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All issues Volume 30 (2023) Parasite, 30 (2023) 13 Full HTML
Open Access
Issue
Parasite
Volume 30, 2023
Article Number 13
Number of page(s) 9
DOI https://doi.org/10.1051/parasite/2023014
Published online 10 May 2023

© S. Ouass et al., published by EDP Sciences, 2023

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Members of the family Anaplasmataceae (Order: Rickettsiales) are obligate intracellular bacteria that can infect a wide range of animals [10, 36]. Historically, two genera, Anaplasma and Ehrlichia, have been described as important tick-borne bacteria of public and veterinary health interest [1, 3, 11, 12, 18, 33]. These genera include Anaplasma phagocytophilum and Ehrlichia chaffeensis that cause human granulocytic anaplasmosis and human monocytic ehrlichiosis, respectively and Ehrlichia ruminantium that causes heartwater (cowdriosis) in African cattle [1, 11, 12, 18, 33]. Recent advances in the genomics of these major pathogens have shed new light on the intracellular lifestyle of Anaplasmataceae and the mechanisms by which they induce and evade the innate immune response [2, 15, 17, 25]. Current epidemiological surveys on Anaplasmataceae are now uncovering emergent tick-borne pathogens such as Neoehrlichia mikurensis, causing neoehrlichiosis in Europe and Asia [31, 38], and Ca. Anaplasma sparouinense, causing Sparouine anaplasmosis in South America [13]. Other pathogens in the family Anaplasmataceae include Aegyptianella pullorum, which has only been observed once in domestic turkeys [35], and Neorickettsia spp. of digeneans (Platyhelminthes), which can be transmitted to the vertebrate hosts of digeneans causing Sennetsu fever in humans (Neorickettsia sennetsu) and Potomac fever in horses (Neorickettsia risticii) [41, 42]. However, the family Anaplasmataceae also includes bacteria that cannot infect vertebrates: Ca. Xenohaliotis californiensis and Ca. Xenolissoclinum pacificiensis, both described from marine invertebrates [16, 21], and members of the Wolbachia genus, specific to terrestrial arthropods and filarial nematodes [19, 23].

An intriguing and little known member of the Anaplasmataceae family is the putative genus Ca. Allocryptoplasma. It was created to name a bacterium originally described as Ca. Cryptoplasma californiense [14], but further corrected to Ca. Allocryptoplasma californiense (the generic name Cryptoplasma was already in use in the nomenclature of protozoa) [30]. In 2015, Candidatus Allocryptoplasma californiense was described in western black-legged ticks, Ixodes pacificus, collected throughout California [14]. Candidatus Allocryptoplasma californiense was detected in 5% of I. pacificus ticks tested statewide, but its prevalence was as high as 21% at one site [14]. Early sequence analysis indicated that Ca. Allocryptoplasma californiense is phylogenetically distinct from known genera and species from the Anaplasmataceae family, although genetically related to an Anaplasma-like bacterium detected in Asian longhorned ticks, Haemaphysalis longicornis, in Korea and China [14, 29, 32]. Further examination of partial 16S rRNA gene sequences showed high levels of homology to Ca. Allocryptoplasma californiense for Anaplasmataceae bacteria detected in castor bean ticks, Ixodes ricinus, in France [34], Italy [27], Slovakia [20], Serbia [4], Tunisia and Morocco [37]. Diverse strains of Ca. Allocryptoplasma were also detected in Haemaphysalis parmata and Amblyomma tholloni ticks collected in a wild chimpanzee habitat in Uganda [22], Amblyomma dissimile in Brazil [28] and in harvest mites of lizards, Neotrombicula autumnalis, in Italy [27]. While these bacteria have never been isolated, DNA of similar Ca. Allocryptoplasma sp. and Anaplasma-like bacteria were also detected in vertebrates which could act as primary hosts for their maintenance and enzootic circulation in Europe. These bacteria were detected in striped field mice, Apodemus agrarius [40], and green lizards, Lacerta viridis [20], in Slovakia, and in common wall lizards, Podarcis muralis, Italian wall lizards, Podarcis siculus, and western green lizards, Lacerta bilineata, in Italy [27]. Although the genus Ca. Allocryptoplasma has not been validly published to date, its detection in tick species of public and veterinary health interest, mammals and reptiles suggests that it could be a major genus of tick-borne pathogens.

Despite the potential medical and veterinary importance of the genus Ca. Allocryptoplasma, the phylogenetic relationship and pattern of genetic variation between Ca. Allocryptoplasma californiense, potential Ca. Allocryptoplasma spp., closely related Anaplasma-like bacteria and other members of Anaplasmataceae family remain unclear. Here, we address these issues by characterizing genetic variation of Ca. Allocryptoplasma infections and by conducting phylogenetic inferences to reconstruct their evolutionary histories within the family Anaplasmataceae. While 16S rRNA, groEL, rpoB and gltA gene sequences were used to describe Ca. Allocryptoplasma californiense in I. pacificus ticks [14], only partial 16S rRNA gene sequences have been used as exclusive markers for description of other Ca. Allocryptoplasma sp. and Anaplasma-like bacteria [4, 20, 22, 27–29, 32, 37, 40]. However, the use of 16S rRNA gene sequences as an exclusive taxonomic marker was recently shown to be inadequate for inferring a reliable phylogeny within the Anaplasmataceae since the tree topology is often poorly resolved and usually unstable because of insufficient sequence polymorphism [7]. We therefore developed, in the present study, a generic multi-locus sequence typing approach, examining the DNA sequence variation of Ca. Allocryptoplasma spp. at five genes (16S rRNA, groEL, rpoB, gltA and sucA) and applied this typing to the strains we detected in four tick species sampled in Europe, South America, and Africa. Using this multi-locus typing approach and data available on public databases, we further examined the phylogenetic placement of Ca. Allocryptoplasma californiense and other Ca. Allocryptoplasma spp. within the family Anaplasmataceae.

Materials and methods

Ethics

The use of ticks from French Guiana was approved by the French Ministry of the Environment under the reference #TREL19028117S/156, in compliance with the Access and Benefit Sharing procedure implemented by the Loi pour la Reconquête de la Biodiversité. The research in Uganda was conducted in the context of the Memorandum of Understanding “Museum National d'Histoire Naturelle/Uganda Wildlife Authority/Makerere University SJ 445-12”, in accordance with the Uganda Wildlife Authority and the Uganda National Council for Science and Technology.

Tick collection

A collection of 26 individual DNA templates obtained from 26 specimens of four tick species was used (Table S1). DNA was extracted from individual ticks using a DNeasy Blood & Tissue Kit (QIAGEN), following the manufacturer’s instructions. For each DNA template, tick identification and infection by Ca. Allocryptoplasma sp. have been formally characterized in previous investigations by molecular and morphological characteristics (for ticks) and high-throughput 16S rDNA sequencing approach for microbiota profiling (for Ca. Allocryptoplasma sp.) (Table S1). The four infected tick species examined in this study belong to the Ixodidae family (hard ticks), and include Amblyomma coelebs (one specimen), A. tholloni (12 specimens), H. parmata (three specimens) and I. ricinus (10 specimens) (Table S1). All tick specimens were questing (non-engorged) nymphs or adults collected from vegetation in South America (A. coelebs), Africa (A. tholloni, H. parmata), and Europe (I. ricinus) (Table S1).

Multi-locus typing of Ca. Allocryptoplasma infection

Ca. Allocryptoplasma sp. were genotyped through nested or semi-nested PCR assays and by sequencing of five housekeeping genes (16S rRNA [1187 bp], rpoB [496 bp], sucA [548–616 bp], groEL [572 bp], and gtlA [606-977 bp]). For 16S rRNA, we used our previously published primers (listed in Table S2), which were previously designed for Ehrlichia typing but were further found to be effective in amplifying Ca. Allocryptoplasma sp. [22]. For the other four genes, Ca. Allocryptoplasma-specific primers (Table S2) were designed using Anaplasma and Ehrlichia reference genomes available in public databases (GenBank accession numbers: A. phagocytophilum, CP006617; A. marginale, CP001079; A. centrale, CP001759; E. chaffeensis, CP007480; E. ruminantium, CP040111; E. canis, CP000107), and groEL, rpoB and gltA gene sequences (GenBank accession numbers: KP276592–KP276592, KP276600–KP276602, KP276604–KP276606) primarily used to describe Ca. Allocryptoplasma californiense in I. pacificus ticks [14]. These genes are found as single copies in Anaplasma and Ehrlichia reference genomes.

Nested and semi-nested PCR amplifications were performed as follows: the first PCR run with the external primers was performed in a 10 μL volume containing 10–50 ng of genomic DNA, 3 mM of each dNTP (Thermo Scientific), 8 mM of MgCl2 (Roche Diagnostics), 3 μM of each primer, 1 μL of 10× PCR buffer (Roche Diagnostics), and 0.5 U of Taq DNA polymerase (Roche Diagnostics). A 1 μL aliquot of the PCR product from the first reaction was then used as a template for the second round of amplification. The second PCR was performed in a total volume of 25 μL and contained 8 mM of each dNTP (Thermo Scientific), 10 mM of MgCl2 (Thermo Scientific), 7.5 μM of each of the internal primers, 2.5 μL of 10×PCR buffer (Thermo Scientific), and 1.25 U of Taq DNA polymerase (Thermo Scientific). All PCR amplifications were performed as follows: initial denaturation at 93 °C for 3 min, 35 cycles of denaturation (93 °C, 30 s), annealing (T m = 52 °C), extension (72 °C, 1 min), and a final extension at 72 °C for 5 min. Positive (DNA of A. tholloni specimens infected by Ca. Allocryptoplasma sp.) and negative (water) controls were included in each PCR assay. Following visualization via electrophoresis in 1.5% agarose gel, positive PCR products were sequenced by Eurofins. Sequence chromatograms were cleaned with Chromas Lite (http://www.technelysium.com.au/chromas_lite.html), and alignments were performed using ClustalW, implemented in the MEGA software package (https://www.megasoftware.net/). Alleles of Ca. Allocryptoplasma sp. were determined on the basis of sequence identity in nucleotide alignments for 16S rRNA, sucA, groEL, rpoB and gltA gene sequences. New sequences obtained in this study were deposited in GenBank with accession numbers OQ724839–OQ724862 and OQ724538–OQ724629.

Molecular phylogenetic analyses

Phylogenetic analyses were based on sequence alignments of 16S rRNA, sucA, groEL, rpoB and gltA gene sequences obtained from analyses of allelic profiles. Sequences of other Anaplasmataceae obtained from GenBank (including representative members of the Anaplasma, Ehrlichia, Neorickettsia, Wolbachia, Neoehrlichia, Ca. Xenohaliotis and Ca. Xenolissoclinum) were also included in the phylogenetic analyses. Sequences of other Rickettsiales (including representative members of the families Rickettsiaceae, Rickettsia rickettsii, and Midichloriaceae, Ca. Midichloria mitochondrii) were used as outgroups. The Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/blast/Blast.cgi) was used to find additional sequences available on GenBank showing nucleotide similarities with Ca. Allocryptoplasma gene sequences. The Gblocks program with default parameters was used to obtain non-ambiguous sequence alignments [6]. All sequence alignments were also checked for putative recombinant regions using the RDP3 analysis package [26]. Tree-based phylogenetic analyses were performed using the maximum-likelihood (ML) method using the MEGA software package (https://www.megasoftware.net/). The evolutionary models that best fit the sequence data were determined using the Akaike information criterion. Clade robustness was assessed by bootstrap analysis using 1,000 replicates.

Statistical analyses

We tested whether the levels of nucleotide divergence between members of the genus Ca. Allocryptoplasma range in threshold values typically observed in other Anaplasmataceae genera. To this end, we computed the nucleotide pairwise identities for each 16S rRNA, sucA, groEL, rpoB and gltA sequence dataset (1) between Ca. Allocryptoplasma strains found in I. pacificus, I. ricinus, A. tholloni, A. coelebs and H. parmata, (2) between representative members of the genus Anaplasma (GenBank accession numbers: A. phagocytophilum, CP006617; A. platys, CP046391; A. capra, JAOTBF010000000, A. ovis, CP015994; A. marginale, CP001079; A. centrale, CP001759), and (3) between representative members of the genus Ehrlichia (E. chaffeensis, CP007480; E. ruminantium, CP040111; E. canis, CP000107; E. muris, CP0069017; E. minasensis, QOHLL010000000). We further compared intrageneric nucleotide pairwise identities using Wilcoxon signed-rank tests and sequential Bonferroni correction implemented in R (http://www.r-project.org).

Results

Diversity of Ca. Allocryptoplasma in ticks

We attempted to amplify the 16S rRNA, sucA, groEL, rpoB and gltA Ca. Allocryptoplasma gene sequences from the 26 DNA templates belonging to four tick species (Table 1). The five Ca. Allocryptoplasma genes were successfully amplified for A. tholloni (12 DNA templates) and I. ricinus (10), while four Ca. Allocryptoplasma genes (16S rRNA, groEL, rpoB and gltA, but not sucA) were amplified for A. coelebs (1) and H. parmata (3) (Table 1). The sequences were easily readable without double peaks, indicating that there was no coinfection of Ca. Allocryptoplasma strains in all specimens except two A. tholloni specimens. These two specimens showed double peaks for their sucA, groEL and rpoB gene sequences, suggesting that coinfection with at least two distinct genetic variants of Ca. Allocryptoplasma may be present in coinfection: double peaks were observed at nucleotide positions that we found variable between the two distinct genetic variants detected in single infection in the 10 other A. tholloni specimens (see below). The two A. tholloni specimens with possible Ca. Allocryptoplasma coinfection were removed from further analyses.

Table 1

Allelic profile of the five polymorphic genes for Ca. Allocryptoplasma from the four tick species examined in this study (Ixodes ricinus, Amblyomma coelebs, A. tholloni, and Haemaphysalis parmata) and from Ca. Allocryptoplasma californiense infecting Ixodes pacificus available in GenBank. Letters a–g represent the different alleles for each Ca. Allocryptoplasma gene. Dashes indicate an absence of gene PCR amplification.

On the basis of 16S rRNA, sucA, groEL, rpoB and gltA gene sequences, we characterized two to six distinct alleles depending on the gene (16S rRNA: 98.7–99.4% pairwise nucleotide identity; sucA: 78.3–99.8%, groEL: 81.5–99.4%, rpoB: 84.0–99.5%, gltA: 77.5%), leading to the identification of six genetically different Ca. Allocryptoplasma strains in I. ricinus, A. tholloni, A. coelebs and H. parmata (Table 1). The highly conserved nature of the 16S rRNA gene prevented molecular distinction of closely related Ca. Allocryptoplasma strains, since several Ca. Allocryptoplasma strains, identical on the basis of their 16S rRNA gene sequences, could be distinguished through variation in their sucA, groEL and rpoB gene sequences (Table 1). Indeed, two genetically distinct strains of Ca. Allocryptoplasma, although identical on the basis of their 16S rRNA gene sequences, were detected in H. parmata (Table 1) with high levels of nucleotide identity (16S rRNA: 100% pairwise nucleotide identity; sucA: 99.8%, groEL: 99.4%, rpoB: 98.6%). Two distinct Ca. Allocryptoplasma strains were also detected in A. tholloni (Table 1; 16S rRNA: 100% pairwise nucleotide identity; sucA: 98.1%, groEL: 99.1%, rpoB: 99.5%, gltA: 100%). Of the six Ca. Allocryptoplasma strains, each is specific to its respective tick species, and none is shared by two or more tick species (Table 1).

None of the 16S rRNA, sucA, groEL, rpoB and gltA gene sequences of Ca. Allocryptoplasma strains identified in I. ricinus, A. tholloni, A. coelebs and H. parmata were identical to those of Ca. Allocryptoplasma californiense infecting I. pacificus (Table 1), although showing high levels of nucleotide identity (16S rRNA: 98.8-99.2% pairwise nucleotide identity; groEL: 79.4–92.4%, rpoB: 84.9–93.6%, gltA: 75.1–93.5%). None of the sucA, groEL, rpoB and gltA gene sequences observed in this study were 100% identical to other Anaplasmataceae sequences available in GenBank. However, based on partial 16S rRNA gene sequences, the Ca. Allocryptoplasma strain found in I. ricinus in this study is 100% identical to the Ca. Allocryptoplasma strain previously found in I. ricinus in France (GenBank accession number: GU734325) [34], Italy (MT829287–MT829288) [27], Serbia (MW900167) [4], Tunisia and Morocco (AY672415–AY672420) [37]. The 16S rRNA gene sequence of the Ca. Allocryptoplasma of I. ricinus is also 100% identical to infection detected in harvest mites of lizards, N. autumnalis, in Italy (MT829286) [27], in striped field mice, Ap. agrarius (EF121953–EF121954) [40], and green lizards, L. viridis (MG924904) [20], in Slovakia, and in common wall lizards, P. muralis (MT829283, MT829285), Italian wall lizards, P. siculus (MT829289), and western green lizards, L. bilineata (MT829284), in Italy [27]. Furthermore, based on partial 16S rRNA gene sequences, the Ca. Allocryptoplasma strains found in I. ricinus, A. tholloni, A. coelebs and H. parmata strain are 96.0–99.0% identical to strains found in other ticks, including H. longicornis (JN715833, GU075699-GU07504) and A. dissimile (MG437272). Further nucleotide BLAST searches found high nucleotide identity (97.5–100%) with the 16S rRNA sequence of an undescribed Anaplasmataceae bacterium (MZ351089) characterized in A. hebraeum in Eswatini [24], and with the unpublished sequence of a Ca. Allocryptoplasma strain (OM884475) in I. scapularis in Florida.

Phylogeny of Ca. Allocryptoplasma

ML analyses based on 16S rRNA, sucA, groEL, rpoB and gltA nucleotide sequences were conducted to examine the phylogenetic proximity between Ca. Allocryptoplasma strains and other Anaplasmataceae (Figs. 1 and 2). We observed no sign of recombination in the dataset (all p > 0.20 for the GENECONV and RDP recombination detection tests). All phylogenetic reconstructions showed that the Ca. Allocryptoplasma strains, including the type species Ca. Allocryptoplasma californiense, delineate a robust monophyletic clade within the family Anaplasmataceae (Figs. 1 and 2). The closest relative of the genus Ca. Allocryptoplasma is the genus Anaplasma: ML analyses based on 16S rRNA, sucA, groEL, rpoB and gltA nucleotide sequences consistently showed that Ca. Allocryptoplasma and Anaplasma are sister genera, while other genera (Ehrlichia, Neoehrlichia) are more distantly related (Figs. 1 and 2).

thumbnail Figure 1

Phylogeny of the family Anaplasmataceae constructed using maximum-likelihood (ML) estimations based on (A) 16S rRNA sequences with a total of 1157 unambiguously aligned bp (best-fit approximation for the evolutionary model: K2+G+I), and (B) on short-length 16S rRNA sequences of Ca. Allocryptoplasma with a total of 202 unambiguously aligned bp (best-fit approximation for the evolutionary model: K2+G+I). All genera of the family Anaplasmataceae, including representative species, are indicated. *, Ca. Allocryptoplasma sequences obtained in this study (GenBank accession numbers OQ724839–OQ724862). GenBank accession numbers of other sequences used in analyses are shown on the phylogenetic trees. Numbers at nodes indicate percentage support of 1000 bootstrap replicates. Only bootstrap values >70% are shown. The scale bar is in units of substitution/site.

thumbnail Figure 2

Phylogeny of the family Anaplasmataceae constructed using maximum-likelihood (ML) estimations based on (A) groEL gene sequences (529 unambiguously aligned bp; best-fit approximation for the evolutionary model: T92+G), (B) on rpoB gene sequences (410 unambiguously aligned bp; best-fit approximation for the evolutionary model: GTR+G+I), (C) on gltA gene sequences (289 unambiguously aligned bp; best-fit approximation for the evolutionary model: HKY+G+I), and (D) on sucA gene sequences (563 unambiguously aligned bp; best-fit approximation for the evolutionary model: GTR+G+I). All genera of the family Anaplasmataceae, including representative species, are indicated. *, Ca. Allocryptoplasma sequences obtained in this study (GenBank accession numbers OQ724538–OQ724629). GenBank accession numbers of other sequences used in analyses are shown on the phylogenetic trees. Numbers at nodes indicate percentage support of 1000 bootstrap replicates. Only bootstrap values >70% are shown. The scale bar is in units of substitution/site.

Phylogenetic reconstructions showed that the different Ca. Allocryptoplasma strains found in the same tick species always cluster together. Indeed, the two Ca. Allocryptoplasma strains of A. tholloni are more closely related to each other than to any other Ca. Allocryptoplasma strain (Figs. 1 and 2). A similar pattern was observed for the two Ca. Allocryptoplasma strains found in H. parmata (Figs. 1 and 2). The Ca. Allocryptoplasma strain of I. ricinus is more closely related to Ca. Allocryptoplasma californiense of I. pacificus and they form together a robust subclade within the genus Ca. Allocryptoplasma (Figs. 1 and 2). The Ca. Allocryptoplasma strains of A. tholloni, A. coelebs and H. parmata often cluster in ML analyses based on 16S rRNA, sucA, groEL, rpoB and gltA nucleotide sequences (Figs. 1 and 2).

Further ML analyses of closely-related 16S rRNA sequences, including sequences from Ca. Allocryptoplasma available on GenBank regardless of length (Figs. 1A–1B), confirmed that the Ca. Allocryptoplasma strains of other tick species (I. scapularis, H. longicornis, A. dissimile, A. hebraeum), harvest mites (N. autumnalis), striped field mice (Ap. agrarius) and several lizard species (L. viridis, L. bilineata, P. muralis and P. siculus) also cluster within the genus Ca. Allocryptoplasma. The Ca. Allocryptoplasma strain of I. scapularis is closely related to Ca. Allocryptoplasma californiense of I. pacificus, and the Ca. Allocryptoplasma strain of H. longicornis to the one of H. parmata we characterized in this study (Figs. 1A–1B). On the basis of 16S rRNA short-length sequences, the same Ca. Allocryptoplasma strain was present in I. ricinus from France, Italy, Tunisia and Morocco, as well as in N. autumnalis harvest mites from Italy, striped field mice from Slovakia, and lizards from Italy and Slovakia (Fig. 1B).

Genetic diversity in the genus Ca. Allocryptoplasma

To characterize genetic diversity within the genus Ca. Allocryptoplasma, we computed the nucleotide pairwise identities between each Ca. Allocryptoplasma strains based on 16S rRNA, sucA, groEL, rpoB and gltA nucleotide sequences (Fig. 3). For 16S rRNA and gltA, nucleotide pairwise identities within the genus Ca. Allocryptoplasma range in threshold values observed within the genera Anaplasma and Ehrlichia (Wilcoxon tests, all p > 0.02, not significant after sequential Bonferroni procedures) (Fig. 3). For sucA, groEL and rpoB, nucleotide pairwise identities within the genus Ca. Allocryptoplasma were higher than within the genus Ehrlichia (Wilcoxon tests, all p < 0.008) but lower than within the genus Anaplasma (Wilcoxon tests, all p < 0.002) (Fig. 3). This means that Ca. Allocryptoplasma has intrageneric genetic diversity similar to values observed in other Anaplasmataceae genera.

thumbnail Figure 3

Intrageneric nucleotide pairwise identities of Ca. Allocryptoplasma, Anaplasma and Ehrlichia for the 16S rRNA, groEL, rpoB and gltA gene sequences. Wilcoxon tests: **p < 0.005; ***p < 0.001; N.S., not significant after sequential Bonferroni corrections.

Discussion

In this study, we demonstrated that substantial genetic diversity of Ca. Allocryptoplasma is present in ticks in most continents. On the basis of multi-locus gene sequences, we describe six novel distinct genetically different Ca. Allocryptoplasma strains in I. ricinus, A. tholloni, A. coelebs, and H. parmata. Combining our present study with the current literature sequences indicates that there is molecular evidence of Ca. Allocryptoplasma DNA in at least nine tick species in Europe, Asia, Africa, and North and South America, and also in mice and lizards in Europe [4, 14, 20, 22, 27–29, 32, 34, 37, 40]. Phylogenetic and genetic analyses further confirm that these Ca. Allocryptoplasma cluster into a monophyletic clade, divergent from all other genera of the family Anaplasmataceae, although more closely related to the genus Anaplasma. Their detections in ticks of significant medical or veterinary interest suggest that Ca. Allocryptoplasma is an emergent genus of tick-borne pathogens of general concern.

Three complementary lines of argument indicate that Ca. Allocryptoplasma is a putative genus similar to validated genera of the family Anaplasmataceae. The first argument lies in the clustering of all Ca. Allocryptoplasma strains or species within a unique well-supported clade, a pattern consistently observed in phylogenetic trees based on 16S rRNA, sucA, groEL, rpoB and gltA gene sequences. The second concerns the level of genetic diversity in the genus Ca. Allocryptoplasma that ranges around values typically observed within the genera Anaplasma and Ehrlichia. This means that the genetic divergence observed between two members of the genus Ca. Allocryptoplasma is on average similar to divergence between two species belonging to another Anaplasmataceae genus. Since only one putative species, Ca. Allocryptoplasma californiense [14], has been described within Ca. Allocryptoplasma, the observed genetic divergence suggests that additional putative species could be described from genetic data. Finally, analyses of multi-locus gene sequences shows that Ca. Allocryptoplasma is a sister genus of Anaplasma, i.e., they are more closely related to each other than to any other genus. With the genera Ehrlichia and Neoehrlichia, Ca. Allocryptoplasma and Anaplasma, form a monophyletic subgroup of the Anaplasmataceae family specifically associated with ticks and vertebrates.

Tick species often differ in the strains of Ca. Allocryptoplasma they harbor. On the basis of multi-locus typing sequences, none of the strains of Ca. Allocryptoplasma is shared between different tick species. The best example is found in H. parmata and A. tholloni, both collected from the same location in Uganda, but each harboring its specific strains of Ca. Allocryptoplasma. On the basis of short 16S rRNA sequences, worthy of note is that identical strains of Ca. Allocryptoplasma were observed in two cases: one strain in two African tick species, A. hebraeum and A. tholloni, and another in two North American tick species, I. pacificus and I. scapularis. However, the 16S rRNA gene sequences show insufficient sequence polymorphism [7], and identical strains of Ca. Allocryptoplasma on the basis of their 16S rRNA gene sequences may differ in their sequences for other genes as we observed here in A. tholloni and H. parmata. Since strains of Ca. Allocryptoplasma found in A. hebraeum and I. pacificus have not been examined for other gene sequences [24], their exact similarity with the strains found in A. tholloni and I. scapularis, respectively remain uncertain. The multi-locus typing approach can thus reveal greater genetic diversity of Ca. Allocryptoplasma than expected by previous studies due to this methodological issue.

The risk of acquiring a Ca. Allocryptoplasma infection is currently unknown, but the detection of members of the genus Ca. Allocryptoplasma in ticks of significant medical or veterinary interest is of concern. In the Northern Hemisphere, I. ricinus, I. pacificus and I. scapularis are the tick species most commonly biting humans [8, 9], and field specimens of these species harbor Ca. Allocryptoplasma [4, 14, 20, 27, 34, 37]. In Western Europe and North Africa, Ca. Allocryptoplasma was detected over most of the distribution range of I. ricinus [4, 20, 27, 34, 37], suggesting that the infection circulates widely in field populations of this tick species. Ixodes ricinus, as I. pacificus and I. scapularis, are host generalist tick species [8, 9] and they may readily transmit Ca. Allocryptoplasma to a range of mammals, birds and reptiles, as suggested by the Ca. Allocryptoplasma DNA detected in striped field mice [40] and lizards [20, 27]. In Asia, H. longicornis is a livestock pest that transmit pathogens relevant to human and animal health, but it is also an introduced, and now established, exotic species in the western Pacific Region and the USA [43]. In South America, A. coelebs is specialized on mammals [5] and can readily bite humans [39], but A. dissimile feed exclusively on reptiles and amphibians [5]. In Africa, H. parmata is a global generalist, while A. tholloni tends to feed on elephants, but both can bite Hominidae, at least occasionally [22]. Humans are therefore exposed to bites of Ca. Allocryptoplasma-infected ticks without the risk of infection being documented to date. Currently, there is no specific tests to diagnose Ca. Allocryptoplasma infections, suggesting than animal and human cases are undiagnosed, as often shown for novel human pathogens in the family Anaplasmataceae [13, 31, 38]. In this context, new means of detecting and characterizing Ca. Allocryptoplasma are now necessary to improve our understanding of Ca. Allocryptoplasma epidemiology.

To conclude, we have identified Ca. Allocryptoplasma as a clade of genetically diverse, but phylogenetically related bacteria. Our results based on the multi-locus typing scheme suggest that Ca. Allocryptoplasma may be a novel valid genus similar to Anaplasma and Ehrlichia in the family Anaplasmataceae. The repeated detection of Ca. Allocryptoplasma in ticks on most continents confirms that infection persists widely through its circulation in at least nine tick species. Additional studies of Ca. Allocryptoplasma are needed to determine its transmission cycle and to establish whether these tick-borne bacteria are relevant to human and animal health.

Acknowledgments

We are grateful to Marie Buysse, Florian Binetruy and Rachid Koual, and to the team of the Sebitoli Chimpanzee Project for technical support.

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Cite this article as: Ouass S, Boulanger N, Lelouvier B, Insonere J-L-M, Lacroux C, Krief S, Asalu E, Rahola N & Duron. 2023. Diversity and phylogeny of the tick-borne bacterial genus Candidatus Allocryptoplasma (Anaplasmataceae). Parasite 30, 13.

Supplementary material

Table S1: List and origin of tick species used in this study for multi-locus typing of Ca. Allocryptoplasma.

Table S2: Genes and primers used in polymerase chain reaction (PCR) assays for multi-locus typing of Ca. Allocryptoplasma.

Access here

All Tables

Table 1

Allelic profile of the five polymorphic genes for Ca. Allocryptoplasma from the four tick species examined in this study (Ixodes ricinus, Amblyomma coelebs, A. tholloni, and Haemaphysalis parmata) and from Ca. Allocryptoplasma californiense infecting Ixodes pacificus available in GenBank. Letters a–g represent the different alleles for each Ca. Allocryptoplasma gene. Dashes indicate an absence of gene PCR amplification.

In the text

All Figures

thumbnail Figure 1

Phylogeny of the family Anaplasmataceae constructed using maximum-likelihood (ML) estimations based on (A) 16S rRNA sequences with a total of 1157 unambiguously aligned bp (best-fit approximation for the evolutionary model: K2+G+I), and (B) on short-length 16S rRNA sequences of Ca. Allocryptoplasma with a total of 202 unambiguously aligned bp (best-fit approximation for the evolutionary model: K2+G+I). All genera of the family Anaplasmataceae, including representative species, are indicated. *, Ca. Allocryptoplasma sequences obtained in this study (GenBank accession numbers OQ724839–OQ724862). GenBank accession numbers of other sequences used in analyses are shown on the phylogenetic trees. Numbers at nodes indicate percentage support of 1000 bootstrap replicates. Only bootstrap values >70% are shown. The scale bar is in units of substitution/site.
In the text
thumbnail Figure 2

Phylogeny of the family Anaplasmataceae constructed using maximum-likelihood (ML) estimations based on (A) groEL gene sequences (529 unambiguously aligned bp; best-fit approximation for the evolutionary model: T92+G), (B) on rpoB gene sequences (410 unambiguously aligned bp; best-fit approximation for the evolutionary model: GTR+G+I), (C) on gltA gene sequences (289 unambiguously aligned bp; best-fit approximation for the evolutionary model: HKY+G+I), and (D) on sucA gene sequences (563 unambiguously aligned bp; best-fit approximation for the evolutionary model: GTR+G+I). All genera of the family Anaplasmataceae, including representative species, are indicated. *, Ca. Allocryptoplasma sequences obtained in this study (GenBank accession numbers OQ724538–OQ724629). GenBank accession numbers of other sequences used in analyses are shown on the phylogenetic trees. Numbers at nodes indicate percentage support of 1000 bootstrap replicates. Only bootstrap values >70% are shown. The scale bar is in units of substitution/site.
In the text
thumbnail Figure 3

Intrageneric nucleotide pairwise identities of Ca. Allocryptoplasma, Anaplasma and Ehrlichia for the 16S rRNA, groEL, rpoB and gltA gene sequences. Wilcoxon tests: **p < 0.005; ***p < 0.001; N.S., not significant after sequential Bonferroni corrections.
In the text

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