'Candidatus Neoehrlichia mikurensis' in Europe.

'Candidatus Neoehrlichia mikurensis' is an uncultured emerging bacterium that is provisionally included in the family Anaplasmataceae. In Europe, it is transmitted by Ixodes ricinus ticks. Rodents are the reservoirs. It is widely distributed in mammals (both wild and domestic) and birds. It causes an inflammatory disease in humans with underlying diseases, but the microorganism also affects immunocompetent individuals in which asymptomatic infection has been recognized. A high degree of suspicion and the use of molecular tools are needed for the correct diagnosis. Efforts to cultivate it and to investigate its pathogenesis should be a priority.

Isolation of Rickettsia amblyommatis in HUVEC line.

Rickettsia amblyommatis, formerly named Rickettsia amblyommii and 'Candidatus Rickettsia amblyommii' is an intracellular bacterium belonging to the spotted fever group Rickettsia. It is highly prevalent in Amblyomma americanum and in other Amblyomma spp. throughout the Western Hemisphere. R. amblyommatis has been cultivated in chicken fibroblast, primary embryonated chicken eggs, Vero cells and arthropod-derived cells. Because of the affinity of rickettsiae to invade vascular endothelial cells, we tried to isolate R. amblyommatis from a nymph of Amblyomma cajennense s.l. collected in Saltillo (Coahulia, Mexico) using human umbilical vein endothelial cells (HUVEC). One tick half was analysed by ompA PCR and was found to be positive for R. amblyommatis. The other half was selected for in vitro culture of Rickettsia spp. It was triturated in 1 mL of endothelial cell growth medium with 1% antibiotic-antimycotic solution, and the homogenate was inoculated into a HUVEC line. Culture was maintained at 33°C in endothelial cell growth medium plus 2 mM l-glutamine and 2% fetal calf serum, with 5% CO2. The medium was changed weekly. Culture was checked by Gimenez stain for Rickettsia-like intracellular organisms. After 48 days of incubation, Rickettsia-like organisms were observed in HUVEC. PCR assays and sequencing of ompA gene in the culture suspension showed 100% identity with R. amblyommatis. This isolate was successfully established in HUVEC, and it has been deposited in the collection of the Center of Rickettsioses and Arthropod-Borne Diseases, Infectious Diseases Department, Hospital San Pedro-Center of Biomedical Research from La Rioja, Logroño, Spain. The HUVEC line is a useful tool for the isolation of R. amblyommatis.

A comparative test of ixodid tick identification by a network of European researchers.

This study reports the results of a comparative test of identification of ticks occurring in Western Europe and Northern Africa. A total of 14 laboratories were voluntarily enrolled in the test. Each participant received between 22 and 25 specimens of adult and nymphal ticks of 11 species: Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hy. marginatum, Ixodes ricinus, I. hexagonus, Rhipicephalus annulatus, R. bursa, R. rossicus, and/or R. sanguineus s.l. Ticks were morphologically identified by three of the co-authors and the identification confirmed by a fourth co-author who used molecular methods based on several genes. Then ticks were randomly selected and blindly distributed among participants, together with a questionnaire. Only specimens collected while questing and, if possible, in the same survey, were circulated. Because of the random nature of the test, a participant could receive several specimens of the same species. Species in the different genera had variable misidentification rates (MR) of 7% (Dermacentor), 14% (Ixodes), 19% (Haemaphysalis), 36% (Hyalomma), and 54% (Rhipicephalus). Within genera, the MR was also variable ranging from 5.4% for I. ricinus or 7.4% for D. marginatus or D. reticulatus to 100% for R. rossicus. The test provided a total misidentification rate of 29.6% of the species of ticks. There are no significant differences in MR according to the sex of the tick. Participants were requested to perform a second round of identifications on the same set of ticks, using only purposely prepared keys (without illustrations), circulated to the enrolled participants, including 2 species of the genus Dermacentor, 8 of Haemaphysalis, 10 of Hyalomma, 23 of Ixodes, and 6 of Rhipicephalus. The average MR in the second round was 28%: 0% (Dermacentor), 33% (Haemaphysalis), 30% (Hyalomma) 18% (Ixodes), and 50% (Rhipicephalus). Species which are not reported in the countries of a participating laboratory had always highest MR, i.e. purely Mediterranean species had highest MR by laboratories in Central and Northern Europe. Participants expressed their concerns about a correct identification for almost 50% of the ticks of the genera Hyalomma and Rhipicephalus. The results revealed less than total confidence in identifying the most prominent species of ticks in the Western Palearctic, and underpin the need for reference libraries for specialists involved in this task. Results also showed that a combination of certain genes may adequately identify the target species of ticks.

Detection of tick-borne Anaplasma bovis, Anaplasma phagocytophilum and Anaplasma centrale in Spain.

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.

Septic shock in a patient infected with Rickettsia sibirica mongolitimonae, Spain.

In 1996, the first human case of infection by Rickettsia sibirica subsp. mongolitimonae was described in France. Subsequently, other human cases were reported in the same country. The acronym LAR (lymphangitis-associated rickettsiosis) has been proposed to designate this disease because lymphangitis is one of the main clinical manifestations. Later, a few more cases were described in Portugal, South Africa, Egypt, Greece and Spain. We report a case of R. sibirica mongolitimonae infection as a cause of septic shock in a Spanish patient living in La Rioja (northern Spain). In addition, the broad clinical spectrum of this tick-borne disease is discussed.