Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people

doi:10.21203/rs.3.rs-2234764/v1

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Research Article

Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people

https://doi.org/10.21203/rs.3.rs-2234764/v1

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published 28 Feb, 2023

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Background

Tick-borne pathogen, Anaplasma phagocytophilum is currently described as a single species; however, studies using genetic markers can distinguish groups of variants associated with different hosts, pathogenicity, zoonotic potential, and biotic and geographic niches. The objective of our study was to investigate the genetic diversity of A. phagocytophilum from Ixodes ricinus ticks attached to people.

Methods

In collaboration with a commercial diagnostic company, a total of 52 DNA samples were obtained from ticks that tested positive for A. phagocytophilum by qPCR. The genetic profile of each sample was described using the groEL and ankA gene. Identification of the tick species was confirmed by partial sequencing of the COI subunit and the portion of the TrospA gene.

Results

All 52 ticks were identified as I. ricinus. Two protocols of nested PCR amplifying 1293 bp and 407 bp fragments of the groEL gene of A. phagocytophilum yielded the amplicons of the expected size for all 52 samples. Among all sequences, we identified 10 unique genetic variants of the groEL gene belonging to ecotype I and ecotype II. The analysis targeting the ankA gene was successful in 46 of 52 ticks. Among all sequences, we identified 21 unique genetic variants phylogenetically belonging to three clusters.

Conclusions

Our results indicate that ticks attached to people harbor distant genetic variants of A. phagocytophilum, some of which are not recognized as zoonotic. Further studies are needed to determine the risk of human infection by genetic variants other than those designated as zoonotic.

Anaplasma phagocytophilum

Ixodes ricinus

anaplasmosis

genetic diversity

infectious diseases

Anaplasma phagocytophilum is a Gram-negative bacterium responsible for human granulocytic anaplasmosis (HGA) in people, tick-borne fever (TBF) in ruminants, equine granulocytic anaplasmosis (EGA) in horses, and granulocytic anaplasmosis (GA) in dogs and cats [1]. In the Palearctic region, the dominant genetic variants of A. phagocytophilum are transmitted by I. ricinus and I. persulcatus, while I. scapularis and I. pacificus are known vectors in the Nearctic region [2, 3].

Genetic variants of A. phagocytophilum in North America and Europe differ in host preference and clinical symptoms, with TBF and EGA dominant in Europe and HGA dominant in North America [4, 5]. The genetic diversity of European strains of A. phagocytophilum of the different origin was demonstrated by phylogenetic analyses of genes such as groEL [6–8], ankA [4], and msp4 [9]. Different target genes led to different names of the genetic variants and consequently different terminology, such as ecotype (groEL), cluster (ankA and groEL), and genotype (msp4) [4, 10].

The heat shock operon groESL contains two genes encoding the chaperone proteins groES and groEL, as well as the intergenic region. A 5' fragment of the groEL gene has been widely used for genotyping A. phagocytophilum, especially those from Europe [11–13]. Currently, based on groEL, four ecotypes and eight clusters with different pathogenicity and geographical origin are distinguished [7, 8].

The variable ankA gene encodes the ankyrin repeat-containing protein AnkA (153–160 kDa). The ankA gene differentiates variants corresponding with the species of their animal hosts and exhibits higher sequence variability compared to that of 16S rDNA, the groEL, and msp4 genes [3]. Association of genetic variants was found among vertebrate hosts, tick vectors, and geographic locations. Regardless of the gene used for analysis, infected humans, whether in Europe or the Americas, appear to share related strains belonging to the same genetic group [5, 14].

The number of HGA cases reported in the USA has been steadily increasing since the disease became reportable; from 348 in 2000 to 5,762 cases in 2017 (https://www.cdc.gov/anaplasmosis/stats/index.html). In Europe, the total annual number of HGA cases has not exceeded 300 [2, 15]. Several HGA cases were reported also in Canada, Russia, China, Taiwan, South Korea, and Japan (reviewed by Rar et al. 2021). According to the National Institute of Public Health in the Czech Republic, 53 cases of HGA were reported between 2007 and 2017; however, in the seroprevalence study from 2014, specific antibodies were detected in 34 of 314 individuals tested [16]. Furthermore, recently, 12.6% of 103 patients with clinical symptoms persisting after antibiotic treatment of diagnosed Lyme disease were positive for A. phagocytophilum IgG antibodies [17]. Similar rates of seroprevalence were reported in other European countries including Norway, Sweden, and Poland, where Anaplasma antibodies were detected in 11.0%, 12.0% and 11.8% population, respectively (17). The disease is likely greatly underdiagnosed due to nonspecific flu-like symptoms such as fever, headache, and myalgias that usually resolve without treatment [1]. If the immune system fails and the infection is left untreated, the disease can cause life-threatening symptoms such as respiratory failure, severe gastrointestinal bleeding, renal failure, and liver damage [19]. Although surveillance reports of A. phagocytophilum in tick vectors are increasing worldwide, the epidemiological risk of infection by this pathogen is underestimated [20]. Detection of pathogens in blood-feeding ticks shows the risk of human exposure better than studies on foraging ticks [21]. In this context, the main objective of our study was to investigate the genetic diversity of A. phagocytophilum in ticks attached to people in the Czech Republic nationwide.

Tick identification

The total of 52 DNA samples of ticks positive for A. phagocytophilum by qPCR [22] and attached to people were acquired in collaboration with a commercial diagnostic company offering the pathogen detection in ticks sent by general public (Fig. 1). Tick species, stage development and sex, were determined by the specialist from the collaborating institution. Samples were collected in years 2011–2020, individually homogenized, and nucleic acids were isolated by the ExiPrep Plus Viral DNA/RNA Kit using an automated isolator Exiprep 16Plus (Bioneer, Republic of Korea).

The tick species identification was confirmed by partial sequencing of the cytochrome c oxidase I subunit gene (COI) and the intron part of the TROSPA gene as described by Noureddine et al. (2011). The amplification of the COI gene was performed in the total volume of 20.0 µl using Phusion Green Hot Start II High-Fidelity PCR Master Mix (ThermoFisher Scientific) following the manufacturer instructions with annealing temperature of 55°C. The amplification of the TROSPA gene was done by 2x PCR BIO Taq Mix Red (PCR Biosystems) in the total volume of 25.0 µl following the manufacturer instructions with the annealing temperature 65°C. Amplicons were visualized, processed, and sequenced as described below. Sequence identity was determined by BLASTn analyses of the GenBank database at NCBI.

Amplification of groEL and ankA

To determine the groEL ecotype, nested PCR was performed targeting a 407 bp portion of the variable fragment of groEL as described previously [7, 23]. Whenever possible, a longer fragment of groEL (1297 bp) was amplified following the nested PCR protocol [6] using slightly modified primers [24]. Subsequently, the samples were analysed using two nested PCR assays aimed at the ankA gene (Additional file 1: Table S1). The first assay (herein called protocol 1) amplified ~ 530 bp fragment of the ankA gene, and the second one (protocol 2) ~ 500 bp. All PCRs were performed using the commercial master mix (2×PCRBIO Taq Mix Red, PCR Biosystems, UK) following the manufacturer instructions. A total volume of 25.0 µl was prepared for each reaction, comprising 12.5 µl of the master mix, 10.0 pmol of each primer, 2.0 µl of template DNA or 1.0 µl of PCR product from the first round in the case of nested PCR, and PCR water for the remaining volume. All PCR products were visualized on the 1.5% agarose gel with the Midori Green Advance system (Nippon Genetics Europe, Germany). Products of the expected size were purified using the Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech Ltd., Taiwan), and sequenced at the Macrogen capillary sequencing services (Macrogen Europe, the Netherlands) using the amplification primers.

Sequence and phylogenetic analysis

All sequences were assembled, edited, and analysed using the Geneious Prime® software version 2022.0.1 and compared to those available in the GenBank database by BLASTn analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). For individual ticks, wherever possible, sequences from two separate assays, targeting overlapping regions of the ankA gene, were assembled into a single sequence (~ 800 bp). One sample that yielded mixed chromatogram signals, was cloned using the pGEM®-T Easy Vector System (Promega, USA). Cloned plasmid DNA was purified from the bacterial culture using the GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, USA) and sequenced using the universal SP6 primer.

The phylogeny was computed separately for groEL and ankA. For the groEL gene phylogenetic analysis was performed on 87 sequences from the GenBank representing all four described ecotypes [8] and eight clusters [7] together with 10 unique sequences from this study and two sequences of A. platys used as an outgroup. The ankA gene phylogenetic analysis was performed on 74 sequences from the GenBank representing different clusters together with 21 unique sequences from this study and sequences of A. marginale as an outgroup.

Due to an uneven length of sequences, the alignments were calculated in two steps by MAFFT algorithm, using “Auto” strategy for sequences > 1000 nt and function –add for implementing sequences < 1000 nt to the alignment. The phylogenetic trees were inferred by the maximum likelihood method by IQTREEv. 1.6.5 [25]. The best-fit evolution model was selected based on the Bayesian information criterion (BIC) computed by implemented ModelFinder [26]. Branch supports were assessed by the ultrafast bootstrap (UFBoot) approximation [27] and by the SH-like approximate likelihood ratio test (SH-aLRT) [28]. Trees were visualized and edited in FigTree v1.4.1 and Inkscape 0.91. The map of localities was constructed in the Datawrapper online software (https://app.datawrapper.de/).

Ticks were identified by morphology and by sequencing of TROSPA and COI genes (data not shown). All 52 ticks were identified as I. ricinus and no other species including I. inopintaus were found. Two protocols of nested PCR, amplifying 1293 bp and 407 bp fragments of the groEL gene, yielded the amplicons of expected size for all 52 samples; 19 (6.3%) adults (14 females and 5 males, respectively) and 31 (10.3%) nymphs (Table 1). For two ticks, data on sex and stage development were not available. In total, 8 short and 44 long sequences of groEL were obtained. Inspection of chromatograms did not reveal any multiple peaks suggestive of mixed infections. Among all sequences, we identified 10 unique genetic variants with 97.9–99.9% sequence identity. The main variant (GT15) was detected in 39 individuals (75%), followed by four other variants, each detected in two ticks. The remaining five genetic variants were represented by a single individual (Fig. 1, Table 2). BLAST analysis was performed for each genetic variant. Representative sequences were deposited to the NCBI GenBank database under the accession numbers OP265397-OP265406.

Table 1

Genetic variants of *Anaplasma phagocytophilum* detected in ticks attached to people in the Czech Republic.
Stage	Sex	No.	groEL		ankA
			No. of sequences	ecotype	No. of sequences	clusters
Adult	F	14	14	I, II	14	1, 4
Adult	M	5	5	I, II	5	1, 4
Nymph		31	31	I, II	23	1, 2, 4, 7
unknown		2	2	I, II	2	1, 4

Table 2

Genetic variants of *A. phagocytophilum* detected in ticks attached to people in the Czech Republic
groEL ecotype	groEL variant	No. of sequences	ankA variant	No. of sequences	ankA cluster
I	GT15	39	GT152	6	1
			GT75	4
			GT47	6
			GT32	4
			GT54	2
			GT77	2
			GT148	1
			GT152	1
			GT240	1
			GT267	1
			GT276	1
	GT139	2	GT139	2
	GT300	1	GT300	5^a
	GT294	1	GT294	3^a
	GT213	1	GT213	1 (2)^b	7
	GT135	1	GT135	1	4
II	GT250	2	GT250	1	4
	GT58	2	GT58	1	2
	GT58	2	GT270	1
	GT184	2	GT184	1
	GT115	1	GT115	1
^a samples identical with GT15 groEL genetic variant
^b sample with mixed chromatogram, two sequence variants obtained after cloning

The analysis targeting the ankA gene was successful in 46 of 52 ticks. PCR yielded products of the expected size in 38 (455–530 bp) and 33 (470–574 bp) ticks using protocol 1 and 2, respectively (Fig. 1, Table 2). In 24 ticks, products from both assays were obtained allowing the assembly of longer sequence. Inspection of chromatograms of the ankA gene, revealed multiple peaks indicating the mixed infection in one tick (GT213). Sequencing of 5 clones from this individual tick yielded two A. phagocytophilum variants with 98.7% of identity (differences observed in 7 nucleotides; 3 silent and 4 missense mutations). Among all sequences, we identified 21 unique genetic variants with 62.0–99.9% sequence identity. Representative sequences from the study were deposited to the NCBI GenBank database under the accession numbers OP265407-OP265426.

Phylogenetic analysis

In our study, we followed the classification of A. phagocytophilum based on the partial groEL gene sequences, introduced by Jahfari et al. (2014) and extended by Jaarsma et al. (2019). In the phylogenetic analysis, three well supported clades were distinguished. The largest clade, consists of two ecotypes, I and II (Fig. 2). The ecotype I is monophyletic and contains wide range of isolates from USA and Europe from humans, horses, dogs, and domestic ruminants as well as wild boar and urban wildlife. Ecotype II consists of two monophyletic clusters with a high level of bootstrap support. However, ecotype II is not monophyletic and clusters III and IV clearly differ in geographical distribution, host, and vector species preference. Sequences obtained from specimens from cluster III originating from Europe and were found in I. ricinus and deer. Cluster IV contains specimens from Asia, including isolates from a human, I. persulcatus, and rodents (Additional file 2, Figure S1). All sequences from our study fell into this largest clade (Fig. 2). Forty-five isolates were in two clusters belonging to the ecotype I and remaining 6, belong to cluster III within the ecotype II (Figs. 2, S1).

The second well distinguishable clade consists of isolates of ecotype III with clusters V and VI. Cluster V is represented by sequences from rodents and I. trianguliceps from Europe and Western Siberia; cluster VI is composed of sequences from rodents and I. persulcatus from Asia. These two clusters are closely related to cluster VIII [sensu 7], representing isolates from I. ventalloi exclusively. The last clade is represented by a small number of sequences in ecotype IV / cluster VII. None of the genetic variants detected in our study belonged to ecotype III and IV.

To describe the results of phylogenetic analysis based on the ankA gene, we followed the classification used by Rar et al. (2021). In our analysis, three well-supported clades were formed. The first clade consists of 6 clusters originating from Europe, Asia and the United States (Figs. 3, Additional file 3: Figure S2). The specimens originating from the USA are present in clusters 11 and 12. These clusters are sister groups to ankA cluster 1. European cluster 1 is the most diverse and contains all of the sequences from humans, small, medium and large mammals, and I. ricinus. Thirty-nine of our sequences were placed in this cluster 1 (Table 2). The Asian group is referred to as cluster 8 and is represented by the sequences obtained from hybrids of I. persulcatus x I. pavlovskyi. The ankA clusters 2 and 3, also containing A. phagocytophilum of the European origin, are mainly represented by specimens from wild and domestic ruminants and I. ricinus. Four of our sequences were classified in cluster 2.

The second, well supported clade consists of 5 clusters. Cluster 9 is represented by a single sequence from a tick collected from a woodchat shrike and it is related to clusters 4 and 7 with sequences from wild and domestic ruminants and I. ricinus from Europe (Fig. 3, Additional file 3: Figure S2). Two of our sequences belong to cluster 4, and two sequences from a single cloned sample fall into cluster 7. The other branch of the second clade is formed by clusters 5 and 10 from Asia and Europe. Sequences belonging to these clusters have been detected in I. persulcatus, I. pavlovskyi, their natural hybrids, and rodents (Fig. 3).

The third and smallest clade contains only a single cluster 6 with sequences from birds and Ixodes spp. from birds (Figs. 3, Additional file 3: Figure S2).

To our knowledge, this is the first elaborated study demonstrating the genetic diversity of A. phagocytophilum in ticks attached to humans based on groEL and ankA together. There are few data on A. phagocytophilum detected in ticks feeding on humans from Poland [29], Italy [30,31], Romania [32] and Scotland [33]. In Europe, A. phagocytophilum is transmitted mainly by I. ricinus. This tick has three feeding developmental stages, exhibiting different host preferences. This affects the range of pathogens ingested during each blood meal, thus later developmental stages are more likely to carry the pathogens. The three-host life strategy of I. ricinus affects the transmission of A. phagocytophilum, which is shown in our study as well as in the study performed on questing ticks [30]. Despite the increasing number of studies on the genetic diversity of A. phagocytophilum, there is still insufficient data to understand the geographic distribution, host preferences, and pathogenicity to humans of the different genetic variants [24]. During the last decade, multilocus approaches gained an attention as a promising tool that can help to better understand epidemiology of A. phagocytophilum infections. In our study, we chose two genes and performed both analyses on the same samples. The association between ankA and groEL clusters was recently suggested by Rar et al. (2021), and concordance between specific clusters/ecotypes was described [3]. In our study, 10 groEL and 21 ankA unique variants were detected, and subsequent phylogenies showed only a partial correlation between groEL ecotypes and ankA clusters. The groEL ecotype I has the broadest host range and I. ricinus is the vector with the wide distribution in the western Palearctic region and includes all genetically characterized strains detected from humans in Europe. Based on our results, the groEL ecotype I genetic variants are represented in ankA clusters 1, 4, and 7, whereas groEL ecotype II corresponds to ankA clusters 2 and 4, supporting the hypothesis put forward by Rar et al. (2021). However, it should be noted that ankA cluster 4 consists of variants of A. phagocytophilum belonging to groEL ecotypes I and II. The ankA gene may be a virulence factor and it has been suggested to be involved in host adaptation underlying diversifying selection [2]. This may have direct implications for the observed genetic variability of this gene in A. phagocytophilum. The other explanation for the observed discrepancy is possible coinfection of different genetic variants of A. phagocytophilum in a tick sample. However, with exception of one tick, double peaks indicative of coinfection were not observed in our study. Recent results characterizing tick-pathogen molecular interactions support the hypothesis that A. phagocytophilum has evolved common mechanisms to establish infections in tick vectors and vertebrate hosts, due to its adaptation to a variety of vector and reservoir species. The mechanisms that A. phagocytophilum uses to infect and replicate in tick and vertebrate hosts are still largely unknown and the interactions between different genetic variants of A. phagocytophilum are not fully explored [34]. In our study, we found ticks infected with A. phagocytophilum the groEL ecotype II/ankA clusters 2, 4, and 7, which are not known to be responsible for HGA in Europe. However, Kim et al. (2013) reported a single clinical case, caused by A. phagocytophilum (in our study: ecotype II/clusterIV) from Sauth Korea, where the pathogen is transmitted by ticks other than I. ricinus. Strains with the zoonotic potential known from Europe, represent only a small fraction of the A. phagocytophilum genetic variants and belong to ankA cluster 1/groEL ecotype I. These monophyletic groups are characterized by a wide host range including domestic and wild animals, often with a synanthropic lifestyle or living closely to human settlements (e.g. foxes, hedgehogs, wild boars). The lack of clinical cases caused by A. phagocytophilum strains other than ecotype I/cluster 1 in Europe, may therefore be related to their low prevalence in the environment, and their ability to infect humans needs to be confirmed.

Notwithstanding the variability noted, A. phagocytophilum is considered a single species. This contrasts with a similar situation in B. burgdorferi s.l., that is subdivided into several operational taxonomic units (referred to as genospecies) with differences in geographic distribution, host species preferences, zoonotic potential, and tissue tropism [35]. The introduction of a comprehensive and unified nomenclature and tools defining genetic lineages of A. phagocytophilum would allow easier communication of results and deeper understanding of the ecology and epidemiology of different genetic variants of this bacterium.

Acknowledgement

Not applicable

Funding

This work was supported by the funding from the Grant Agency of the Czech Republic GACR 21-11661S to LZ and DM; KH was supported by the project Nr. CZ.02.1.01/0.0/0.0/ 16_019/0000787 “Fighting Infectious Diseases” provided by the Ministry of Education Youth and Sports of the Czech Republic.

Availability of data and material (data transparency)

Representative sequences of groEL and ankA were deposited to the NCBI GenBank database under the accession numbers OP265397-OP265406 and OP265407-OP265426, respectively. (They will not be released to the public database until Sep 18, 2023, or until the data or accession numbers appear in print, whichever

is first.)

Authors' contributions

All authors contributed to the study conception and design. Material preparation and data collection were performed by Václav Hönig and Paulina Maria Lesiczka. Data analyses were performed by Paulina Maria Lesiczka. The first draft of the manuscript was written by Paulina Maria Lesiczka, Kristyna Hrazdilova and Ludek Zurek; all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

Not applicable

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Additional file 3: Fig S2 is not available with this version

No competing interests reported.

Additionalfile1TableS1.docx
Additional file 1: Table S1. Primers used in the study.
Additionalfile2FigS1.pdf
Additional file 2:Fig S1. Detailed Maximum likelihood phylogenetic tree based on the groEL gene of A. phagocytophilum

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published 28 Feb, 2023

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Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people

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Abstract

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Background

Materials And Methods

Tick identification

Sequence and phylogenetic analysis

Results

Phylogenetic analysis

Discussion

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Supplementary Information

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