Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2005, p Vol. 43, No /05/$ doi: /jcm Copyright 2005, American Society for Microbiology. All Rights Reserved. Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco M Hammed Sarih, 1 Youmna M Ghirbi, 2 Ali Bouattour, 2 Lise Gern, 3 Guy Baranton, 4 and Danièle Postic 4 * Institut Pasteur du Maroc, Casablanca, Morocco 1 ; Institut Pasteur, Tunis, Tunisia 2 ; Institut de Zoologie, Université de Neuchâtel, Neuchâtel, Switzerland 3 ; and Institut Pasteur, Paris, France 4 Received 6 July 2004/Returned for modification 18 September 2004/Accepted 4 November 2004 A broad-range 16S rrna gene PCR assay followed by partial sequencing of the 16S rrna gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa. * Corresponding author. Mailing address: Laboratoire des Spirochètes, Institut Pasteur, 28 rue du Dr. Roux, Paris Cedex 15, France. Phone: Fax: dpostic@pasteur.fr. M.S. and Y.M. contributed equally to this work. Members of the family Anaplasmataceae are the agents of veterinary diseases distributed worldwide, some of which are now also regarded as emerging human pathogens. They are obligately intracellular bacteria residing in hematopoietic cells and cause two main forms of a human febrile illness: human granulocytic ehrlichiosis (HGE), caused by the HGE agent, and human monocytic ehrlichiosis, caused mostly by Ehrlichia chaffeensis. On the basis of 16S rrna sequence comparisons, members of bacterial species within the family Anaplasmataceae have recently been reorganized into four main groups, each corresponding to an individualized genus (12). The genus Anaplasma comprises agents responsible for zoonotic infections, the granulocytic ehrlichiosis, in both humans (HGE) and animals, mainly horses (Anaplasma equi) and sheep, goats, and cows (Anaplasma phagocytophilum). In the upper midwestern United States, the pathogens are transmitted by Ixodes scapularis (9, 40), whereas they are transmitted by Ixodes persulcatus in Asia (3, 27). Although few human cases have been described (23, 31, 35, 41), Anaplasma strains have been detected in Ixodes ricinus ticks collected in different countries of Western Europe (5, 7, 14, 17, 22, 32, 34, 38). The genus Ehrlichia includes the agent of human monocytic ehrlichiosis (Ehrlichia chaffeensis), mainly transmitted by Amblyomma americanum in the United States (11, 15, 43), and other species, mostly involved in animal infections. To our knowledge, just two human cases have been reported in Europe, based only on serologic evidence (26, 33). However serologic testing cannot consistently distinguish between infections due to distinct species in this group of bacteria. The infection of I. ricinus by E. chaffeensis is poorly documented, and most Ehrlichia DNAs found in this tick were Ehrlichia-like DNAs related to the agent of human monocytic ehrlichiosis. Therefore, the role of I. ricinus as a vector of this pathogen is only suspected to date. Neorickettsia and Wolbachia are the two additional genera in the family Anaplasmataceae. However, ticks have never been implicated in the transmission of the former, and no mammalian infection due to Wolbachia pipientis has ever been documented. Different targets have been designed to demonstrate the presence of DNAs from these bacteria either in ticks or in the blood of various mammalian hosts, including humans. The principal genes used were the 16S rrna (rrs) gene, the eubacterial groesl heat shock operon, the gene encoding P44 proteins, a family of surface proteins capable of eliciting an immunologic response in patients with granulocytic ehrlichiosis, and the epank1 gene, encoding a 153-kDa protein antigen restricted to the Anaplasma genus. By these means, different more or less closely related ehrlichiosis agents have been demonstrated in I. ricinus ticks from Europe. These agents have been poorly investigated in North Africa, and their actual geographic range is unknown. I. ricinus ticks are abundant in localized sites of Maghrebian countries and are very frequently infected by Borrelia lusitaniae (37, 44), a species which had been considered nonpathogenic but was recently isolated from a patient in Portugal (8). Coinfections of I. ricinus involving B. burgdorferi sensu lato and Ehrlichia spp. have frequently been reported in Europe, and the ecoepidemiology of these two pathogens often overlaps. Therefore, our aim was, first, to estimate the prevalence of tick infection by members of the Anaplasmataceae family in North Africa and, second, to establish which members of this family were present in ticks and therefore could constitute a threat either for humans or for animals. 1127

2 1128 SARIH ET AL. J. CLIN. MICROBIOL. MATERIALS AND METHODS Sample collection. Questing I. ricinus ticks were collected on vegetation in Tunisia and Morocco (Fig. 1). In Tunisia two sites were investigated: Jbel el Jouza, located in a lower-humid zone in the Northwest, where rainfall ranges from 600 to 800 mm/year, and Ain Draham, in an upper-humid bioclimatic zone in the Northwest, where rainfall is 800 mm/year (44). In Morocco, the study was conducted at Taza, a humid area in the Middle Occidental Atlas, the sole site investigated which revealed the presence of I. ricinus ticks (37). Sampling at these sites was conducted from November 2002 to March Questing I. ricinus ticks were collected by blanket dragging of the vegetation, as previously reported (37, 44). Additionally, in Tunisia in 2003, Hyalomma ticks removed from bovines and Rhipicephalus ticks removed from dogs were included in the study. All ticks were identified at the species level. Immediately after collection, ticks were stored in 70% ethanol until they were further processed. Preparation of DNA from ticks. Ticks were rinsed in distilled water and dried. DNA was extracted from engorged ticks by using the QIAamp tissue kit (QIA- GEN, Hilden, Germany) according to the manufacturer s instructions. DNA was extracted from free-living ticks by detergent lysis in the presence of proteinase K overnight at 56 C and was then boiled for 15 min. To monitor any contamination during these processes, 1 extraction control (distilled water) for every 10 ticks was added in each extraction experiment. DNAs were stored at 4 C until use as templates for PCR, or at 20 C for longer periods. PCR detection. PCR amplifications were performed in a final volume of 50 l containing 0.2 M each primer, 200 M each deoxynucleoside triphosphate, 1.25 UofTaq polymerase (Q.Bio gene), and 1 Taq buffer (1.5 mm MgCl 2 ). The amplification reaction was carried out in a Touch Down thermal cycler (Hybaid) under the following conditions: initial denaturation at 93 C for 1 min; 35 cycles, each including denaturation at 93 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 30 s. Primers were Ehr521 and Ehr747 (29). Amplification was verified by agarose gel electrophoresis and revealed by ethidium bromide staining. Positive and negative quality controls were included with each experiment. Negative controls included extraction controls as well as controls using distilled water as the template. DNA extracted from Ehrlichia canis, E. chaffeensis, and the HGE agent (kindly provided by P. Brouqui) were used as positive controls. FIG. 1. Sites of tick collection in Morocco (A) and Tunisia (B). DNA sequencing. PCR products were sequenced by Genome Express, Meylan, France. Sequences were determined on both forward and reverse strands in order to obtain maximal data accuracy. Partial rrs sequences determined in this study and sequences recorded from databases were aligned manually by VSM software and analyzed by the neighbor-joining method and by the unweightedpair group method with mathematical averages (UPGMA). Phylogenic trees were drawn with Mega software (19). Nucleotide sequence accession numbers. Sequence data have been deposited in GenBank. Accession numbers for the partial rrs sequences are AY to AY RESULTS AND DISCUSSION A total of 418 questing I. ricinus ticks, collected from vegetation, were tested for Ehrlichia infection by a PCR assay. One hundred ninety-seven ticks (larvae, nymphs, and adults) collected in Tunisia and 221 adult ticks collected in Morocco between November 2002 and March 2003 were included in the study. PCR products of the expected size were obtained from 53 of 324 (16.3%) adult I. ricinus ticks, from 2 of 59 nymphs, and from 1 of 35 larvae. Additionally, 32 Hyalomma detritum, 6 Hyalomma marginatum marginatum, and 14 Hyalomma anatolicum excavatum ticks collected from bovines in Tunisia, as well as 188 Rhipicephalus sanguineus ticks from dogs, were checked for the presence of Ehrlichia DNA. PCR signals were obtained from 18.8% of H. detritum, 7.1% of H. excavatum, and 2.9% of R. sanguineus ticks. No signal was obtained from H. m. marginatum. Moreover, 36 Haemaphysalis sulcata and 47 Haemaphysalis punctata ticks, collected on the vegetation, were analyzed. All were negative for the presence of Ehrlichia DNA. Given the broad genetic diversity among the species in the family Anaplasmataceae, we searched for primer sets able to

3 VOL. 43, 2005 EHRLICHIA IN TICKS IN MAGHREB 1129 amplify DNA from all organisms belonging to this family. We chose this strategy in order to draw up an inventory of different species within this family present in ticks collected in Maghrebian countries. The rrs gene proved to be a useful target. Initially, using sequences available in data banks, we verified that partial rrs sequences were representative of the whole gene. Indeed, the clustering obtained was similar to that reported by others using the sequence of the whole gene, as well as other genes (12). Therefore, we used the primer set Ehr521 Ehr747, which was initially designed for the specific amplification of A. phagocytophilum (29) but actually allowed the amplification of 247 bp in the rrs genes from all organisms of this heterogeneous family. Despite a high sensitivity, the specificity was poor, since all members of the family and also some Rickettsia and Bartonella spp. were detected (17, 24, 39). Therefore, in order to provide objective confirmation of the identification, all PCR products whose DNA concentration was 50 ng/ l were sequenced. The phylogenetic relationships between partial rrs sequences of ehrlichiae from North Africa and sequences available in data banks are shown in Fig. 2. The nucleotide sequences of 36 PCR products from I. ricinus ticks were determined. All sequences matched with sequences of representatives of the family Anaplasmataceae (Table 1 and Fig. 2). Therefore, we extrapolated the prevalence of tick infection, although we are aware of the risk of overestimation when no sequence is available to confirm the amplification specificity. The prevalence of infection of adult I. ricinus ticks in Tunisia (25.2%) was twice as high as the prevalence in Morocco (12.2%). A similar result was noted when the prevalence of infection by B. lusitaniae was studied in these two countries. Furthermore, the density of ticks was also higher in Tunisia than in Morocco (1, 2). In the present study, no significant difference in the infection rate of ticks according to sex was observed. As expected, the prevalence of infection of immature tick stages was lower, 3.4% for nymphs and 2.8% for larvae. Different studies conducted in the United States and in Europe revealed high discrepancies between the prevalences of infection in ticks, which may be associated with geographic or seasonal differences or with tick stage or tick status (unfed or fed, either on healthy animals or on animals with ehrlichiosis). In Europe, reported prevalences of A. phagocytophilum in free-living I. ricinus ticks range from 0.8 to 24% (7, 34). Moreover, 139 I. ricinus ticks were simultaneously studied for infection by B. lusitaniae and members of the family Anaplasmataceae. Ten ticks (7.2%) were infected by both organisms (data not shown). This is in the range of the coinfection rate usually reported in the literature (1, 5). A huge worldwide genetic diversity has been described among members of the family Anaplasmataceae. It is noteworthy that a similar diversity was demonstrated for ticks collected in North Africa, since the sequences determined in this study fell into three of the four groups constituting the family Anaplasmataceae. Most I. ricinus ticks were infected by organisms whose partial rrs sequences fell into the cluster of Anaplasma, although some of them constituted separate and highly divergent branches (Fig. 2). Sequences from two I. ricinus ticks (together with one H. detritum tick) fell into the cluster of the causative agent of HGE, cospecific with those of the veterinary pathogens A. phagocytophilum and A. equi. The partial sequences determined in this study shared 100% identity with the A. phagocytophilum sequence (accession number UO2521) but differed by 2 nucleotides from the sequence determined from an I. ricinus tick collected in France (accession number AF012528) (30), as shown in Fig. 2. Sequences of amplicons derived from 19 I. ricinus ticks in Tunisia (3 of which, IR12, IR25, and IR47, are included in Fig. 2) and from 13 I. ricinus ticks in Morocco (3 of which, MTI199, MTI210, and MTI216, are included in Fig. 2) (Table 1) constitute a separate branch in the Anaplasma cluster. The complete identity of the sequences of organisms from both Tunisian and Moroccan ticks must be underlined. These sequences were 100% identical but differed markedly from A. phagocytophilum sequences (11 nucleotide differences), the most closely related partial rrs sequences. Therefore, they clearly represent a distinct new species, and provisionally we designated this species Anaplasma-like. Two samples, one from an I. ricinus tick (44IR) and one from an H. detritum tick (17HY) in Tunisia, contained sequences that fell into the cluster comprising the agent of monocytic ehrlichiosis, E. chaffeensis, and a pathogen for domestic ruminants, Ehrlichia ruminantium. Partial sequences 44IR and 17HY differed from E. chaffeensis sequences (accession number U86664) by 4 and 7 nucleotides, respectively. Ehrlichia-like organisms related to the monocytic Ehrlichia have frequently been reported in American studies. However, they have also been found in some European countries (17, 38), although more rarely than A. phagocytophilum. Phylogenetic analysis (Fig. 2) showed that sequences from Maghrebian samples clearly differed from those of Ehrlichia-like organisms found in Dutch I. ricinus ticks (accession number AF104680) (38). Whether these organisms should be classified as separate species or only strain variants remains an open question. Surprisingly, one sequence obtained from an I. ricinus tick collected in Morocco was closely related to that of W. pipientis (10 nucleotide differences from W. pipientis [accession number U23709]) (Table 1). Sequences related to Wolbachia have rarely been identified from ticks in European studies (17). This bacterium, identified for the first time from the mosquito Culex pipiens, infects a large range of nematodes and arthropods (18). W. pipientis is mostly involved in the metabolism of its hosts, causing reproductive alterations, and seems to be devoid of direct pathogenicity for humans or animals (4). However, as a symbiont of human pathogens, W. pipientis could be implicated in pathological filariasis (36). No sequence homologous to sequences in the Neorickettsia cluster was found in any tick. This was not surprising, since ticks have never been implicated in the transmission of these agents, which are more specifically associated with helminths (12). Four DNAs extracted from R. sanguineus gave positive PCR signals (Table 1). Three of them were sequenced. They fell into the Anaplasma cluster and exhibited 100% identity with the sequence of Anaplasma platys (accession number AF156784). Interestingly, these R. sanguineus ticks were found on dogs; one was a male and two were females, one unfed and the other partially engorged at the time of collection. These results were not surprising, since A. platys has been reported to parasitize circulating platelets of dogs in Japan and is known to cause infectious cyclic thrombocytopenia, which may be fatal for

4 1130 SARIH ET AL. J. CLIN. MICROBIOL. FIG. 2. UPGMA-rooted tree obtained with MEGA software from partial rrs gene sequences. Accession numbers are given for sequences from data banks. Designations for sequences determined in this study are as follows: MT, isolates from I. ricinus ticks collected in Morocco; IR, RH, and HY, isolates from I. ricinus, Rhipicephalus, and Hyalomma ticks, respectively, collected in Tunisia. A, Anaplasma; A. phago, A. phagocytophilum; E,Ehrlichia; N,Neorickettsia; W,Wolbachia.

5 VOL. 43, 2005 EHRLICHIA IN TICKS IN MAGHREB 1131 TABLE 1. Identification of Anaplasmataceae species from partial 16S rrna gene sequences from different tick species collected in Maghrebian countries a Organism No. of PCR-positive ticks/no. of ticks examined Tunisia I. ricinus Morocco R. sanguineus H. detritum H. a. excavatum Total 29/197 27/221 4/188 6/32 1/14 Anaplasma-like b A. platys 3 A. phagocytophilum 2 1 Ehrlichia-like 1 1 W. pipientis 1 Non-Ehrlichia DNA 1 1 a I. ricinus ticks were free-living; R. sanguineus ticks were collected from dogs in Tunisia; H. detritum and H. a. excavatum ticks were collected from bovines in Tunisia. b Inconclusive sequences. dogs (16). No DNA from E. canis, a species frequently involved in canine infections (2), was detected in ticks collected on dogs in Tunisia. However several species of Anaplasmataceae can be transmitted to a variety of hosts in nature, even though E. canis and E. ruminantium have been isolated exclusively from dogs and cattle, respectively. For example, in Missouri, the agent of Ehrlichia infection in dogs is currently Ehrlichia ewingii (21). It is not yet known whether R. sanguineus could be the vector of A. platys, and the actual role of Rhipicephalus in the transmission of members of Ehrlichia species remains to be confirmed. An extended study is needed to clarify these data from North Africa. Seven DNAs extracted from Hyalomma ticks led to positive signals in PCR and were sequenced (Table 1). One sequence completely matched that of A. phagocytophilum, and another fell into the Ehrlichia cluster (Fig. 2). Three sequences from Hyalomma ticks branched in the cluster Anaplasma but resulted in short sequences (139 to 158 nucleotides) and some discrepancies on the two strands. The data inferred from these sequences were too inconclusive to be included in the phylogenetic analysis. Moreover, the two remaining sequences from Hyalomma ticks did not match any previously known DNA sequence in a FASTA search. Consequently, even though no precise infection rate could be deduced from the analysis of Hyalomma ticks, evidence of the presence of DNA closely related to A. phagocytophilum in at least one H. detritum tick and of DNA closely related to Ehrlichia in another tick was acquired. The presence of A. phagocytophilum in European ticks was reported for the first time in 1997 (42), although its detection from different animal species had been described earlier (28). Afterwards, reports came from North and Northwestern Europe (1, 7, 14, 17, 22, 25, 30, 34, 38), as well as from Central Europe (5, 32) and even Southeastern Europe (6). The reported prevalences of infection showed large differences according to country, study, and tick species. Although the presence of members of the family Anaplasmataceae was reported as early as 1937 in Algeria in bovines, sheep, and dogs (10), data from North Africa have since been rare. Ghorbel et al. (13) reported a positive serology against E. canis for 10% of febrile patients in Tunisia, whereas no sera positive for E. chaffeensis were found in blood donors in Central Tunisia by Letaief et al. (20). The present study indicates that the agent of HGE is present on this continent. In Europe as in the United States, A. phagocytophilum is maintained in a natural cycle between the tick vector, I. scapularis or I. ricinus, respectively, and reservoir hosts. It is very likely that similar relationships also occur in North Africa, and this possibility needs to be further investigated by identifying the reservoir hosts. Our findings prove, for the first time, the presence of members of the family Anaplasmataceae in ticks in North Africa. Additionally, this study demonstrates a high level of genetic diversity among species present on this continent. Moreover, we demonstrate that these pathogens are harbored not only by I. ricinus but also by other tick species, Hyalomma and Rhipicephalus. Some of the Anaplasmataceae species detected in ticks are pathogenic for both humans and livestock. The demonstration of species closely related to the agents of granulocytic and monocytic ehrlichioses suggests that evidence of human and animal infections should be sought. Although people may be bitten by ticks infected by Anaplasmataceae members, the health implications in North Africa remain unknown. Therefore, our findings warrant further investigation in order to clarify the pathogenic potential of diverse Anaplasmataceae species in North Africa and the burden for human and animal populations, as well as the genetic relationships and epidemiology of these species. The respective roles of different tick species in the transmission of the different pathogens remain to be evaluated. Collectively, these data should be considered in medical practice, and ehrlichiosis should be included in the differential diagnosis of febrile illness in North Africa as well as in Europe. ACKNOWLEDGMENTS This work was supported by European Commission grant ICA3-CT We thank K. Dellagi and M. Hassar, Directors of the Pasteur Institutes of Tunisia and Morocco, respectively, for their constant support. We thank P. Brouqui for providing control Ehrlichia DNAs. REFERENCES 1. Baumgarten, B. U., M. Röllinghoff, and C. Bogdan Prevalence of Borrelia burgdorferi and granulocytic and monocytic ehrlichiae in Ixodes ricinus ticks from Southern Germany. J. Clin. Microbiol. 37: Breitschwerdt, E. B., B. C. Hegarty, and S. I. Hancock Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, orbartonella vinsonii. 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