Isabel Lopes de Carvalho

Coinfections of Rickettsia slovaca and Rickettsia helvetica with Borrelia lusitaniae in ticks collected in a Safari Park, Portugal.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff.

Francisella-Like Endosymbiont in Dermacentor reticulatus Collected in Portugal

In Portugal, recent studies have confirmed the presence of Francisella tularensis in Dermacentor reticulatus. Bacterial endosymbionts with significant homology to F. tularensis have been described in several species of ticks. In this work we identified Francisella-like endosymbionts in D. reticulatus ticks (39%), confirming the presence of these bacteria in Portugal. This finding should be considered in future studies using molecular approaches to detect Francisella prevalence in ticks and environmental samples.

Francisella tularensis, Portugal

To the Editor: Tularemia is a zoonosis caused by Francisella tularensis. Recently, tularemia has emerged in new locations, populations, and settings (1). After an outbreak in Spain in 1997 (2), it was expected that the disease would spread toward Portugal, a country with an extended area that borders the affected areas.

Blackbirds Turdus merula as competent reservoirs for Borrelia turdi and Borrelia valaisiana in Portugal: evidence from a xenodiagnostic experiment

To confirm that thrushes, such as blackbirds Turdus merula, play a role as reservoir for some Borrelia genospecies, we performed a xenodiagnostic experiment with blackbirds captured in a mixed wood located in Western Portugal where Borrelia turdi, an uncommon genospecies in Europe, was the most prevalent genospecies associated with birds. Two out of five birds harboured B. turdi infected Ixodes frontalis at the time of capture. Four out of five birds transmitted spirochaetes to Ixodes ricinus xenodiagnostic ticks: two birds transmitted Borrelia valaisiana to 25.7% and 10.5% of ticks, and two transmitted B. turdi to 6.4% and 5.4% of ticks. Our results showed that blackbirds transmit B. valaisiana and B. turdi to I. ricinus feeding larvae, acting as reservoir hosts for these genospecies in nature.

Detection of Borrelia lusitaniae, Rickettsia sp. IRS3, Rickettsia monacensis, and Anaplasma phagocytophilum in Ixodes ricinus collected in Madeira Island, Portugal.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.

Rickettsia lusitaniae sp. nov. isolated from the soft tick Ornithodoros erraticus (Acarina: Argasidae)

In this study a novel Rickettsia from the spotted fever group, isolated from Ornithodoros erraticus soft ticks collected from pigpens in the south of Portugal, is described. After initial screening revealed Rickettsia-positive ticks, isolation attempts were then performed. Successful isolates were achieved by shell-vial technique using Vero E6 cells at 28°C. Molecular characterization of the isolate was performed based on analysis of five rickettsial genes gltA, ompA, ompB, sca1 and htr with their subsequent concatenation along with other rickettsial species resulting in a clustering of the new isolate with Rickettsia felis and Rickettsia hoogstraalii. The degree of nucleotide sequence similarity with other rickettsiae fulfills the criteria for classification of our isolate as a novel species. The name Rickettsia lusitaniae sp. nov. (=CEVDI PoTiRo) is proposed for this new species found in O. erraticus.

The medical and veterinary role of Ornithodoros erraticus complex ticks (Acari: Ixodida) on the Iberian Peninsula

ABSTRACT: Argasid ticks of the Ornithodoros erraticus complex are associated with traditional pig-farming practices on the Iberian Peninsula and are also found elsewhere in North Africa, West Africa, and western Asia. The ticks associated with pig farming on the Iberian Peninsula are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe, and their ecology makes them an extremely effective reservoir of both ASFV and the Borrelia species which cause tick-borne relapsing fever (TBRF) in humans. The recent reappearance of ASFV in the European Union, coupled with evidence that Portuguese tick populations continue to harbor Borrelia despite a lack of confirmed human infections, suggest that these populations merit closer attention. In Portugal, a series of surveys over the last twenty-five years indicates that the number of farm sites with tick infestations has declined and suggest that populations are sensitive to changes in farm management, particularly the use of modern pig housing. Various technologies have been suggested for the control of farm-associated Ornithodoros ticks and related species but, in our opinion, farm management changes are still the most effective strategy for population control. Furthermore, we suggest that this species could probably be eradicated from Iberian pig farms.

Molecular Characterization of a New Isolate of Borrelia lusitaniae Derived from Apodemus sylvaticus in Portugal

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

The importance of lizards and small mammals as reservoirs for Borrelia lusitaniae in Portugal

Borrelia lusitaniae is a pathogen frequent in the Mediterranean area. Apart from lizards, evidence for birds and small mammals as competent reservoirs for this genospecies has been occasional. We collected questing ticks, skin biopsies and Ixodes sp. ticks feeding on lizards, birds and small mammals in a B. burgdorferi s.l. (sensu lato) enzootic area to assess their importance in the maintenance of B. lusitaniae. Borrelia lusitaniae was the most prevalent genospecies in questing ticks and was commonly found in larvae feeding on Psammodromus algirus. One biopsy infected with B. lusitaniae was collected from the tail of one Podarcis hispanica, which suggests systemic infection. Ixodes ricinus larvae feeding on Apodemus sylvaticus were infected with B. lusitaniae but with a lower prevalence. Our results reinforce the importance of lizards as reservoirs for B. lusitaniae, suggesting that P. algirus, in particular, acts as main reservoir for B. lusitaniae in Portugal.

Borrelia garinii and Francisella tularensis subsp. holarctica detected in migratory shorebirds in Portugal

Migratory shorebirds use, among many, the East Atlantic Flyway that links breeding areas as north as Tundra habitats to aquatic wintering grounds in West Africa. As a consequence, they are potentially important in the spread of global zoonotic diseases transmitted by ticks, such as Lyme borreliosis and tularemia—two diseases previously detected in Portugal. In this study, we looked at the infection status of seven populations of shorebirds during their migration, breeding, or wintering in the Portuguese wetlands to access if they carry these pathogens and to discuss their potential risk in the Portuguese wetlands. A total of 212 migratory shorebirds captured in the Tagus and Sado estuaries; key staging and wintering sites in this flyway and important breeding areas for some species were analyzed for the presence of Borrelia burgdorferi sensu lato and Francisella tularensis. In the present study, B. garinii was identified in seven (3%) specimens (five black-tailed godwits Limosa limosa, one common redshank Tringa totanus, and one little stint Calidris minuta), whereas F. tularensis subsp. holarctica was identified in one (0.4%) little stint. To our knowledge, this is the first evidence that shorebirds that migrate through or winter in Portugal transport these pathogens, potentially contributing for their introduction along the flyway, including the Mediterranean region.