Li et al. BMC Veterinary Research (2015) 11:246 DOI 10.1186/s12917-015-0560-0 METHODOLOGY ARTICLE Open Access Development of a pan-Babesia FRET-qPCR and a survey of livestock from five Caribbean islands Jing Li1, Patrick Kelly2, Jilei Zhang1, Chuanling Xu1 and Chengming Wang1* Abstract Background: Babesia spp. are tick-borne protozoan hemoparasites and the second most common blood-borne parasites of mammals, in particular domestic animals. We used the Clustal Multiple Alignment program and 18S rRNA gene sequences of 22 Babesia species from GenBank to develop a PCR that could detect a wide variety of Babesia spp. in a single reaction. The pan-Babesia FRET-qPCR we developed reliably detected B. gibsoni, B. canis, B. vogeli, B. microti, B. bovis, and B. divergens under controlled conditions but did not react with closely related species, mainly Hepatozoon americanum, Theileria equi, and Toxoplasma gondii. Results: When we tested the pan-Babesia FRET-qPCR on DNA of whole blood from 752 cattle, sheep, goats, donkeys and horses from five Caribbean islands, we detected Babesia spp. expected to be present in the animals, mainly B. bovis and B. bigemina in cattle and B. caballi in horses and donkeys. Further, we found that animals were not uncommonly infected with species of Babesia usually associated with other hosts, mainly B. vogeli and B. gibsoni in cattle, sheep and goats, B. rossi in goats, and B. caballi in goats and sheep. Finally, the pan-Babesia FRET-qPCR enabled us to identify unknown species of Babesia in cattle, goats, sheep and donkeys. Conclusions: Overall, 70 % (525/752) of the animals we tested were positive confirming earlier limited studies that infections with Babesia spp. are common in livestock in the Caribbean. Keywords: Babesia spp, FRET-qPCR, Livestock, Caribbean Islands Background in horses and donkeys [12]. Further, B. canis, B. vogeli and Babesia spp. are tick-borne protozoan hemoparasites that B. gibsoni are important causes of morbidity and mortality occur worldwide [1–4]. They are the second most com- in dogs worldwide [13] while B. microti and B. divergens mon blood-borne parasites of mammals, after trypano- are the species that most commonly infect people [8]. somes, with infections occurring commonly in domestic Initially, differentiation of the Babesia spp. was based on animals, in particular cattle, dogs, horses, sheep, and pigs morphological and biological characteristics, and inverte- [5]. Recently, infections with Babesia (babesiosis) have brate and vertebrate host specificity. With the advent of been described in birds [6–8] and have attracted increas- molecular tools, however, these methods have proven to be ing attention as zoonotic infections in people [5, 9]. of limited taxonomic value [8]. A number of nucleic acid- Since the first description of Babesia in cattle by Victor based techniques have been reported which detect Babesia Babes in 1888, over 100 Babesia species have been identi- spp. with high sensitivity and specificity. Most commonly, fied [8]. Many cause significant economic losses in live- these assays have a narrow spectrum and specifically iden- stock, mainly B. bovis and B. bigemina in cattle [10], B. tify B. microti, B. divergens,orgroupsofBabesia spp. asso- motasi and B. ovis in small ruminants [11] and B. caballi ciatedwithspecifichostssuchasdogs[14,15],cattle [16, 17] or sheep [18, 19]. To enable the detection of a wide * Correspondence: [email protected] range of Babesia spp. of veterinary and public health signifi- 1Jiangsu Co-innovation Center for Prevention and Control of Important cance in a single reaction we developed a broad-based Animal Infectious Diseases and Zoonoses, Yangzhou University College of Animal Science and Technology, Yangzhou, Jiangsu 225009, P. R. China qPCR.Further,wetestedourpan-Babesia FRET-qPCR on Full list of author information is available at the end of the article DNAs extracted from whole blood samples collected from © 2015 Li et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. BMC Veterinary Research (2015) 11:246 Page 2 of 8 five livestock species on five Caribbean islands. The results centrifugation and stored at −20 °C until thawed at room of these experiments are described below. temperature and DNA extracted from aliquots (200 μL) using the QIAamp DNA Blood Mini Kit (QIAGEN, Methods Valencia, CA, USA) according to the manufacturer’sin- Whole blood structions. The DNA was eluted in 200 μL washing buffer Whole blood samples (n = 752) were collected into EDTA and shipped to Yangzhou University College of Veterinary from apparently healthy livestock on five Caribbean islands, Medicine of Jiangsu province, China at room temperature including 162 from Dominica (cattle = 77, goats = 70, and where it was frozen at −80 °C until PCRs were performed. sheep = 15), 31 from Grenada (all goats), 93 from Montserrat (cattle = 12, goats = 19, and sheep = 62), 198 Pan-Babesia FRET-qPCR from Nevis (cattle = 43, goats = 114, and sheep = 41) and The PCRs in this study were performed on a Roche 268 from St. Kitts (cattle = 193, goats = 4, sheep = 26, don- Light-Cycler 480-II platform. The HMBS-based quanti- keys = 25, and horses = 20) [20]. The study was reviewed tative PCR was used as an endogenous quality control to and approved by the Institutional Animal Care and Use verify the quality of the DNA in the samples [21]. Committee of the Ross University School of Veterinary Medicine (RUSVM), St Kitts. Primers and probes The 18S rRNA sequences for 22 recognized Babesia DNA extraction spp. of public health significance and/or veterinary im- After collection, the blood samples were transported on portance were obtained from GenBank (Fig. 1): B. microti ice to RUSVM where red blood cells were separated by (AB071177, AB219802), B. leo (AF244911, AY452708), B. Fig. 1 Alignment of the partial 18S rRNA gene amplicons of Babesia spp. and other related species. The upstream primer-1 (in red), upstream primer-2 (in blue), the fluorescein/LCRed 640 probes and the downstream primer are indicated in the top of the boxes. Dots indicate nucleotides identical to the primers and probes, and the dashes denote the deletion of the nucleotides. The upstream primers and two probes are used as the indicated sequences while the downstream primer is used as antisense oligonucleotide. While the probes and downstream primer show minimum mismatch with Babesia spp. and other related species, the upstream primers (−1and−2) have 0–1 nucleotide mismatch with Babesia spp. but 6–16 nucleotide mismatches with the related non- Babesia species Li et al. BMC Veterinary Research (2015) 11:246 Page 3 of 8 rodhaini (DQ641423, AB049999), B. felis (AF244912, Toxoplasma gondii as negative controls. In addition, we AY452707), B. poelea (DQ200887), B. bigemina (JQ723014, used plasmids created to contain the 18S rRNA gene KF112076), B. bovis (HQ264124, HQ264127), B. caballi (Integrated DNA Technologies, Coralville, IA, USA) of (AY534883), B. canis (AY072926, JN982353), B. capreoli B. microti, B. bovis,andB. divergens as positive controls (FJ944828, GQ304526), B. crassa (AY260176, JX542614), B. and T. equi as negative controls. divergens (FJ944822, FJ944826), B. gibsoni (KJ142323), B. To test the sensitivity of the pan-Babesia FRET-PCR hongkongensis (JQ867356), B. kiwiensis (EF55315), B. major we used quantitative standards consisting of amplifica- (JF802040), B. motasi (AY260179, AY533147), B. odocoilei tion products of PCRs for B. gibsoni, B. canis and B. (AY661508, U16369), B. ovata (AY081192, AY603400), B. vogeli identified in a previous study [14, 22]. The ampli- rossi (JN982353), B. vitalii (JN880430, JN880431) and B. cons were gel purified with the QIAquick Gel Extrac- vogeli (HM590440). In addition, the 18S rRNA sequences tion Kit (Qiagen, Valencia, CA), quantified by the of 9 related protozoan species were also obtained from PicoGreen DNA fluorescence assay (Molecular Probes, GenBank: Theileria equi (AB515307, AB515312), T. parva Eugene, OR), and sequenced at the Genomic Sequen- (L02366), Trypanosoma cruzi (AF303659), Toxoplasma gon- cing Laboratory (GenScript, Nanjing, Jiangsu, China). dii (L37415), Neosporo caninum (U63069), Eimeria arnyi The purified amplicons were diluted at 1,000, 100, 10, 1 (AY613853), Cytauxzoon felis (AY679105), Hepatozoon genome copies per PCR reaction in T10E0.1 buffer as americanum (AF176836), Cryptosporidium meleagridis described previously [24], and used as quantitative (AF112574) and C. parvum (L16996). standards. These sequences were aligned using Clustal Multiple In the specificity and sensitivity tests, the PCR prod- Alignment to identify conserved and variable regions ucts were electrophoresed through 1.5 % MetaPhor suitable for primers and probes that could differentiate agarose gels. We calculated the size of the possible PCR the species. The upstream primer-1 (5′-ATG GCT TTG amplicons for the different Babesia species to be CCG GCG ATG TAT CA-3′) was selected because of its between 282 to 293 bp and random samples