Anaplasma phagocytophilum Ecotype Analysis in Cattle from Great Britain

Anaplasma phagocytophilum (A. phagocytophilum) is the aetiological agent of tick-borne fever in cattle and sheep, and granulocytic anaplasmosis in human and dogs. Livestock, companion animal and human infections with A. phagocytophilum have been reported globally. Across England and Wales, two isolates (called ecotypes) have been reported in ticks. This study examined A. phagocytophilum isolates present in livestock and wildlife in Great Britain (GB), with a particular focus on cattle. Clinical submissions (EDTA blood) from cattle (n = 21) and sheep (n = 3) were received by APHA for tick-borne disease testing and the animals were confirmed to be infected with A. phagocytophilum using a PCR targeting the Msp2 gene. Further submissions from roe deer (n = 2), red deer (n = 2) and Ixodes ricinus ticks (n = 22) were also shown to be infected with A. phagocytophilum. Subsequent analysis using a nested PCR targeting the groEL gene and sequencing confirmed the presence of ecotype I in cattle, sheep, red deer and Ixodes ricinus, and ecotype II in roe deer and I. ricinus removed from deer carcasses. Despite the presence of two ecotypes, widely distributed in ticks from England and Wales, only ecotype I was detected in cattle in this study.

Human infection with A. phagocytophilum is considered an emerging zoonotic disease. It was first recorded as a febrile illness and named human granulocytic ehrlichiosis (HGE) in North America in 1994 [21]. Case rates have dramatically increased since, and the disease poses an increasingly important public health threat. The first European cases were documented in Slovenia [22][23][24][25], with further cases reported in other European countries, including Spain [4,26], France [27] and Italy [28]. However, overall disease rates remain substantially lower in Europe than in the United States. The rarity of human infection with A. phagocytophilum in GB has been considered to be due to the absence of zoonotic isolates, also described as ecotypes [18]. Recent studies have documented A. phagocytophilum presence in GB, including a prevalence survey of questing ticks in Wales (12.1%), Northern England (4.7%), Southern England (1.8%) and central England (0.5%) [17]. Ecotype I has been recorded in GB sheep (O. aries) [18] associated with specific areas and is considered more likely to infect humans [17]. Gandy et al. [17] and Bianchessi et al. [18] examined questing ticks and sheep, respectively. However, cases of bovine anaplasmosis [29,30] and co-infection with Babesia and A. phagocytophilum resulting in severe disease have been recorded in GB and Europe [16,31,32]. Bovine anaplasmosis is a transient and mild infection characterized by clinical signs such as fever, anorexia, weakness and secondary infection due to immunosuppression [33]. High morbidity associated with abortion and a drop in milk production, resulting in economic losses, have also been reported in dairy cattle diagnosed with bovine anaplasmosis [32][33][34]. However, information regarding the potential role of A. phagocytophilum in the pathogenesis and diagnosis of abortions and stillbirth in cattle is not clearly known [34].
To establish the evolutionary relationship of Anaplasma species, a selection of appropriate molecular gene marker is essential. PCR assays designed to amplify 16S rRNA [13], highly conserved major surface protein-2 (Msp2 or p44), citrate synthase (gltA) [35,36], ankA [37] and heat shock protein (groEL) [17,18] genes have all proven to be useful. In addition, PCR and sequencing assays designed to target multiple genes (multilocus sequence genotyping) have been used effectively for A. phagocytophilum genotyping [37,38]. However, the groEL gene has demonstrated to be a valuable and useful genetic marker to identify and establish distinct genetic clades of A. phagocytophilum in the absence of multilocus sequence genotyping [18,39,40]. Information on A. phagocytophilum ecotypes present in GB cattle is currently unavailable. The aim of this study was to investigate A. phagocytophilum isolates present in GB cattle from samples (initially tested using Msp2 gene qPCR) obtained from Southern England, Wales and Scotland. Additional samples obtained from sheep, deer and I. ricinus were also examined.

Sample Collection, Polymerase Chain Reaction (PCR) and Sequencing
Whole engorged ticks (collected from cattle and roe deer only) and EDTA blood samples collected from cattle (Bos taurus), sheep (O. aries) and both roe (Capreolus capreolus), and red (Cervus elaphus) deer submitted from nine locations (for tick-borne pathogen testing) in Great Britain, including England (Devon, Cornwall, Norfolk, Cumbria, Somerset, and Dorset), Scotland and Wales (Pembrokeshire and Powys), were examined. Samples were tested for A. phagocytophilum following DNA extraction using QIAamp DNeasy kit (QIAgen, Manchester, UK). Msp2 gene-specific TaqMan qPCR assay [41] was used to initially screen samples for the presence of A. phagocytophilum DNA. Where this was detected, a partial groEL gene heminested PCR assay was applied [39]. PCR products obtained were separated on agarose gel (1.5%) prepared with SYBR safe nucleic acid staining dye (Thermo Fisher Scientific, UK) and run at 100 volts for 60 min. PCR controls consisted of A. phagocytophilum DNA (positive) [16] and nuclease-free water (negative). The groEL gene PCR products were sequenced using primers Ephpl groEL (569) and Eph groEL (1142). Morphological identification and molecular barcoding of tick samples were carried out, following published methods [42]. Details of all PCR primers, master mix reaction volumes and thermal cycling conditions are provided in the Supplementary file (Supplementary Materials Table S4).

Sequence Editing, Assembly and Phylogenetic Analysis
Quality assessment, removal of primer sequence, editing, alignment and generation of consensus sequence from Sanger sequence obtained was carried out using SeqMan Pro in DNAStar Lasergene v15, followed by NCBI online BLASTn search. A. phagocytophilum groEL gene DNA sequences generated were deposited in NCBI GenBank under accession number OQ436965-OQ437012. A total of 328 A. phagocytophilum groEL gene nucleotide sequences, including sequences generated from this study, downloaded from GenBank database and published in the recent literature [18,43,44], were used for phylogenetic analysis. A maximum likelihood phylogenetic tree was constructed in IQ-TREE v2.0.7 [45], using 10,000 bootstrap approximations and implementation of UFBoot2 within IQ-TREE to evaluate nodes [46]. IQ-TREE v2.0.7 automatically selected the best nucleotide substitution model (HKY+F+R2) [47] with the lowest Bayesian information criterion (BIC) [48]. The precision of the phylogenetic tree generated was improved by the inclusion of the Mod-elFinder option in command-line for the selection of the best fitting nucleotide substitution model [48]. The tree constructed was visualized and annotated in FigTree v1.4.4. to produce a radial phylogram for simple presentation.

Results
A total of fifty samples that previously tested positive for the presence of A. phagocytophilum via the Msp2 gene qPCR assay were assessed for A. phagocytophilum genotyping, following groEL gene PCR assay.  Table S2). Although all nucleotide sequences obtained were >90% identical to other A. phagocytophilum sequences available in the GenBank database, 17/48 of sequences obtained were >99% identical to sequences (accession numbers: KJ832487, KJ832484) originating from Bos taurus (France) and O. aries (AF548385) from Norway. Maximum likelihood phylogenetic analysis demonstrated that 45/48 sequences generated in this study clustered with A. phagocytophilum sequences identified as ecotype I, while the remaining three (3/48) clustered with ecotype II (Figure 1, Supplementary Materials Figure S1 [18]. This set of three A. phagocytophilum sequences were generated from C. capreolus blood/tick samples and showed a greater identity (NCBI online BLASTn search) to A. phagocytophilum sequences (accession number KM215256, JN005745, DQ779568 and AF383226) detected from C. capreolus from Poland and Switzerland, classified as ecotype II in the recent literature [43].

Figure 1.
A simplified radial phylogram displayed following a maximum likelihood phylogenetic tree constructed with 10,000 bootstrap approximation using A. phagocytophilum groEL gene sequences generated from this study (n = 48) and representative ecotypes (n = 328) from previous studies [18,43]. Clades,branches and branch tips in black represents ecotype I, red represents ecotype II, green represents ecotype III and blue represents ecotype IV, while taxa in magenta under ecotypes I and II represent nucleotide sequences reported from this study. Branch tips highlighted in grey under ecotype I clade represents A. phagocytophilum ecotype I sequences reported from Great Britain cattle. Roman numerals I, II, III and IV represents ecotypes. The phylogeny is rooted at midpoint and drawn to display circular node shapes at bootstrap support values ≥ 90.

Figure 1.
A simplified radial phylogram displayed following a maximum likelihood phylogenetic tree constructed with 10,000 bootstrap approximation using A. phagocytophilum groEL gene sequences generated from this study (n = 48) and representative ecotypes (n = 328) from previous studies [18,43]. Clades, branches and branch tips in black represents ecotype I, red represents ecotype II, green represents ecotype III and blue represents ecotype IV, while taxa in magenta under ecotypes I and II represent nucleotide sequences reported from this study. Branch tips highlighted in grey under ecotype I clade represents A. phagocytophilum ecotype I sequences reported from Great Britain cattle. Roman numerals I, II, III and IV represents ecotypes. The phylogeny is rooted at midpoint and drawn to display circular node shapes at bootstrap support values ≥ 90.

Discussion
Following the renaming of A. phagocytophilum [1], genotypes with a diverse host range have emerged. The emergence of multiple genotypes has created complexity in the understanding of pathogenicity and clinical disease manifestation in various animal hosts and potential for zoonotic transmission in humans. Bovine anaplasmosis caused by A. phagocytophilum is associated with a severe clinical disease in cattle globally, and risk factors such as the season of the year, the presence of tick vectors, latitude and farm hygiene practice have been associated with disease [49]. However, this study aimed at establishing A. phagocytophilum ecotypes using a small number of samples confirmed to be positive via the Msp2 qPCR. To the best of our knowledge, this is the first study to demonstrate the presence of ecotype I in cattle and red deer in GB. Bovine anaplasmosis and co-infection with B. divergens and A. phagocytophilum characterized by severe disease and mortality has been reported in GB [16,32] and other parts of mainland Europe [29][30][31]. Although this study did not test for Babesia species, B. divergens is known to share the same vector (I. ricinus) as A. phagocytophilum and co-infection can increase the severity of disease [16]. I. ricinus is a three-host tick, both larval and nymphal stages of I. ricinus feed on small to medium-sized animals while adults prefer larger animals, including cattle. The presence of A. phagocytophilum (ecotype I) in all engorged ticks collected from cattle is likely to originate from a previous blood meal (during larval/nymphal lifecycle stages from other animal hosts) or present in the blood meal obtained from infested cattle. This report supports previous findings that infected animals can play a significant role in the enzootic transmission of A. phagocytophilum [50]. Additional studies are required to provide a clear understanding of the pathogenicity and clinical manifestation of A. phagocytophilum in cattle, and assist clinicians in diagnosis, treatment and control. The presence of ecotype I in sheep and both ecotypes (I and II) in I. ricinus further supports recent studies reporting A. phagocytophilum in sheep [18] and questing ticks collected from GB recreational areas [17], while the report of ecotype II from roe deer in this study supports previous reports from Central Europe [51,52]. Cervids (red and roe deer) are wildlife reservoir hosts for various genotypes of A. phagocytophilum known to be transmitted by I. ricinus [53][54][55]. Although roe deer have been demonstrated to be co-infected with multiple A. phagocytophilum variants, these variants are not considered pathogenic to domestic animals and humans. However, genotypes reported from red deer are pathogenic to domestic animals [51]. The presence of ecotype I in red deer from this study suggest further investigations are needed to understand the role of red deer in A. phagocytophilum transmission in GB. In addition, the geographical clustering of ecotype I sequences reported in this study partly supports previous findings in Europe [56]. Ecotype I reported in this study clustered within the same geographical subclade with European wild and domestic animals, equine and human zoonotic ecotypes I sequences, suggesting zoonotic risk and the likelihood of human infection as previously described. The separate geographical clustering of the American human, domestic and wild animal ecotype I may likely suggest differences in potentials for zoonotic infections between the European and American ecotype I as recently described [18]. This report provides additional information on the A. phagocytophilum isolates infecting cattle in GB.

Conclusions
In conclusion, this study reports that only A. phagocytophilum ecotype I has been detected in GB cattle and provides further evidence for the presence of ecotypes I and II in red and roe deer, respectively. The presence of ecotype I in cattle and I. ricinus infesting cattle corroborates such reports in previous studies. These findings constitute the first report of A. phagocytophilum ecotype I in GB cattle, despite the presence of ecotypes I and II in the indigenous tick population [17]. However, this report should be interpreted with caution owing to the smaller sample size assessed in this study. The presence of A. phagocytophilum ecotype I in sheep was also confirmed in this study as previously reported in GB [18]. The epidemiology and clinical disease manifestation of the A. phagocytophilum genotypes in GB cattle require further investigation as A. phagocytophilum and/or co-infection with B. divergens in cattle is routinely reported and likely to result in severe disease and potential mortality. While the full zoonotic potentials of ecotype I is not well understood, it has been associated with human granulocytic anaplasmosis. Preventive measures, including public education on tick control and limiting the exposure of animal and humans to tick bites, are suggested measures in the absence of adequate information on A. phagocytophilum disease pathogenicity and epidemiology in GB cattle.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens12081029/s1. Figure S1: A full polar cladogram displayed, following maximum likelihood phylogenetic tree constructed at 10,000 bootstrap approximations using A. phagocytophilum groEL gene nucleotide sequences (48) generated from this study and representative ecotypes (328) reported in the previous literature; Table S1: GenBank accession number and list of A. phagocytophilum nucleotide sequences obtained from GB livestock, I. ricinus and deer species; Table S2: A summary of NCBI online custom BLASTn search containing a list of GB nucleotide sequences obtained from GB livestock, I. ricinus and deer species, GenBank accession numbers and percentage identity, established ecotypes based on the recent literature, host and country of origin; Table S3: List of GenBank nucleotide accession numbers used for NCBI online BLASTn search; Table S4