To confirm that B. venatorum is present in the United Kingdom and to identify putative vertebrate hosts, we collected blood from sheep (n = 93) and cattle (n = 107) at 2 farms in northeastern Scotland (Appendix Figure), selected because of previous reports of tickborne disease, including red water in cattle (Babesia divergens infection) and tickborne fever (Anaplasma phagocytophilum infection) and louping ill (ovine encephalomyelitis) in sheep. We also collected blood postmortem from culled wild red deer at site A (n = 24) and 6 surrounding areas (n = 60; Appendix Figure). To provide temporal information, we sampled 34 sheep at site B in both June and November 2014 and sampled 12 sheep in either June or November. The study was approved by the Ethics and Welfare Committee of the University of Glasgow School of Veterinary Medicine (Ref. 15a/13).

We prepared smears from sheep blood and stained them with May-Grünwald Giemsa stain. We extracted DNA using a Wizard Genomic DNA Purification Kit (Promega, https://www.promega.com) with prior homogenization and incubation with proteinase K (Invitrogen, https://www.thermofisher.com). We amplified the informative hypervariable V4 region of the 18S SSU rRNA gene using nested PCR and previously validated primers/conditions (outer: BT1-F 5′-GGTTGATCCTGCCAGTAGT and BTH-1R 5′-TTGCGACCATACTCCCCCCA [13]; inner: RLB-F2 5′-GACACAGGGAGGTAGTGACAAG and RLB-R2 5′-CTAAGAATTTCACCTCTGACAGT [14]). We separated amplicons of the predicted size using agarose gel electrophoresis, purified them using a QIAquick PCR purification kit (QIAGEN, https://www.qiagen.com), and had them sequenced commercially (Eurofins Genomics, https://www.eurofinsgenomics.com). We deposited the sequences into GenBank (accession ns. MK641004–18) and compared them with the National Center for Biotechnology Information nonredundant database using BLAST (https://blast.ncbi.nlm.nih.gov). We aligned high-scoring hits and constructed a neighbor-joining tree using ClustalW (https://www.genome.jp/tools-bin/clustalw). We assessed tree stability with 1,000 bootstrapping replicates and visualized it using FigTree version 1.4.2 (https://github.com/rambaut/figtree/releases). We included the bovine 18S SSU rRNA sequence as a root.

Figure 1

Figure 1. Representative images of small intracellular Babesia (arrows) identified in sheep erythrocytes from several sites in northeastern Scotland, UK. Both paired proforms and ring forms are visible. Images were taken at ×1,000...

Figure 2

Figure 2. A neighbor-joining tree of 18S small subunit rRNA amplicon sequences obtained from sheep at sites A and B in northeastern Scotland, UK. Blue shading indicates sequences obtained in this study. Previously...

Initial blood smears from sheep revealed the presence of a small Babesia species displaying ring and pyriform morphology in 3 samples (Figure 1). Further PCR and sequencing revealed 11 positive samples that were identical to 30 Babesia 18S SSU rRNA sequences in the National Center for Biotechnology Information database annotated as Babesia sp. EU1 or B. venatorum (Figure 2). These sequences had been amplified from human patients in Austria, Italy, and China. These data demonstrate that B. venatorum is indeed present in the United Kingdom and that domestic sheep are a host.

At the site sampled on 2 separate occasions, most (85%) ewes were negative in June and November 2014 and none were positive at both times. Four (12%) were negative in June and became positive in November, whereas 1 was positive on first sampling but negative on the second. These findings indicate that B. venatorum infection is persistent but dynamic within the sheep population; new animals are infected over time, and previously infected animals become PCR negative.

A separate group of 4 sequences distinct from B. venatorum was also obtained in the study and showed high identity to the sheep apicomplexan parasite Sarcocystis tenella (Figure 2). No B. venatorum infections were detected in the cattle population, despite sharing pasture with infected sheep, and no B. venatorum infections were detected in any of the culled red deer.

This study confirms that B. venatorum is present in the United Kingdom, but it remains unclear how the parasite entered the country, because there was no history of imported animals at either farm surveyed. However, the survey sites are situated near the main landing areas for migratory birds coming to the United Kingdom from continental Europe, particularly Norway, and B. venatorum has been found in ticks collected from the environment and in migratory birds in Scandinavia (15). We postulate that birds could act as an import vector for ticks carrying B. venatorum.

The presence of B. venatorum in the United Kingdom represents a new risk to humans working, living, or hiking in areas harboring infected ticks and livestock, particularly sheep. As such, local health and veterinary professionals will need to be aware of the disease if the risk for tickborne disease in the United Kingdom is to be fully understood. Current UK medical inclusion criteria for babesiosis focus on identifying cattleborne B. divergens. Going forward, consideration of B. venatorum, through careful morphologic description of blood smears and sequencing of informative regions of the 18S SSU rRNA gene, will be necessary for accurate diagnosis and correctly targeted treatment regimens.

Our study has revealed that sheep are a natural host for B. venatorum in the United Kingdom. Previously, roe deer were believed to be the main vertebrate host for this parasite in Europe (9,10). It is unclear why B. venatorum has not previously been detected in sheep, although it may be that infection in this host species occurs only in particular foci or is limited to the United Kingdom. Thus, ongoing active surveillance of Babesia species in UK livestock would be useful to fully understand the prevalence and transmission of the disease. Such information may be critical for controlling the spread of babesiosis, because sheep are routinely transported large distances (including across international borders) and are closely associated with tick habitats. Our study also suggests that the role that livestock play in B. venatorum transmission in continental Europe should be reassessed.

In summary, we have demonstrated that B. venatorum is present in the UK sheep population. This finding represents a novel potential threat to animal and human health and demonstrates that livestock may act as a major host for B. venatorum, affecting the spread of babesiosis across Europe.