Ana M. Palomar

Crimean-Congo Hemorrhagic Fever Virus in Ticks, Southwestern Europe, 2010

To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV; family Bunyaviridae, genus Nairovirus) causes outbreaks of severe hemorrhagic fever in humans, with case-fatality rates <30% (1,2). The disease was initially recognized by Russian scientists in the 1940s (3), and the virus was first isolated in the Democratic Republic of Congo some years later (4). CCHFV is reported throughout broad regions of Africa, Europe, the Middle East, and Asia. Reports linking transmission of the virus with an infected vector have involved ticks of the genus Hyalomma (5). It appears that maintenance of active foci of CCHFV in the field is dependent on Hyalomma spp., even within periods of silent activity. Several vertebrates are involved in the natural transmission cycle (6). Transmission of CCHFV to humans occurs through tick bites, direct contact with blood or tissues of infected animals, person-to-person spread, or by nosocomial infection (1). In southeastern Europe, the Balkans are the known western limit for CCHFV (7). This finding is of special interest because Hyalomma marginatum, the main tick vector in the western Paleartic (an ecozone that includes temperate and cold areas of Eurasia and North Africa and several archipelagos and islands in the Atlantic and Pacific Oceans), is common throughout the Mediterranean Basin (7), where clinical cases of the disease or the virus have not been reported. Unsupported claims of the effects of climate on virus distribution have been reported but never empirically demonstrated (8). We report the detection of CCHFV in ticks collected in southwestern Europe. A total of 117 semi-engorged adult H. lusitanicum ticks were collected from 28 adult red deer (Cervus elaphus) in November 2010, at a site (39.63°N, 7.33°W) in Caceres, Spain. Live ticks were transported to the special pathogens laboratory at Hospital San Pedro–CIBIR in Logrono (northern Spain), classified, and frozen at −80°C. For RNA extraction, specimens were washed in 70% ethanol and then in Milli-Q water (Milli-Q Advantage water system; Millipore Iberica, S.A., Madrid, Spain) that had been autoclaved. Each tick was cut lengthwise; half was used for additional processing and the remainder was stored. Before use, each half was crushed in sterile conditions. RNA was individually extracted by using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and frozen at −80°C. The RNA was distributed in 12 pools and retrotranscribed by using the Omniscript RT kit (QIAGEN) according to the manufacturer’s instructions and then frozen at −20°C. Nested PCRs were performed by using specific primers for the small segment of CCHFV as described (9). Negative controls (with template DNA but without primers and with primers and containing water instead of template DNA) were included in all assays. For the second round of PCRs, 2 of 12 pools showed amplicons of the expected size (211 bp). Only 1 amplicon could be sequenced. MEGA5 (www.megasoftware.net) was used to compare the sequence with representative small segment sequences of CCHFV available in GenBank (Figure). (Aligned sequences are available from the authors.) Pools of cDNA were submitted to the Spanish National Center of Microbiology (Madrid), where results were confirmed. The CCHFV sequence we report showed 98% genetic similarity (204/209 bp) with sequences recorded for CCHFV in Mauritania and Senegal, on the western coast of Africa. Figure Evolutionary relationships of Crimean-Congo hemorrhagic fever virus strains from Spain and other representative sites. Evolutionary history was inferred by using the unweighted pair group method with arithmetic mean. The optimal tree is shown (sum of ... This finding suggests the circulation of CCHFV in southwestern Europe. The close affinity of the strain from Spain with strains circulating in western Africa and the lack of similarity with isolates from eastern Europe suggest the introduction of this virus from nearby countries of northern Africa. Migratory movements of birds could explain the presence of the virus in southwestern Europe because birds are common hosts of immature H. marginatum, which was reportedly introduced into Europe through annual migratory flights along the western coast of Africa (10). Because of the lack of genetic similarities among virus strains, trade movements of domestic or wild ungulates from eastern Europe do not support our finding. We cannot state whether this virus was circulating previously or if other strains are present in the area because CCHFV detection in the western Mediterranean region has not been previously addressed. H. lusitanicum ticks exist as relatively isolated populations in a narrow strip from Sicily to Portugal. The Mediterranean rabbit and ungulates, the main hosts for immature and adult H. lusitanicum ticks, respectively, are residents of the collection area; however, the movement of these animals through trade has not occurred for several years. Thus, H. lusitanicum ticks could not serve as a spreading vector in the western Mediterranean region. The CCHFV strain from southwestern Europe has been found in ticks restricted to hosts that cannot spread long distances. Therefore, although it would be unlikely, given the strain’s similarity with CCHFV isolates from Senegal and Mauritania, we should not exclude the possibility of an ancient existence for this strain. Additional data collected in the Mediterranean Basin are necessary to establish the actual range of CCHFV.

Role of Birds in Dispersal of Etiologic Agents of Tick-borne Zoonoses, Spain, 2009

We amplified gene sequences from Anaplasma phagocytophilum, Borrelia garinii, B. valaisiana, B. turdi, Rickettsia monacensis, R. helvetica, R. sibirica sibirica, and Rickettsia spp. (including Candidatus Rickettsia vini) in ticks removed from birds in Spain. The findings support the role of passerine birds as possible dispersers of these tick-borne pathogens.

Crimean-Congo Hemorrhagic Fever Virus in Ticks from Migratory Birds, Morocco

Crimean-Congo hemorrhagic fever virus was detected in ticks removed from migratory birds in Morocco. This finding demonstrates the circulation of this virus in northwestern Africa and supports the hypothesis that the virus can be introduced into Europe by infected ticks transported from Africa by migratory birds.

Rickettsia Species in Ticks Removed from Humans in Istanbul, Turkey

A total of 167 ticks collected from humans in Istanbul (Turkey) in 2006 were screened for Rickettsia species, and nested PCRs targeting gltA and ompA rickettsial fragment genes were carried out. Rickettsia monacensis (51), R. aeschlimannii (8), R. conorii subsp. conorii (3), R. helvetica (2), R. raoultii (1), R. africae (1), R. felis (1), and other Rickettsia spp. (2), were detected. To our knowledge, these Rickettsia species (except R. conorii) had never been reported in ticks removed from humans in Turkey. The presence of R. africae also had not been previously described, either in Hyalomma ticks or in any European tick species. In addition, R. aeschlimannii and R. felis had not been found associated with Rhipicephalus bursa specimens. The presence of human pathogenic Rickettsia in ticks removed from humans provides information about the risk of tick-borne rickettsioses in Turkey.

Anaplasma spp. in Wild Mammals and Ixodes ricinus from the North of Spain

Our objectives were to investigate the presence of Anaplasma spp. infection in red deer, wild boars, and Ixodes ricinus removed from deer surveyed in La Rioja, as well as to analyze the presence of Anaplasma spp. in I. ricinus from different Spanish regions--ours included. A total of 21 deer and 13 wild boar blood samples as well as 295 I. ricinus removed from deer, vegetation, or asymptomatic people were tested by polymerase chain reaction targeting Anaplasma spp. 16S rRNA gene and groESL heat shock operon. Twelve deer blood samples were found to be infected with Anaplasma centrale (n = 7) or Anaplasma phagocytophilum (n = 5). No wild boar blood samples gave positive polymerase chain reaction results. Further, A. phagocytophilum was detected in 12 out of 89 I. ricinus removed from deer and in 18 out of 168 I. ricinus collected over vegetation in the North of Spain. Anaplasma spp. was not detected in any of the 38 I. ricinus removed from people. Nucleotide sequences for 16S rRNA gene showed substancial heterogeneity. The etiological agent of human anaplasmosis was found in two deer blood samples, an adult tick from deer, and a nymph from vegetation. The 16S rRNA sequences for 12 out of 35 samples matched the sequence of the Ap-variant 1 strain previously described in the United States, and the remaining 19 positive samples (deer blood and I. ricinus) showed variations with unknown significance. Although the groEL DNA sequences varied among analyzed strains, the deduced amino acid sequences did not change for any of them. This study suggests that deer population from La Rioja harbors strains of A. phagocytophilum similar to that pathogen for humans and other of unknown pathogenicity. Further, it seems that the Ap-variant 1 is circulating among I. ricinus ticks from the North of Spain more frequently than the A. phagocytophilum strain associated to human anaplasmosis.

Genetic characterization of Candidatus Rickettsia vini, a new rickettsia amplified in ticks from La Rioja, Spain

A total of 222 ticks removed from birds in La Rioja (Spain) were screened for spotted fever group rickettsia species using ompA PCR assays. Rickettsia monacensis (n=1) and R. sibirica (n=1) were detected. Apart from that, 27 out of 29 Ixodes spp. DNA extracts that tested positive for ompA did not match with any validated spotted fever group rickettsia. Multilocus sequence typing for 16S rRNA, gltA, ompB, sca4, and 17-kDa antigen genes was performed, and R. heilongjiangensis was found to be the nearest validly published spotted fever group rickettsia. Based on genetic criteria agreed by experts, this genotype can be classified as a new Candidatus Rickettsia sp. and was named Candidatus Rickettsia vini.

Detection of tick-borne ‘Candidatus Neoehrlichia mikurensis’ and Anaplasma phagocytophilum in Spain in 2013

Background‘Candidatus Neoehrlichia mikurensis’ is a tick-borne bacteria implicated in human health. To date, ‘Ca. Neoehrlichia mikurensis’ has been described in different countries from Africa, Asia and Europe, but never in Spain. However, according to the epidemiological features of the main vector in Europe, Ixodes ricinus, its circulation in our country was suspected.MethodsA total of 200 I. ricinus ticks collected in the North of Spain were analyzed. DNAs were extracted and used as templates for PCRs targeting fragment genes for Anaplasma/Ehrlichia detection. The amplified products were sequenced and analyzed.Results‘Ca. Neoehrlichia mikurensis’ was amplified in two specimens. Furthermore, Anaplasma phagocytophilum was detected in 61 samples analyzed.ConclusionsThe detection of ‘Ca. Neoehrlichia mikurensis’ in I. ricinus ticks from Spain indicates its circulation and the potential risk of contracting a human infection in this country.

Investigation of tick-borne bacteria (Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Borrelia spp.) in ticks collected from Andean tapirs, cattle and vegetation from a protected area in Ecuador

BackgroundIxodid ticks play an important role in the transmission and ecology of infectious diseases. Information about the circulation of tick-borne bacteria in ticks is lacking in Ecuador. Our aims were to investigate the tick species that parasitize Andean tapirs and cattle, and those present in the vegetation from the buffer zone of the Antisana Ecological Reserve and Cayambe-Coca National Park (Ecuador), and to investigate the presence of tick-borne bacteria.MethodsTick species were identified based on morphologic and genetic criteria. Detection of tick-borne bacteria belonging to Rickettsia, Anaplasma, Ehrlichia and Borrelia genera was performed by PCRs.ResultsOur ticks included 91 Amblyomma multipunctum, 4 Amblyomma spp., 60 Rhipicephalus microplus, 5 Ixodes spp. and 1 Ixodes boliviensis. A potential Candidatus Rickettsia species closest to Rickettsia monacensis and Rickettsia tamurae (designated Rickettsia sp. 12G1) was detected in 3 R. microplus (3/57, 5.3%). In addition, Anaplasma spp., assigned at least to Anaplasma phagocytophilum (or closely related genotypes) and Anaplasma marginale, were found in 2 A. multipunctum (2/87, 2.3%) and 13 R. microplus (13/57, 22.8%).ConclusionsThis is the first description of Rickettsia sp. in ticks from Ecuador, and the analyses of sequences suggest the presence of a potential novel Rickettsia species. Ecuadorian ticks from Andear tapirs, cattle and vegetation belonging to Amblyomma and Rhipicephalus genera were infected with Anaplasmataceae. Ehrlichia spp. and Borrelia burgdorferi sensu lato were not found in any ticks.

Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines.

Highlights • First in vitro culture system for Anaplasma centrale.• A. centrale infected and grew in two out of 32 tick cell lines tested.• Potential for safer and more ethical bovine anaplasmosis vaccine.

Neotrombicula inopinata (Acari: Trombiculidae) - a possible causative agent of trombiculiasis in Europe

BackgroundFor over a decade, the presence of trombiculid mites in some mountain areas of La Rioja (Northern Spain) and their association with seasonal human dermatitis have been recognized. This work aimed to establish the species identity of the agent causing trombiculiasis in the study area.MethodsTrombiculid larvae (chigger mites) were collected from vegetation in the Sierra Cebollera Natural Park and in Sierra La Hez during an outbreak of human trombiculiasis in 2010. Three specimens collected from a bird were also examined. Identification was made using morphological and morphometric traits based on the most recent taxonomic sources. A comparison of those mites with specimens of the same species collected throughout Europe was performed by means of cluster analysis with multiscale bootstrap resampling and calculation of approximately unbiased p-values.ResultsAll collected mites were identified as Neotrombicula inopinata (Oudemans, 1909). Therefore, this species is the most likely causative agent of trombiculiasis in Spain, not Neotrombicula autumnalis (Shaw, 1790), as it was generally assumed. No chigger was identified as N. autumnalis in the study area. Neotrombicula inopinata clearly differs from N. autumnalis in the presence of eight or more setae in the 1st and 2nd rows of dorsal idiosomal setae vs. six setae in N. autumnalis. Comparison of N. inopinata samples from different locations shows significant geographic variability in morphometric traits. Samples from Western and Eastern Europe and the Caucasus formed three separate clusters.ConclusionSince the taxonomical basis of many studies concerning N. autumnalis as a causative agent of trombiculiasis is insufficient, it is highly possible that N. inopinata may be hiding behind the common name of “harvest bug” in Europe, together with N. autumnalis.