Maria Margarida Santos-Silva

Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe

Many factors are involved in determining the latitudinal and altitudinal spread of the important tick vector Ixodes ricinus (Acari: Ixodidae) in Europe, as well as in changes in the distribution within its prior endemic zones. This paper builds on published literature and unpublished expert opinion from the VBORNET network with the aim of reviewing the evidence for these changes in Europe and discusses the many climatic, ecological, landscape and anthropogenic drivers. These can be divided into those directly related to climatic change, contributing to an expansion in the tick’s geographic range at extremes of altitude in central Europe, and at extremes of latitude in Scandinavia; those related to changes in the distribution of tick hosts, particularly roe deer and other cervids; other ecological changes such as habitat connectivity and changes in land management; and finally, anthropogenically induced changes. These factors are strongly interlinked and often not well quantified. Although a change in climate plays an important role in certain geographic regions, for much of Europe it is non-climatic factors that are becoming increasingly important. How we manage habitats on a landscape scale, and the changes in the distribution and abundance of tick hosts are important considerations during our assessment and management of the public health risks associated with ticks and tick-borne disease issues in 21st century Europe. Better understanding and mapping of the spread of I. ricinus (and changes in its abundance) is, however, essential to assess the risk of the spread of infections transmitted by this vector species. Enhanced tick surveillance with harmonized approaches for comparison of data enabling the follow-up of trends at EU level will improve the messages on risk related to tick-borne diseases to policy makers, other stake holders and to the general public.

The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens

This work is an updated revision of the available information on Portuguese ixodid tick species. It includes data on tick biology, ecology, taxonomy and host/pathogen-associations. The current list of Portuguese ixodid ticks comprises twenty species: Dermacentor marginatus (Sulzer, 1776), Dermacentor reticulatus (Fabricius, 1794), Haemaphysalis hispanica Gil Collado, 1938, Haemaphysalis inermis Birula, 1895, Haemaphysalis punctata Canestrini & Fanzago, 1878, Hyalomma lusitanicum Koch, 1844, Hyalomma marginatum Koch, 1844, Ixodes acuminatus Neumann, 1901, Ixodes bivari Dias, 1990, Ixodes canisuga Johnston, 1849, Ixodes frontalis (Panzer, 1798), Ixodes hexagonus Leach, 1815, Ixodes ricinus (Linnaeus, 1758), Ixodes simplex Neumann, 1906, Ixodes ventalloi Gil Collado, 1936, Ixodes vespertilionis Koch, 1844, Rhipicephalus (Boophilus) annulatus (Say, 1821), Rhipicephalus bursa Canestrini & Fanzago, 1878, Rhipicephalus pusillus Gil Collado, 1938, and Rhipicephalus sanguineus (Latreille, 1806).

Ticks Parasitizing Wild Birds in Portugal: Detection of Rickettsia aeschlimannii, R. helvetica and R. massiliae

From January 2002 to December 2004, 152 ticks were collected from 40 wild birds recovered in Santo André Natural Reserve and Monsanto Forestal Park, Portugal mainland. Five ticks species were identified from 22 species of birds, and new host record were provided for some species. In addition, 32 (21%) ticks were screened by PCR to detect infections with agents belonging to order Rickettsiales: Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Rickettsia spp. PCR amplicons were obtained in 5 (15.6%) tick samples. Rickettsia DNA exhibiting gltA sequences similar to those of Rickettsia aeschilimannii, R. helvetica and R. massiliae were identified in Hyalomma marginatum, Ixodes ventalloi and in Rhipicephalus turanicus, respectively. This is the first report of rickettsiae infections in ticks collected from wild birds in Portugal. Giving the results presented above wild birds play an important role in the maintenance and dissemination of several tick species and associated rickettsiae.

Ultrastructural study of the infection process of Rickettsia conorii in the salivary glands of the vector tick Rhipicephalus sanguineus.

This work was designed to study the infection process of Rickettsia conorii in the salivary glands of experimentally infected Rhipicephalus sanguineus ticks. One hundred six uninfected engorged nymphs were intracelomically inoculated with approximately 2 x 10(3) plaque-forming units of a rickettsial suspension. After the molt, unfed and fed adults were dissected, and the salivary glands were extracted and processed for transmission electron microscopy observation. Three different uninfected control groups were used for (1) evaluating the impact of the inoculation procedure, (2) establishing the feeding period of infected ticks, and (3) ultrastructural characterization of the salivary glands. Overall, 75.5% (80 of 106) of the nymphs inoculated with rickettsiae died during the molt or soon after hatching into adult instars; 50% (12 of 24) of the remaining infected adults showed severe malformations compromising their viability. In apparently healthy specimens, time of engorgement was longer. The contrast with the negative control groups was statistically significant, suggesting that R. conorii exerts a strong negative effect on the vector ticks. The ultrastructural study showed that in the salivary glands of infected ticks, rickettsial growth occurs preferentially in central, peripheral, and interstitial acini cells.

Evidence of Bartonella spp., Rickettsia spp. and Anaplasma phagocytophilum in domestic, shelter and stray cat blood and fleas, Portugal

Cats are reservoirs of several infectious agents and potential sources of infection to humans. Examples of these are B. henselae and B. clarridgeiae, agents of cat scratch disease (CSD). The transmission occurs mainly by the scratch of contaminated cat claws. However, the possibility of direct transmission by cat fleas should not be excluded. Moreover, it is known that the presence of cat fleas (Ctenocephalides felis) is essential for the maintenance of the infection within cat populations. Cats may also be involved in the maintenance cycle of other flea-borne agents such as Rickettsia felis that cause human disease. To our knowledge no previous studies have been performed to detect the presence of Bartonella spp., R. felis and A. phagocytophilum in Portuguese cat fleas. This study also evaluated the prevalence of antibodies against Bartonella spp., Rickettsia spp. and A. phagocytophilum and the detection of Bartonella bacteraemia by PCR in cat blood.

Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in Portugal.

Ticks are obligatory blood-sucking arthropod (Acari:Ixodida) ectoparasites of domestic and wild animals as well as humans. The incidence of tick-borne diseases is rising worldwide, challenging our approach toward diagnosis, treatment and control options. Rhipicephalus bursa Canestrini and Fanzago, 1877, a two-host tick widely distributed in the Palearctic Mediterranean region, is considered a multi-host tick that can be commonly found on sheep, goats and cattle, and occasionally on horses, dogs, deer and humans. R. bursa is a species involved in the transmission of several tick-borne pathogens with a known impact on animal health and production. The aim of this study was to estimate R. bursa prevalence in Portugal Mainland and circulating pathogens in order to contribute to a better knowledge of the impact of this tick species. Anaplasma marginale and Theileria spp. were detected and classified using phylogenetic analysis. This is the first report of Theileria annulata and Theileria equi detection in R. bursa ticks feeding on cattle and horses, respectively, in Portugal. This study contributes toward the identification of currently circulating pathogens in this tick species as a prerequisite for developing future effective anti-tick control measures.

Importation of a Hyalomma lusitanicum tick into the UK on a dog

A SINGLE, fully engorged female Hyalomma lusitanicum tick was imported into the UK on a dog recently returned from Portugal during May 2016 and submitted to Public Health Englands Tick Surveillance Scheme (TSS). H lusitanicum exists in a narrow geographical range from Portugal to Sicily (Estrada-Pena and others 2012), where its distribution is restricted due to its strict biotic niche (Estrada-Pena and Venzal 2007). Immature stages parasitise wildlife (Santos-Silva and others 2011) and adults are mostly associated with domestic and wild ungulates (Valcarcel and others 2015). However, records on dogs have been reported (Apanaskevich and others 2008), including in Portugal where biting of humans by this species has also been reported (Santos-Silva and …

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.

Anaplasma marginale and Theileria annulata in questing ticks from Portugal

Ticks are ubiquitous arthropods and vectors of several pathogenic agents in animals and humans. Monitoring questing ticks is of great importance to ascertain the occurrence of pathogens and the potential vector species, offering an insight into the risk of disease transmission in a given area. In this study 428 host-seeking ticks, belonging to nine species of Ixodidae and collected from 17 of the 23 Portuguese mainland subregions, were screened for several tick-borne agents with veterinary relevance: Anaplasmamarginale, Anaplasma ovis, Anaplasma centrale, Babesia spp., Coxiella burnetii and Theileria spp. Prevalence was assessed by PCR and amplified amplicons sequenced for validation of results. Twenty ticks, in a total of 428, were found positive: one Ixodes ventalloi for Theileria annulata and four Dermacentor marginatus, one Haemaphysalis punctata, five Ixodes ricinus, five I. ventalloi, and four Rhipicephalus sanguineus sensu lato for A. marginale. According to the reviewed literature, this is the first report of A. marginale and T. annulata detection in I. ventalloi. Furthermore, the amplification of A. marginale DNA in several tick species suggests a broad range for this agent in Portugal that might include other uncommon species as R. sanguineus s.l. This work provides new data towards a better understanding of tick-pathogen associations and also contributes to the surveillance of tick-borne agents in geographic areas with limited information.

Detection of antibodies against Anaplasma phagocytophilum in Algerian mice (Mus spretus), Portugal.

The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agents enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p > 0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.