# Single genotype of Anaplasma phagocytophilum identified from ticks, Camargue, France.

Amélie Chastagner, Xavier Bailly, Agnès Leblond, Sophie Pradier, Gwenaël Vourc'h

► To cite this version:

Amélie Chastagner, Xavier Bailly, Agnès Leblond, Sophie Pradier, Gwenaël Vourc'h. Single genotype of Anaplasma phagocytophilum identified from ticks, Camargue, France.. Emerging Infectious Diseases, 2013, 19 (5), pp.825-7. 10.3201/eid1905.121003. hal-00972552

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https://hal.science/hal-00972552v1

Submitted on 29 May 2020

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
This study was partly supported
by grants-in-aid from the Ministry of
Health, Labour and Welfare of Japan
(H22-Shokuhin-Ippan-012, H23-Shinko-
Shitei-020, H24-Shokuhin-Ippan-007, and
H24-Shokuhin-Ippan-008).

Hidemasa Izumiya,
Jun Terajima, Shouji Yamamoto,
Makoto Ohnishi,
Haruo Watanabe, Akemi Kai,
Takayuki Kurazono,
Masumi Taguchi, Tetsuo Asai,
Masato Akiba, Yuko Matsumoto,
and Yutaka Tamura

Author affiliations: National Institute of Infectious Diseases, Tokyo, Japan (H. Izumiya, J. Terajima, S. Yamamoto, M. Ohnishi, H. Watanabe); Tokyo Metropolitan Institute of Public Health, Tokyo (A. Kai); Saitama Institute of Public Health, Saitama, Japan (T. Kurazono); Osaka Prefectural Institute of Public Health, Osaka, Japan (M. Taguchi); National Veterinary Assay Laboratory, Tokyo (T. Asai); National Institute of Animal Health, Ibaraki, Japan (M. Akiba); Yokohama City Institute of Health, Kanagawa, Japan (Y. Matsumoto); and Rakuno Gakuen University, Hokkaido, Japan (Y. Tamura)

DOI: http://dx.doi.org/10.3201/eid1905.121395

References

1. Threlfall EJ. Epidemic *Salmonella typhimurium* DT 104-a truly international multiresistant clone. J Antimicrob Chemother. 2000;46:7-10. http://dx.doi.org/10.1093/jac/46.1.7

2. Briggs CE, Fratamico PM. Molecular characterization of an antibiotic resistance gene cluster of *Salmonella typhimurium* DT104. Antimicrob Agents Chemother. 1999;43:846-9.

3. van Belkum A. Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA). FEMS Immunol Med Microbiol. 2007;49:22-7. http://dx.doi.org/10.1111/j.1574-695X.2006.00173.x

4. Lindstedt BA, Vardund T, Aas L, Kapperud G. Multiple-locus variable-number tandem-repeats analysis of *Salmonella enterica* subsp. *enterica* serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. J Microbiol Methods. 2004;59:163-72. http://dx.doi.org/10.1016/j.mimet.2004.06.014

5. Larsson JT, Torpdahl M, Petersen RF, Sorensen G, Lindstedt BA, Nielsen EM. Development of a new nomenclature for *Salmonella typhimurium* multilocus variable number of tandem repeats analysis (MLVA). Euro Surveill. 2009;14:19174 http://www.eurosurveillance.org/View-Article.aspx?ArticleId=19174

6. Anderson ES, Ward LR, Saxe MJ, de Sa **JD. Bacteriophage-typing** designations of *Salmonella* typhimurium. J Hyg (Lond). 1977;78:297-300. http://dx.doi.org/10.1017/S0022172400056187

7. Hermans AP, Abee T, Zwietering MH, Aarts HJ. Identification of novel *Salmonella enterica* serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization. Appl Environ Microbiol. 2005;71:4979-85. http://dx.doi.org/10.1128/AEM.71.9.4979-4985.2005

Address for correspondence: Hidemasa
Izumiya, 1-23-1 Toyama, Shinjuku-ku, Tokyo,
162-8640, Japan; email: izumiya@nih.go.jp

Single Genotype
of Anaplasma
phagocytophilum
identified from
Ticks, Camargue,
France

To the Editor: Granulocytic anaplasmosis is a tickborne zoonosis caused by *Anaplasma phagocytophilum* bacteria, which are emerging in Europe. Besides infecting humans, *A. phagocytophilum* infect a wide range of wild and domestic mammals (1). In Europe, the *Ixodes ricinus* tick is the main vector for the bacteria, but *A. phagocytophilum* has also been detected in association with *Rhipicephalus* and *Dermacentor* spp. ticks (2). The climate and biotopes of the Mediterranean region are particularly favorable for several species of ticks and, therefore, for tickborne diseases.

Although *I. ricinus* ticks are rare
or absent in the Mediterranean Ba-
sin, serosurveys performed on equine

populations in Camargue, southern
France, indicated an *A. phagocytophi-
lum* seroprevalence of ≈10% (3). To
investigate the prevalence and diver-
sity of *A. phagocytophilum* bacteria in
ticks in Camargue, we collected quest-
ing ticks from horse pastures and feed-
ing ticks from horses.

Ticks feeding on horses were collected in randomly selected stables during 2007 (84 stables), 2008 (72 stable), and 2010 (19 stables). The stables were chosen among those where evidence of *A. phagocytophilum* seroconversion in horses had been previously found (3). In 2008 and 2010, questing ticks were collected by the dragging method in 19 pastures, around bushes, and in areas where horses spent the most time. Surveys were conducted in the spring, which represents the peak activity time of *Ixodes* ticks.

A total of 406 adult ticks were
collected, representing 6 species:
*Rhipicephalus bursa*, *R. sanguineus*,
*R. turanicus*, *R. pusillus*, *Dermacen-
tor marginatus*, and *Hyalomma mar-
ginatum*. Tick species were identified
by morphologic criteria and molecu-
lar analyses based on mitochondrial
12S rDNA sequences (4). Total DNA
was extracted from the ticks by us-
ing the NucleoSpin Tissue Kit (Ma-
cherey-Nagel, Düren, Germany) (5).
*A. phagocytophilum* was detected by
nested PCR targeting the 16S rDNA
(online Technical Appendix 1, ww-
wnc.cdc.gov/EID/article/19/5/12-
1003-Techapp1.pdf).

Of the 406 ticks, 40 were infected
with *A. phagocytophilum*. The infected
group included ticks from all 6 collect-
ed species except *R. pusillus*. Infection
rates among the species ranged from 0
to 22% (online Technical Appendix 2,
wwwnc.cdc.gov/EID/article/19/5/12-
1003-Techapp2.pdf). The prevalence
of *A. phagocytophilum* infection did
not differ significantly between spe-
cies (logistic regression model, p
= 0.76) but was higher among quest-
ing ticks than feeding ticks (p<0.001;
odds ratio 1.15).
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

Figure. Phylogenetic networks of *Anaplasma phagocytophilum* based on *msp4* (A) and *ankA* (B) genes and built with SplitsTree4 (version 4.11.3; http://splittree.org/) by the Neighbor-Net method. The sequences of the genotype described in Camargue, France, is framed. Sequences found in the 2 networks are in **boldface**: *A. phagocytophilum* amplified from ticks collected in Combrailles, Auvergne region, France (JX197116–JX197126 and JX197265–JX197270), a human isolate (strain Webster, EU857674 and GU236811)), an American roe deer isolate (strain CRT, JX197261 and JX197113), and an *American horse isolate* (strain MRK, AY530196 and AF153716). Scale bars indicate number of nucleotide substitutions per site.

We amplified 6 loci by nested PCR (online Technical Appendix 1) to characterize *A. phagocytophilum* genetic diversity in positive samples: *ankA*, *msp4*, *pleD*, *typA*, and intergenic regions *hemE–APH_0021* and *APH_1099–APH_1100* (National Center for Biotechnology Information annotation). The GenBank accession numbers for the nucleotide sequences are JX197073–JX197368. No polymorphism was found among the 6 loci tested in the 40 *A. phagocytophilum*–positive ticks. The genotype identified was 100% identical to the reference sequence (NC_007797) for loci *msp4*, *pleD*, and *typA* and for intergenic regions *hemE–APH_0021* and *APH_1099–APH_1100*. The *ankA* sequence was 96% similar (487 nt) to the reference sequence. The relevance of

these loci as markers of diversity was verified (online Technical Appendix 3, wwwnc.cdc.gov/EID/article/19/5/12-1003-Techapp3.pdf).

To study the phylogenetic relationships between cognate sequences, we included in our analysis all sequences available in GenBank for genes *ankA* and *msp4*. To account for recombination events that affect *ankA* and *msp4* (data not shown) in phylogenetic analyses, we used Neighbor-Net networks (Figure). Phylogenetic analysis of *msp4* (Figure, panel A) indicated that the genotype of *A. phagocytophilum* from ticks in Camargue was included in a clade that also includes genotypes that infect humans and horses in the United States.

The diversity of *ankA* sequences has been described as 4 phylogenetic

clusters (6). All sequences obtained in our study were included in cluster I, particularly in a branch composed exclusively of sequences of *A. phagocytophilum* isolated from humans in the United States (Figure, panel B).

Previous studies investigating *A. phagocytophilum* have revealed a genetic diversity that is thought to have been caused by sympatric epidemiologic cycles involving different vectors and reservoir hosts (1,6,7). In 5 species of ticks (40 ticks total) that we collected from a 250-km² area in southern France, we found only 1 genotype of *A. phagocytophilum*, which we determined to be phylogenetically close to genotypes found in the United States. Sequences phylogenetically related to bacteria in the United States were also observed in Sardinia (8) and Sicily (9).
The low diversity we found could
be explained by a recent introduction of
the bacteria into the area [although *A. phagocytophilum*—seropositive horses
have been found in the area since 2001
(3)] or by a selective sweep linked to
the particular ticks and host reservoir in
Camargue. The 5 species of ticks that
we found positive for *A. phagocytophilum* have been described as potential
vectors of *A. phagocytophilum* in the
Mediterranean Basin (2,10). Among
the tick species in our investigation, *R. bursa* and *R. sanguineus* ticks are the
2 main carriers of *A. phagocytophilum*,
and these ticks are likely to feed on hu-
mans and, thus, pose a risk of infection
to the local population. Further studies
are needed to address the potential ef-
fect of *A. phagocytophilum*—infected
ticks on human health in this area and,
more specifically, the relationship be-
tween genotype and pathogenicity.

Acknowledgments

We thank Curtis Nelson and Friederike
von Loewenich for generously providing the
DNA of strains HGE1, CRT, and Webster;
Véronique Bachy for actively helping us to
obtain samples from domestic animals; Mag-
alie René-Martellet and Frédéric Beugnet for
helping with the morphological identifica-
tion of ticks; Gillian Martin for proofreading
the manuscript; Nelly Dorr for creating the
databases used in this study; and Françoise
Rieu-Lesme and Sébastien Masséglia who
were involved in laboratory work.

Amélie Chastagner,
Xavier Bailly, Agnès Leblond,
Sophie Pradier,<fnref n="1" />
and Gwenaël Vourc'h

Author affiliations: National Center for Bio-
technology Information (INRA), Saint Ge-
nès Champanelle, France (A. Chastagner,
X. Bailly, A. Leblond, G. Vourc'h); University
of Lyon, Marcy l'Etoile, France (A. Leblond);
Ecole Nationale Vétérinaire d'Alfort, Mai-
sons-Alfort, France (S. Pradier); and INRA
ENVAANSES, Maisons-Alfort (S. Pradier)

DOI: http://dx.doi.org/10.3201/eid1905.121003

**References**

1. Rikihisa Y. Mechanisms of obligatory intracellular infection with *Anaplasma phagocytophilum*. *Clin Microbiol Rev.* 2011;24:469–89. http://dx.doi.org/10.1128/CMR.00064-10

2. de la Fuente J, Naranjo V, Ruiz-Fons F, Vicente J, Estrada-Pena A, Almazan C, et al. Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus) in central Spain. Eur J Wildl Res. 2004;50:187–96. http://dx.doi.org/10.1007/s10344-004-0060-1

3. Leblond A, Pradier S, Pittel PH, Fortier G, Boireau P, Chadoeuf J, et al. An epidemiological survey of equine anaplasmosis (*Anaplasma phagocytophilum*) in southern France [in French]. Rev Sci Tech. 2005;24:899–908.

4. Beati L, Keirans JE. Analysis of the sys-
tematic relationships among ticks of the
genera *Rhipicephalus* and *Boophilus*
(Acari: Ixodidae) based on mitochondrial
12S ribosomal DNA gene sequences and
morphological characters. J Parasitol.
2001;87:32–48.

5. Halos L, Jamal T, Vial L, Maillard R, Suau A, Le Menache A, et al. Determination of an efficient and reliable method for DNA extraction from ticks. Vet Res. 2004;35:709–13. http://dx.doi.org/10.1051/vetres:2004038

6. Scharf W, Schauer S, Freyburger F, Petrovec M, Schaarschmidt-Kiener D, Liebisch G, et al. Distinct host species correlate with *Anaplasma phagocytophilum ankA* gene clusters. J Clin Microbiol. 2011;49:790–6. http://dx.doi.org/10.1128/JCM.02051-10

7. Bown KJ, Lambin X, Ogden NH, Begon M, Telford G, Woldehiwet Z, et al. Delin-
eating *Anaplasma phagocytophilum* eco-
types in coexisting, discrete enzootic cy-
cles. Emerg Infect Dis. 2009;15:1948–54.
http://dx.doi.org/10.3201/eid1512.090178

8. Alberti A, Zobba R, Chessa B, Addis MF, Sparagano O, Parpaglia MLP, et al. Equine and canine *Anaplasma phagocytophilum* strains isolated on the island of Sardinia (Italy) are phylogenetically related to pathogenic strains from the United States. Appl Environ Microbiol. 2005;71:6418–22. http://dx.doi.org/10.1128/AEM.71.10.6418-6422.2005

9. de la Fuente J, Torina A, Caracappa S, Tumino G, Furla R, Almazan C, et al. Serologic and molecular characterization of *Anaplasma* species infection in farm animals and ticks from Sicily. Vet Parasitol. 2005;133:357–62. http://dx.doi.org/10.1016/j.vetpar.2005.05.063

10. Toledo A, Olmeda AS, Escudero R, Jado I, Valcarcel F, Casado-Nistal MA, et al. Tick-borne zoonotic bacteria in ticks collected from central Spain. Am J Trop Med Hyg. 2009;81:67–74.

Address for correspondence: Amélie
Chastagner, INRA, UR346 Epidémiologie
Animale, Centre de Recherches de
Clermont-Ferrand/Theix, F-63122 Saint
Genès Champanelle, France; email: amelie.
chastagner@clermont.inra.fr

West Nile Virus
Lineage 2 Strain
in Greece, 2012

**To the Editor:** West Nile virus (WNV) has been in Europe at least since the 1960s (1). Before 2010, WNV epidemics in Europe were caused mainly by lineage 1 strains. However, in 2010, a major WNV epidemic in Central Macedonia, Greece, was caused by a lineage 2 strain (Nea Santa-Greece-2010) (2).

This strain also circulated during
2011 (3), causing a second epidemic
among humans throughout the country
(4). Although the virus was closely
related to the goshawk-Hungary-2004
strain circulating in Hungary, Austria,
and Italy (5–7), severe epidemics
occurred only in Greece; 273 cases
of West Nile neuroinvasive disease
(WNND) in humans were reported
during the 2 seasons (2,4).

A third epidemic occurred in 2012, and 109 WNND cases were reported (8). Until mid-August, most cases were in central (Attica; 29 cases) and northeastern Greece (East Macedonia and Thrace; 10 cases). In contrast, during the same period, only 3 cases were confirmed at the location of the 2010 epidemic epicenter, in Central Macedonia (9). This situation led to the question of whether the Nea Santa-Greece-2010 strain was responsible for the third epidemic in Greece.

<footnote n="1">¹Current affiliation: Ecole Nationale
Vétérinaire de Toulouse, Toulouse, France.</footnote>