":"Vet. Parasitol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1186\/s13071-015-1106-9","article-title":"Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya","volume":"8","author":"Aboge","year":"2015","journal-title":"Parasit. Vectors"},{"key":"ref_36","first-page":"406","article-title":"Molecular and seroepidemiological survey of Babesia bovis and Babesia bigemina infections in cattle and water buffaloes in the central region of Vietnam","volume":"31","author":"Li","year":"2014","journal-title":"Trop. Biomed."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/j.vetpar.2011.12.038","article-title":"Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria","volume":"187","author":"Terkawi","year":"2012","journal-title":"Vet. Parasitol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"995","DOI":"10.1292\/jvms.12-0425","article-title":"Epidemiological survey of Babesia bovis and Babesia bigemina infections of cattle in Philippines","volume":"75","author":"Yu","year":"2013","journal-title":"J. Vet. Med. Sci."},{"key":"ref_39","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."}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/3\/3\/23\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T19:30:50Z","timestamp":1760211050000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/3\/3\/23"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,9,13]]},"references-count":39,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2016,9]]}},"alternative-id":["vetsci3030023"],"URL":"https:\/\/doi.org\/10.3390\/vetsci3030023","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,9,13]]}}}