Medical and Veterinary Entomology (2015) 29, 349–353
doi: 10.1111/mve.12124
S H O R T C O M M U N I C AT I O N
Detection of tick-borne Anaplasma bovis, Anaplasma phagocytophilum and Anaplasma centrale in Spain A. M. P A L O M A R 1 , A. P O R T I L L O 1 , P. S A N T I B Á Ñ E Z 1 , D. M A Z U E L A S 2 , L. R O N C E R O 2 , L. G A R C Í A- Á L V A R E Z 1 , S. S A N T I B Á Ñ E Z 1 , Ó. G U T I É R R E Z 3 and J. A. O T E O 1 1
Centre of Rickettsiosis and Arthropod-Borne Diseases, Hospital San Pedro-CIBIR, Logroño, La Rioja, Spain, 2 Abies, Environment Resources, Inc., Logroño, La Rioja, Spain and 3 Aranzadi Society of Sciences, San Sebastián, Guipúzcoa, Spain
Abstract. The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co-infected with ‘Candidatus Midichloria mitochondrii’) and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and ‘Ca. Midichloria mitochondrii’ in I. frontalis. Key words. Anaplasma spp., Haemaphysalis punctata, Ixodes frontalis, Ixodes ricinus, bird, cattle, ticks.
The genus Anaplasma includes six species: Anaplasma phagocytophilum, Anaplasma marginale, Anaplasma centrale, Anaplasma bovis, Anaplasma ovis and Anaplasma platys. All of these are tick-borne, obligatory, intracellular, Gram-negative bacteria responsible for important diseases in humans and/or domestic animals (Rar & Golovljova, 2011). Anaplasma phagocytophilum causes human granulocytic anaplasmosis, as well as anaplasmosis in ruminants, horses, dogs and cats; A. marginale, A. centrale, A. bovis and A. ovis cause anaplasmosis in domestic and wild ruminants, and A. platys causes cyclic thrombocytopenia in dogs (Rar & Golovljova, 2011). At the time of writing, all but A. bovis had been documented in Spain, and cases of human, bovine and canine anaplasmosis had been reported
(Sainz et al., 1999; Oteo et al., 2000; De La Fuente et al., 2005, 2008; Portillo et al., 2011). Different species of ticks are hosts and vectors of different Anaplasma species. Moreover, vertebrate hosts are essential in the lifecycle of these bacteria because they are not transovarially transmitted. Wild mammals such as deer and rodents represent their main reservoirs, and other mammals, such as agricultural livestock, are, at least, amplifiers of the bacteria (Portillo et al., 2011). In addition, birds infected with Anaplasma spp. have been reported (De La Fuente et al., 2005). Although their role as reservoirs is still unclear, birds (carriers of infected ticks) may be responsible for the spread of these infectious agents (Palomar et al., 2012).
Correspondence: Dr José A. Oteo, Departamento de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, 98-7a NE, 26006 Logroño, La Rioja, Spain. Tel.: +34 941 298993; Fax: +34 941 298667; E-mail: [email protected] © 2015 The Royal Entomological Society
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Fig. 1. Tick sampling sites in La Rioja and Burgos (Hacinas); the source of ticks (birds, cattle or vegetation) is indicated for each site. Numbers of ticks collected from each source were: birds: Santa Eulalia (see Table 1); cattle: Ezcaray (one female), Hacinas (nine males, 11 females), Jubera (12 males, 11 females), Soto en Cameros (one male, one female), Munilla (two males, 11 females), Tobía (one male, 11 females); vegetation: Lumbreras (25 larvae, one nymph, seven males, one female), Peroblasco (10 males, 17 females), Tobía (seven males, eight females), Torrecilla en Cameros (one nymph, two males, three females), Villalba de Rioja (two nymphs, seven males, 19 females).
The aim of this study was to investigate the presence of Anaplasma spp. in ticks removed from birds in La Rioja in northern Spain. Subsequently, because of the finding of A. bovis (not previously reported in Spain) in Haemaphysalis punctata ticks removed from birds, the presence of Anaplasma spp. was investigated in this tick species collected from other sources (cattle and vegetation). From 2009 to 2011, weekly bird banding was carried out in Santa Eulalia, La Rioja, Spain (Fig. 1). Bird specimens captured in good physical condition were checked for ticks. Arthropods were removed with tweezers and placed in Eppendorf tubes on which the ring number, bird species and date were recorded. Arthropods were classified using taxonomic keys and molecular methods, as reported in a previous study of ticks from birds (Palomar et al., 2012). In addition, H. punctata specimens collected from cattle and vegetation during routine sampling in nearby areas from January 2012 to August 2013 were selected (Fig. 1). Samples were stored at −80 ∘ C until RNA or DNA extraction was carried out. DNA was extracted from individual ticks except those individuals morphologically classified as belonging to the genus Hyalomma, using two incubations of 20 min each with ammonium hydroxide (1 mL of 25% ammonia and 19 mL of MQ water) at 100 ∘ C and 90 ∘ C, respectively. For Hyalomma spp. samples that had been included in viral screening studies (Palomar, unpublished data, 2014), RNA was individually purified and retrotranscribed using RNeasy Mini and Omniscript RT kits, respectively (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions.
Tick DNA and cDNA samples were used as templates for polymerase chain reaction (PCR) assays, targeting the mitochondrial 16S rRNA gene for tick classification (456 bp) (Black & Piesman, 1994). Detection of the tick mitochondrial 16S rRNA gene in all samples studied confirmed that the extraction and retrotranscription procedures were efficient. In addition, two nested and one single PCR assay with primers targeting the 16S rRNA gene (932/546 bp), the groESL heat shock operon (1350/1297 bp) and the p44 major surface protein gene msp2 (380 bp) (Portillo et al., 2011; Palomar et al., 2012) were used as a first screen for Anaplasma spp. For better characterization of the bacteria and to confirm the circulation of species not previously reported in the area, positive samples for at least one of these PCR targets were subsequently tested using PCR primers for EHR and GEP regions of the 16S rRNA gene (345 bp and 341 bp, respectively) (Eddlestone et al., 2007; Palomar et al., 2012). Each assay was previously tested with different concentrations of DNA and cDNA extracts from A. phagocytophilum (Webster strain), kindly provided by Dr Didier Raoult (Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France) through Dr Stephen Dumler (Johns Hopkins Hospital, Baltimore, MD, U.S.A.). Similar results with the DNA and cDNA genomic extracts (data not shown) showed the sensitivity of both approaches to be comparable. Two negative controls, one of them containing water instead of template DNA and the other with template DNA but without primers, as well as a positive control of A. phagocytophilum (Webster strain), were included in all PCR assays. Amplification products
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
Detection of Anaplasma spp. in Spain 351 Table 1. Ticks removed from birds in Santa Eulalia (42∘ 12′ N, 02∘ 12′ W) from 2009 to 2011. Tick species (n = 336) Bird species (n = 121)
Ixodes frontalis
Ixodes ricinus
Hyalomma marginatum
Haemaphysalis punctata
Ixodes spp.
Aegithalos caudatus (n = 6) Carduelis chloris (n = 2) Cyanistes caeruleus (n = 4) Emberiza cirlus (n = 1) Erithacus rubecula (n = 15) Ficedula hypoleuca (n = 1) Fringilla coelebs (n = 6) Hippolais polyglotta (n = 1) Luscinia megarhynchos (n = 4) Parus major (n = 3) Passer domesticus (n = 8) Phylloscopus collybita (n = 2) Serinus serinus (n = 1) Sturnus unicolor (n = 2) Sylvia atricapilla (n = 27) Sylvia communis (n = 2) Sylvia melanocephala (n = 1) Turdus merula (n = 24) Turdus philomelos (n = 11)
10 (7 N, 3 F) 0 2 (1 L, 1 F) 0 43 (36 L, 7 N) 0 0 0 1 (1 N) 1 (1 L) 0 1 (1 L) 0 0 59 (52 L, 4 N, 3 F) 1 (1 N) 1 (1 N) 39 (21 L, 6 N, 1 M, 11 F) 16 (10 L, 5 N, 1 F) 174 (122 L, 32 N, 1 M, 19 F)
0 0 0 0 8 (7 L, 1 N) 0 1 (1 N) 2 (2 L) 1 (1 N) 0 0 1 (1 L) 0 0 2 (2 L) 0 0 2 (2 N) 0 17 (12 L, 5 N)
0 1 (1 N) 6 (6 N) 0 0 1 (1 L) 1 (1 L) 0 5 (1 L, 4 N) 3 (1 L, 2 N) 2 (1 L, 1 N) 0 0 0 0 0 0 15 (8 L, 7 N) 0 34 (13 L, 21 N)
0 1 (1 L) 0 5 (5 L) 0 0 20 (19 L, 1 N) 0 4 (4 N) 0 13 (3 L, 10 N) 0 1 (1 L) 2 (1 L, 1 N) 0 0 0 62 (52 L, 10 N) 0 108 (82 L, 26 N)
1 (1 N) 0 0 0 0 0 0 0 0 0 0 0 0 1 (1 L) 1 (1 L) 0 0 0 0 3 (2 L, 1 N)
F, female; L, larva; M, male; N, nymph.
were sequenced and nucleotide sequences were compared with those available in GenBank using a blast (Basic Local Alignment Search Tool) search (http://www.ncbi.nlm.nih.gov/blast). A total of 336 ticks were collected from 121 birds belonging to 19 species of the order Passeriformes (Table 1). Ticks were classified within the genera Ixodes (57.7%), Haemaphysalis (32.1%) and Hyalomma (10.2%), and as belonging to the species Ixodes frontalis (n = 174), Ixodes ricinus (n = 17), H. punctata (n = 108), Hyalomma marginatum (n = 34), and Ixodes spp. (n = 3) (Table 1). In addition, 181 H. punctata specimens (71 from cattle and 110 from vegetation) were processed (Fig. 1). Seven (2.1%; two H. punctata, four I. frontalis and one I. ricinus) of the 336 ticks removed from birds tested positive for Anaplasma spp. with at least one of the three targets used for the screening (16S rRNA, groESL and msp2). Thus, one larva and one nymph of H. punctata from, respectively, Common Blackbird (Turdus merula) and Common Nightingale (Luscinia megarhynchos) specimens harboured A. bovis (100% identity) according to the analyses of 16S rRNA gene sequences. These data were corroborated by PCR and sequence analysis of 16S rRNA EHR and GEP (Table 2). In addition, the screening showed that one nymph, two larvae and one female I. frontalis specimens and one I. ricinus nymph removed from specimens of Common Nightingale, Eurasian Blackcap (Sylvia atricapilla), European Robin (Erithacus rubecula) and Common Blackbird were infected with A. phagocytophilum (99.9–100% identity). These results were confirmed for one I. frontalis larva, and showed 99.7% and 100% identity, respectively, with 16S rRNA EHR and GEP sequences of this bacterium. In addition, analysis of the 16S rRNA EHR and GEP sequences for the I. frontalis nymph showed highest identity
(99.3% and 99.1%, respectively) with ‘Candidatus Midichloria mitochondrii’ (Table 2). The initial screening also showed that five of 71 (7.0%) H. punctata removed from cattle harboured Anaplasma spp. Four of these ticks (three males and one female) were found to be infected with A. phagocytophilum (99.8–100% identity). These data were corroborated by the analysis of 16S rRNA GEP sequences obtained for two samples (one male and one female). By contrast, the 16S rRNA GEP sequence derived from the second male tick showed 100% identity with Ehrlichia sp. (Rickettsiales: Anaplasmataceae) closely related to Ehrlichia ewingii. The 16S rRNA sequence of the remaining male tick showed highest identity (99.6%) with A. centrale. This finding was confirmed by analyses of EHR and GEP sequences (100% and 98.6% identity, respectively, with A. centrale) (Table 2). In addition, four of 110 (3.6%) H. punctata collected from vegetation showed positive results for Anaplasma spp. Two nymphs and one male tick showed the presence of A. bovis 16S rRNA, EHR and GEP sequences (100% identity) (Table 2). By contrast, sequences of the 16S rRNA gene (screening) and GEP region from one male specimen showed the highest identity (99.4% and 98.6%, respectively) with A. centrale (Table 2). In this study, the presence of A. bovis, A. phagocytophilum and A. centrale, as well as ‘Ca. M. mitochondrii’ and Ehrlichia sp. closely related to E. ewingii, is reported in ticks from La Rioja (northern Spain). This is the first evidence of the presence of A. bovis in Spain and also in ticks in Europe. Previously, in Europe A. bovis had been detected in blood samples from livestock in Italy (Ceci et al., 2014). In Japan, A. bovis was detected in Ixodes turdus ticks removed from migratory birds [Yellow-browed Bunting (Emberiza chrysophrys)] (Kang
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
EU781706 (100%) EU781706 (100%) JN181071 (99.8%) ND ND JN181071 (100%) AB211164 (99.4%) AB211164 (99.6%) Jubera Jubera Jubera Jubera Tobía Jubera B. taurus B. taurus B. taurus B. taurus Vegetation B. taurus A. centrale (2)
Sta. Eulalia T. merula
I. frontalis (1 L) I. ricinus (1 N) H. punctata (1 M) H. punctata (1 M) H. punctata (1 M) H. punctata (1 F) H. punctata (1 M) H. punctata (1 M)
ND EU781706 (100%) I. frontalis (1 F) I. frontalis (1 L) Sta. Eulalia Sta. Eulalia S. atricapilla E. rubecula
A. phagocytophilum (9)
∗Sequences corresponding to bacteria species other than those shown in the table: HF568843 and CP002130, ‘Candidatus Midichloria mitochondrii’; HQ697589, Ehrlichia sp. A., Anaplasma; B., Bos; E., Erithacus; H., Haemaphysalis; I., Ixodes; L., Luscinia; ND, not detected; S., Sylvia; T., Turdus.
ND ND HQ697589∗(100%) ND JN181071 (99.4%) JN181071 (99.4%) AF283007 (98.6%) AF283007 (98.6%) ND ND ND ND ND ND ND AB211164 (100%) JQ669948 (100%) ND EU921902 (100%) EU921902 (100%) EU921902 (100%) ND ND ND
ND EU781706 (100%) ND CP006616 (99.7%) JQ669948 (100%) JQ669948 (100%)
KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) HF568843∗(99.3%) ND ND ND JQ669948 (100%)
ND ND ND KF031393 (99.9%) ND KF031393 (100%) ND ND ND ND ND ND ND ND KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) EU781706 (100%) H. punctata (1 L) H. punctata (1 N) H. punctata (2 N,1 M) I. frontalis (1 N) Sta. Eulalia Sta. Eulalia Villalba de Rioja Sta. Eulalia T. merula L. megarhynchos Vegetation L. megarhynchos A. bovis (5)
16S rRNA EHR msp2 groESL 16S rRNA
GenBank accession no. (% identity)
Tick species (n, stage) Area Origin Microorganisms (positive samples, n)
Table 2. Anaplasma species detected in this study.
KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) CP002130∗(99.1%)
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16S rRNA GEP
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et al., 2013). Moreover, A. bovis has been detected in Haemaphysalis spp., including Haemaphysalis coccina and Haemaphysalis longicornis, from Asia (Kawahara et al., 2006; Rar & Golovljova, 2011), but has not previously been reported from H. punctata. Anaplasma bovis infection may be asymptomatic in ruminants, but it may also cause a wide variety of clinical symptoms including debility, fever, diarrhoea, weight loss and even death (Rar & Golovljova, 2011). In the present study, A. bovis was found in ticks removed from migratory birds, including a presaharian Common Blackbird and a transaharian Common Nightingale. These birds are known to migrate from Central Europe to reach the North of Spain in autumn. In spring, the Common Blackbird migrating from North Africa and the Common Nightingale migrating from West and Central Africa also arrive in the study area. There are two possible explanations for the presence of A. bovis in ticks removed from migratory birds. Firstly, the presence of A. bovis and H. punctata in Africa (Rar & Golovljova, 2011) supports the proposal that this bacterium may be imported from Africa to Spain by migratory birds. Secondly, H. punctata is a common tick in the studied area and A. bovis may be established there. The finding of A. bovis in three specimens of H. punctata collected from vegetation in an area 74 km west of the bird banding location supports the second hypothesis. To date, neither A. phagocytophilum nor ‘Ca. M. mitochondrii’ have been reported previously in I. frontalis ticks. ‘Candidatus M. mitochondrii’ is an endosymbiotic intramitochondrial alphaproteobacterium associated with a wide range of arthropod hosts that include hard ticks. Migratory birds carrying I. ricinus infected with A. phagocytophilum have been reported close to the study area (Palomar et al., 2012). Anaplasma phagocytophilum has been amplified from birds in Spain (De La Fuente et al., 2005), although the role of birds as reservoirs of A. phagocytophilum has not been demonstrated. In addition, in the present study A. phagocytophilum was detected in H. punctata from cattle. Previous examination of I. ricinus removed from the same cattle showed a high prevalence of A. phagocytophilum infection (Palomar et al., 2014). The A. phagocytophilum 16S rRNA sequences obtained in the present study from H. punctata shared 100% identity with the most prevalent strain found in I. ricinus (Palomar et al., 2014), and were also identical to A. phagocytophilum from I. ricinus removed from birds in Norway (GenBank accession no. JN181071). Anaplasma phagocytophilum was previously detected in H. punctata in Spain (Barandika et al., 2008), although the role of this tick species as a vector has not been demonstrated. The GEP region of the 16S rRNA amplified from one of the A. phagocytophilum-positive male H. punctata from cattle was homologous (100% identical to HQ69758) to an Ehrlichia sp. closely related to E. ewingii originally detected in H. longicornis from Japan (Matsumoto et al., 2011). These results indicate that this tick was co-infected with A. phagocytophilum and an Ehrlichia sp., although further investigation should be performed to determine the identity of the Ehrlichia sample. In the present study, the analysis of msp2 gene and 16S rRNA sequences distinguishes two clusters of A. phagocytophilum strains: one cluster appears in Ixodes spp. collected from birds, and the other appears in H. punctata collected from cattle
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
Detection of Anaplasma spp. in Spain 353 (Table 2). To date, neither of these clusters have been implicated as causative agents of human disease. Anaplasma centrale is a mildly pathogenic bacterium closely related to A. marginale (Rar & Golovljova, 2011). According to Rar & Golovljova (2011), the tick Rhipicephalus simus (Ixodida: Ixodidae) can transmit A. centrale, and H. longicornis has been suggested as a potential vector in Japan (Kawahara et al., 2006). As Table 2 demonstrates, the sequences of A. centrale obtained in the present study showed the highest level of identity with those isolated in Japan. The present study is the first report of A. centrale in H. punctata and provides the first evidence of this bacterium in ticks from Europe. Similar sequences were detected in deer blood samples from the same study area (Portillo et al., 2011). Therefore, it is necessary to be cautious in the serological diagnosis of anaplasmosis in cattle in Spain as cross-reactivity between A. centrale and A. marginale may lead to an inaccurate diagnosis of the causative agent. The detection of A. bovis, A. phagocytophilum and A. centrale in H. punctata specimens indicates the possible importance of this tick species in the epidemiology of these tick-borne pathogens. Further investigations should be performed to clarify the role of H. punctata as a potential vector of different Anaplasma species. The presence of A. bovis in Western Europe supports its implication in anaplasmosis of livestock in this region. Once again, the role of birds as dispersers of infected ticks is demonstrated.
Acknowledgements The first author was awarded a grant by the Fundación Rioja Salud (FRS/PIF-01/10). The authors thank Didier Raoult (Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France) and J. Stephen Dumler (The Johns Hopkins Hospital, Baltimore, MD, U.S.A) for supplying positive controls, Lourdes Romero (Center of Rickettsiosis and Arthropod-Borne Diseases, Hospital San Pedro-CIBIR, LA Rioja, Spain) for the provision of polymerase chain reaction validations, and Rufino Álamo (Territorial Health Service and Social Welfare of the Junta de Castilla y León, Valladolid, Spain) and Gerardo Domínguez (Slaughterhouse of Burgos, Burgos, Spain) for providing tick samples from Hacinas. The authors also wish to express their appreciation to Dr Lesley Bell-Sakyi (Pirbright Institute, Woking, U.K.) for reviewing the English language of this manuscript. References Barandika, J.F., Hurtado, A., García-Sanmartín, J., Juste, R.A., Anda, P. & García-Pérez, A.L. (2008) Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain. Vector-Borne and Zoonotic Diseases, 8, 829–835. Black, W.C. & Piesman, J. (1994) Phylogeny of hard and soft tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.
Proceedings of the National Academy of Sciences of the United States of America, 91, 10034–10038. Ceci, L., Iarussi, F., Greco, B., Lacinio, R., Fornelli, S. & Carelli, G. (2014) Retrospective study of haemoparasites in cattle in Southern Italy by reverse line blot hybridization. Journal of Veterinary Medical Science, 76, 869–875. De La Fuente, J., Naranjo, V., Ruiz-Fons, F. et al. (2005) Potential vertebrate reservoir hosts and invertebrate vectors of Anaplasma marginale and A. phagocytophilum in central Spain. Vector-Borne and Zoonotic Diseases, 5, 390–401. De La Fuente, J., Ruiz-Fons, F., Naranjo, V., Torina, A., Rodríguez, O. & Gortázar, C. (2008) Evidence of Anaplasma infections in European roe deer (Capreolus capreolus) from southern Spain. Research in Veterinary Science, 84, 382–386. Eddlestone, S.M., Diniz, P.P., Neer, T.M. et al. (2007) Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs. Journal of Veterinary Internal Medicine, 21, 1237–1242. Kang, J.G., Kim, H.C., Choi, C.Y. et al. (2013) Molecular detection of Anaplasma, Bartonella, and Borrelia species in ticks collected from migratory birds from Hong-do Island, Republic of Korea. Vector-Borne and Zoonotic Diseases, 13, 215–225. Kawahara, M., Rikihisa, Y., Lin, Q. et al. (2006) Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Applied and Environmental Microbiology, 72, 1102–1109. Matsumoto, K., Takeuchi, T., Yokoyama, N. et al. (2011) Detection of the new Ehrlichia species closely related to Ehrlichia ewingii from Haemaphysalis longicornis in Yonaguni Island, Okinawa, Japan. Veterinary Medicine and Science, 73, 1485–1488. Oteo, J.A., Blanco, J.R., Martínez de Artola, V. & Ibarra, V. (2000) First report of human granulocytic ehrlichiosis from southern Europe (Spain). Emerging Infectious Diseases, 6, 430–432. Palomar, A.M., Santibáñez, P., Mazuelas, D., Roncero, L., Santibáñez, S., Portillo, A. & Oteo, J.A. (2012) Role of birds in dispersal of etiologic agents of tick-borne zoonoses, Spain, 2009. Emerging Infectious Diseases, 18, 1188–1191. Palomar, A.M., García-Álvarez, L., Santibáñez, S., Portillo, A. & Oteo, J.A. (2014) Detection of tick-borne ‘Candidatus Neoehrlichia mikurensis’ and Anaplasma phagocytophilum in Spain in 2013. Parasites and Vectors, 7, 57. Portillo, A., Pérez-Martínez, L., Santibáñez, S., Santibáñez, P., Palomar, A.M. & Oteo, J.A. (2011) Anaplasma spp. in wild mammals and Ixodes ricinus from the north of Spain. Vector-Borne and Zoonotic Diseases, 11, 3–8. Rar, V. & Golovljova, I. (2011) Anaplasma, Ehrlichia, and ‘Candidatus Neoehrlichia’ bacteria: pathogenicity, biodiversity, and molecular genetic characteristics, a review. Infection, Genetics and Evolution, 11, 1842–1861. Sainz, A., Amusategui, I. & Tesouro, M.A. (1999) Ehrlichia platys infection and disease in dogs in Spain. Journal of Veterinary Diagnostic Investigation, 11, 382–384. Accepted 21 February 2015 First published online 25 June 2015
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doi: 10.1111/mve.12124
S H O R T C O M M U N I C AT I O N
Detection of tick-borne Anaplasma bovis, Anaplasma phagocytophilum and Anaplasma centrale in Spain A. M. P A L O M A R 1 , A. P O R T I L L O 1 , P. S A N T I B Á Ñ E Z 1 , D. M A Z U E L A S 2 , L. R O N C E R O 2 , L. G A R C Í A- Á L V A R E Z 1 , S. S A N T I B Á Ñ E Z 1 , Ó. G U T I É R R E Z 3 and J. A. O T E O 1 1
Centre of Rickettsiosis and Arthropod-Borne Diseases, Hospital San Pedro-CIBIR, Logroño, La Rioja, Spain, 2 Abies, Environment Resources, Inc., Logroño, La Rioja, Spain and 3 Aranzadi Society of Sciences, San Sebastián, Guipúzcoa, Spain
Abstract. The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co-infected with ‘Candidatus Midichloria mitochondrii’) and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and ‘Ca. Midichloria mitochondrii’ in I. frontalis. Key words. Anaplasma spp., Haemaphysalis punctata, Ixodes frontalis, Ixodes ricinus, bird, cattle, ticks.
The genus Anaplasma includes six species: Anaplasma phagocytophilum, Anaplasma marginale, Anaplasma centrale, Anaplasma bovis, Anaplasma ovis and Anaplasma platys. All of these are tick-borne, obligatory, intracellular, Gram-negative bacteria responsible for important diseases in humans and/or domestic animals (Rar & Golovljova, 2011). Anaplasma phagocytophilum causes human granulocytic anaplasmosis, as well as anaplasmosis in ruminants, horses, dogs and cats; A. marginale, A. centrale, A. bovis and A. ovis cause anaplasmosis in domestic and wild ruminants, and A. platys causes cyclic thrombocytopenia in dogs (Rar & Golovljova, 2011). At the time of writing, all but A. bovis had been documented in Spain, and cases of human, bovine and canine anaplasmosis had been reported
(Sainz et al., 1999; Oteo et al., 2000; De La Fuente et al., 2005, 2008; Portillo et al., 2011). Different species of ticks are hosts and vectors of different Anaplasma species. Moreover, vertebrate hosts are essential in the lifecycle of these bacteria because they are not transovarially transmitted. Wild mammals such as deer and rodents represent their main reservoirs, and other mammals, such as agricultural livestock, are, at least, amplifiers of the bacteria (Portillo et al., 2011). In addition, birds infected with Anaplasma spp. have been reported (De La Fuente et al., 2005). Although their role as reservoirs is still unclear, birds (carriers of infected ticks) may be responsible for the spread of these infectious agents (Palomar et al., 2012).
Correspondence: Dr José A. Oteo, Departamento de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, 98-7a NE, 26006 Logroño, La Rioja, Spain. Tel.: +34 941 298993; Fax: +34 941 298667; E-mail: [email protected] © 2015 The Royal Entomological Society
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Fig. 1. Tick sampling sites in La Rioja and Burgos (Hacinas); the source of ticks (birds, cattle or vegetation) is indicated for each site. Numbers of ticks collected from each source were: birds: Santa Eulalia (see Table 1); cattle: Ezcaray (one female), Hacinas (nine males, 11 females), Jubera (12 males, 11 females), Soto en Cameros (one male, one female), Munilla (two males, 11 females), Tobía (one male, 11 females); vegetation: Lumbreras (25 larvae, one nymph, seven males, one female), Peroblasco (10 males, 17 females), Tobía (seven males, eight females), Torrecilla en Cameros (one nymph, two males, three females), Villalba de Rioja (two nymphs, seven males, 19 females).
The aim of this study was to investigate the presence of Anaplasma spp. in ticks removed from birds in La Rioja in northern Spain. Subsequently, because of the finding of A. bovis (not previously reported in Spain) in Haemaphysalis punctata ticks removed from birds, the presence of Anaplasma spp. was investigated in this tick species collected from other sources (cattle and vegetation). From 2009 to 2011, weekly bird banding was carried out in Santa Eulalia, La Rioja, Spain (Fig. 1). Bird specimens captured in good physical condition were checked for ticks. Arthropods were removed with tweezers and placed in Eppendorf tubes on which the ring number, bird species and date were recorded. Arthropods were classified using taxonomic keys and molecular methods, as reported in a previous study of ticks from birds (Palomar et al., 2012). In addition, H. punctata specimens collected from cattle and vegetation during routine sampling in nearby areas from January 2012 to August 2013 were selected (Fig. 1). Samples were stored at −80 ∘ C until RNA or DNA extraction was carried out. DNA was extracted from individual ticks except those individuals morphologically classified as belonging to the genus Hyalomma, using two incubations of 20 min each with ammonium hydroxide (1 mL of 25% ammonia and 19 mL of MQ water) at 100 ∘ C and 90 ∘ C, respectively. For Hyalomma spp. samples that had been included in viral screening studies (Palomar, unpublished data, 2014), RNA was individually purified and retrotranscribed using RNeasy Mini and Omniscript RT kits, respectively (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions.
Tick DNA and cDNA samples were used as templates for polymerase chain reaction (PCR) assays, targeting the mitochondrial 16S rRNA gene for tick classification (456 bp) (Black & Piesman, 1994). Detection of the tick mitochondrial 16S rRNA gene in all samples studied confirmed that the extraction and retrotranscription procedures were efficient. In addition, two nested and one single PCR assay with primers targeting the 16S rRNA gene (932/546 bp), the groESL heat shock operon (1350/1297 bp) and the p44 major surface protein gene msp2 (380 bp) (Portillo et al., 2011; Palomar et al., 2012) were used as a first screen for Anaplasma spp. For better characterization of the bacteria and to confirm the circulation of species not previously reported in the area, positive samples for at least one of these PCR targets were subsequently tested using PCR primers for EHR and GEP regions of the 16S rRNA gene (345 bp and 341 bp, respectively) (Eddlestone et al., 2007; Palomar et al., 2012). Each assay was previously tested with different concentrations of DNA and cDNA extracts from A. phagocytophilum (Webster strain), kindly provided by Dr Didier Raoult (Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France) through Dr Stephen Dumler (Johns Hopkins Hospital, Baltimore, MD, U.S.A.). Similar results with the DNA and cDNA genomic extracts (data not shown) showed the sensitivity of both approaches to be comparable. Two negative controls, one of them containing water instead of template DNA and the other with template DNA but without primers, as well as a positive control of A. phagocytophilum (Webster strain), were included in all PCR assays. Amplification products
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
Detection of Anaplasma spp. in Spain 351 Table 1. Ticks removed from birds in Santa Eulalia (42∘ 12′ N, 02∘ 12′ W) from 2009 to 2011. Tick species (n = 336) Bird species (n = 121)
Ixodes frontalis
Ixodes ricinus
Hyalomma marginatum
Haemaphysalis punctata
Ixodes spp.
Aegithalos caudatus (n = 6) Carduelis chloris (n = 2) Cyanistes caeruleus (n = 4) Emberiza cirlus (n = 1) Erithacus rubecula (n = 15) Ficedula hypoleuca (n = 1) Fringilla coelebs (n = 6) Hippolais polyglotta (n = 1) Luscinia megarhynchos (n = 4) Parus major (n = 3) Passer domesticus (n = 8) Phylloscopus collybita (n = 2) Serinus serinus (n = 1) Sturnus unicolor (n = 2) Sylvia atricapilla (n = 27) Sylvia communis (n = 2) Sylvia melanocephala (n = 1) Turdus merula (n = 24) Turdus philomelos (n = 11)
10 (7 N, 3 F) 0 2 (1 L, 1 F) 0 43 (36 L, 7 N) 0 0 0 1 (1 N) 1 (1 L) 0 1 (1 L) 0 0 59 (52 L, 4 N, 3 F) 1 (1 N) 1 (1 N) 39 (21 L, 6 N, 1 M, 11 F) 16 (10 L, 5 N, 1 F) 174 (122 L, 32 N, 1 M, 19 F)
0 0 0 0 8 (7 L, 1 N) 0 1 (1 N) 2 (2 L) 1 (1 N) 0 0 1 (1 L) 0 0 2 (2 L) 0 0 2 (2 N) 0 17 (12 L, 5 N)
0 1 (1 N) 6 (6 N) 0 0 1 (1 L) 1 (1 L) 0 5 (1 L, 4 N) 3 (1 L, 2 N) 2 (1 L, 1 N) 0 0 0 0 0 0 15 (8 L, 7 N) 0 34 (13 L, 21 N)
0 1 (1 L) 0 5 (5 L) 0 0 20 (19 L, 1 N) 0 4 (4 N) 0 13 (3 L, 10 N) 0 1 (1 L) 2 (1 L, 1 N) 0 0 0 62 (52 L, 10 N) 0 108 (82 L, 26 N)
1 (1 N) 0 0 0 0 0 0 0 0 0 0 0 0 1 (1 L) 1 (1 L) 0 0 0 0 3 (2 L, 1 N)
F, female; L, larva; M, male; N, nymph.
were sequenced and nucleotide sequences were compared with those available in GenBank using a blast (Basic Local Alignment Search Tool) search (http://www.ncbi.nlm.nih.gov/blast). A total of 336 ticks were collected from 121 birds belonging to 19 species of the order Passeriformes (Table 1). Ticks were classified within the genera Ixodes (57.7%), Haemaphysalis (32.1%) and Hyalomma (10.2%), and as belonging to the species Ixodes frontalis (n = 174), Ixodes ricinus (n = 17), H. punctata (n = 108), Hyalomma marginatum (n = 34), and Ixodes spp. (n = 3) (Table 1). In addition, 181 H. punctata specimens (71 from cattle and 110 from vegetation) were processed (Fig. 1). Seven (2.1%; two H. punctata, four I. frontalis and one I. ricinus) of the 336 ticks removed from birds tested positive for Anaplasma spp. with at least one of the three targets used for the screening (16S rRNA, groESL and msp2). Thus, one larva and one nymph of H. punctata from, respectively, Common Blackbird (Turdus merula) and Common Nightingale (Luscinia megarhynchos) specimens harboured A. bovis (100% identity) according to the analyses of 16S rRNA gene sequences. These data were corroborated by PCR and sequence analysis of 16S rRNA EHR and GEP (Table 2). In addition, the screening showed that one nymph, two larvae and one female I. frontalis specimens and one I. ricinus nymph removed from specimens of Common Nightingale, Eurasian Blackcap (Sylvia atricapilla), European Robin (Erithacus rubecula) and Common Blackbird were infected with A. phagocytophilum (99.9–100% identity). These results were confirmed for one I. frontalis larva, and showed 99.7% and 100% identity, respectively, with 16S rRNA EHR and GEP sequences of this bacterium. In addition, analysis of the 16S rRNA EHR and GEP sequences for the I. frontalis nymph showed highest identity
(99.3% and 99.1%, respectively) with ‘Candidatus Midichloria mitochondrii’ (Table 2). The initial screening also showed that five of 71 (7.0%) H. punctata removed from cattle harboured Anaplasma spp. Four of these ticks (three males and one female) were found to be infected with A. phagocytophilum (99.8–100% identity). These data were corroborated by the analysis of 16S rRNA GEP sequences obtained for two samples (one male and one female). By contrast, the 16S rRNA GEP sequence derived from the second male tick showed 100% identity with Ehrlichia sp. (Rickettsiales: Anaplasmataceae) closely related to Ehrlichia ewingii. The 16S rRNA sequence of the remaining male tick showed highest identity (99.6%) with A. centrale. This finding was confirmed by analyses of EHR and GEP sequences (100% and 98.6% identity, respectively, with A. centrale) (Table 2). In addition, four of 110 (3.6%) H. punctata collected from vegetation showed positive results for Anaplasma spp. Two nymphs and one male tick showed the presence of A. bovis 16S rRNA, EHR and GEP sequences (100% identity) (Table 2). By contrast, sequences of the 16S rRNA gene (screening) and GEP region from one male specimen showed the highest identity (99.4% and 98.6%, respectively) with A. centrale (Table 2). In this study, the presence of A. bovis, A. phagocytophilum and A. centrale, as well as ‘Ca. M. mitochondrii’ and Ehrlichia sp. closely related to E. ewingii, is reported in ticks from La Rioja (northern Spain). This is the first evidence of the presence of A. bovis in Spain and also in ticks in Europe. Previously, in Europe A. bovis had been detected in blood samples from livestock in Italy (Ceci et al., 2014). In Japan, A. bovis was detected in Ixodes turdus ticks removed from migratory birds [Yellow-browed Bunting (Emberiza chrysophrys)] (Kang
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
EU781706 (100%) EU781706 (100%) JN181071 (99.8%) ND ND JN181071 (100%) AB211164 (99.4%) AB211164 (99.6%) Jubera Jubera Jubera Jubera Tobía Jubera B. taurus B. taurus B. taurus B. taurus Vegetation B. taurus A. centrale (2)
Sta. Eulalia T. merula
I. frontalis (1 L) I. ricinus (1 N) H. punctata (1 M) H. punctata (1 M) H. punctata (1 M) H. punctata (1 F) H. punctata (1 M) H. punctata (1 M)
ND EU781706 (100%) I. frontalis (1 F) I. frontalis (1 L) Sta. Eulalia Sta. Eulalia S. atricapilla E. rubecula
A. phagocytophilum (9)
∗Sequences corresponding to bacteria species other than those shown in the table: HF568843 and CP002130, ‘Candidatus Midichloria mitochondrii’; HQ697589, Ehrlichia sp. A., Anaplasma; B., Bos; E., Erithacus; H., Haemaphysalis; I., Ixodes; L., Luscinia; ND, not detected; S., Sylvia; T., Turdus.
ND ND HQ697589∗(100%) ND JN181071 (99.4%) JN181071 (99.4%) AF283007 (98.6%) AF283007 (98.6%) ND ND ND ND ND ND ND AB211164 (100%) JQ669948 (100%) ND EU921902 (100%) EU921902 (100%) EU921902 (100%) ND ND ND
ND EU781706 (100%) ND CP006616 (99.7%) JQ669948 (100%) JQ669948 (100%)
KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) HF568843∗(99.3%) ND ND ND JQ669948 (100%)
ND ND ND KF031393 (99.9%) ND KF031393 (100%) ND ND ND ND ND ND ND ND KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) EU781706 (100%) H. punctata (1 L) H. punctata (1 N) H. punctata (2 N,1 M) I. frontalis (1 N) Sta. Eulalia Sta. Eulalia Villalba de Rioja Sta. Eulalia T. merula L. megarhynchos Vegetation L. megarhynchos A. bovis (5)
16S rRNA EHR msp2 groESL 16S rRNA
GenBank accession no. (% identity)
Tick species (n, stage) Area Origin Microorganisms (positive samples, n)
Table 2. Anaplasma species detected in this study.
KJ659040 (100%) KJ659040 (100%) KJ659040 (100%) CP002130∗(99.1%)
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16S rRNA GEP
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et al., 2013). Moreover, A. bovis has been detected in Haemaphysalis spp., including Haemaphysalis coccina and Haemaphysalis longicornis, from Asia (Kawahara et al., 2006; Rar & Golovljova, 2011), but has not previously been reported from H. punctata. Anaplasma bovis infection may be asymptomatic in ruminants, but it may also cause a wide variety of clinical symptoms including debility, fever, diarrhoea, weight loss and even death (Rar & Golovljova, 2011). In the present study, A. bovis was found in ticks removed from migratory birds, including a presaharian Common Blackbird and a transaharian Common Nightingale. These birds are known to migrate from Central Europe to reach the North of Spain in autumn. In spring, the Common Blackbird migrating from North Africa and the Common Nightingale migrating from West and Central Africa also arrive in the study area. There are two possible explanations for the presence of A. bovis in ticks removed from migratory birds. Firstly, the presence of A. bovis and H. punctata in Africa (Rar & Golovljova, 2011) supports the proposal that this bacterium may be imported from Africa to Spain by migratory birds. Secondly, H. punctata is a common tick in the studied area and A. bovis may be established there. The finding of A. bovis in three specimens of H. punctata collected from vegetation in an area 74 km west of the bird banding location supports the second hypothesis. To date, neither A. phagocytophilum nor ‘Ca. M. mitochondrii’ have been reported previously in I. frontalis ticks. ‘Candidatus M. mitochondrii’ is an endosymbiotic intramitochondrial alphaproteobacterium associated with a wide range of arthropod hosts that include hard ticks. Migratory birds carrying I. ricinus infected with A. phagocytophilum have been reported close to the study area (Palomar et al., 2012). Anaplasma phagocytophilum has been amplified from birds in Spain (De La Fuente et al., 2005), although the role of birds as reservoirs of A. phagocytophilum has not been demonstrated. In addition, in the present study A. phagocytophilum was detected in H. punctata from cattle. Previous examination of I. ricinus removed from the same cattle showed a high prevalence of A. phagocytophilum infection (Palomar et al., 2014). The A. phagocytophilum 16S rRNA sequences obtained in the present study from H. punctata shared 100% identity with the most prevalent strain found in I. ricinus (Palomar et al., 2014), and were also identical to A. phagocytophilum from I. ricinus removed from birds in Norway (GenBank accession no. JN181071). Anaplasma phagocytophilum was previously detected in H. punctata in Spain (Barandika et al., 2008), although the role of this tick species as a vector has not been demonstrated. The GEP region of the 16S rRNA amplified from one of the A. phagocytophilum-positive male H. punctata from cattle was homologous (100% identical to HQ69758) to an Ehrlichia sp. closely related to E. ewingii originally detected in H. longicornis from Japan (Matsumoto et al., 2011). These results indicate that this tick was co-infected with A. phagocytophilum and an Ehrlichia sp., although further investigation should be performed to determine the identity of the Ehrlichia sample. In the present study, the analysis of msp2 gene and 16S rRNA sequences distinguishes two clusters of A. phagocytophilum strains: one cluster appears in Ixodes spp. collected from birds, and the other appears in H. punctata collected from cattle
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 29, 349–353
Detection of Anaplasma spp. in Spain 353 (Table 2). To date, neither of these clusters have been implicated as causative agents of human disease. Anaplasma centrale is a mildly pathogenic bacterium closely related to A. marginale (Rar & Golovljova, 2011). According to Rar & Golovljova (2011), the tick Rhipicephalus simus (Ixodida: Ixodidae) can transmit A. centrale, and H. longicornis has been suggested as a potential vector in Japan (Kawahara et al., 2006). As Table 2 demonstrates, the sequences of A. centrale obtained in the present study showed the highest level of identity with those isolated in Japan. The present study is the first report of A. centrale in H. punctata and provides the first evidence of this bacterium in ticks from Europe. Similar sequences were detected in deer blood samples from the same study area (Portillo et al., 2011). Therefore, it is necessary to be cautious in the serological diagnosis of anaplasmosis in cattle in Spain as cross-reactivity between A. centrale and A. marginale may lead to an inaccurate diagnosis of the causative agent. The detection of A. bovis, A. phagocytophilum and A. centrale in H. punctata specimens indicates the possible importance of this tick species in the epidemiology of these tick-borne pathogens. Further investigations should be performed to clarify the role of H. punctata as a potential vector of different Anaplasma species. The presence of A. bovis in Western Europe supports its implication in anaplasmosis of livestock in this region. Once again, the role of birds as dispersers of infected ticks is demonstrated.
Acknowledgements The first author was awarded a grant by the Fundación Rioja Salud (FRS/PIF-01/10). The authors thank Didier Raoult (Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France) and J. Stephen Dumler (The Johns Hopkins Hospital, Baltimore, MD, U.S.A) for supplying positive controls, Lourdes Romero (Center of Rickettsiosis and Arthropod-Borne Diseases, Hospital San Pedro-CIBIR, LA Rioja, Spain) for the provision of polymerase chain reaction validations, and Rufino Álamo (Territorial Health Service and Social Welfare of the Junta de Castilla y León, Valladolid, Spain) and Gerardo Domínguez (Slaughterhouse of Burgos, Burgos, Spain) for providing tick samples from Hacinas. The authors also wish to express their appreciation to Dr Lesley Bell-Sakyi (Pirbright Institute, Woking, U.K.) for reviewing the English language of this manuscript. References Barandika, J.F., Hurtado, A., García-Sanmartín, J., Juste, R.A., Anda, P. & García-Pérez, A.L. (2008) Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain. Vector-Borne and Zoonotic Diseases, 8, 829–835. Black, W.C. & Piesman, J. (1994) Phylogeny of hard and soft tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.
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