Development of a tool for screening adverse food reactions and food allergy in Portuguese children

Introduction and objectives: A standardised questionnaire may be an excellent tool for epidemiological studies aiming at screening children with suspected food allergies. Thus, the aim of the present study was to develop a screening questionnaire for assessing children with suspected food allergy and to analyse its reproducibility. Materials and methods: A questionnaire of adverse food reactions was developed by literary review of similar questionnaires validated in other countries as well as less well defined, non-validated Portuguese questionnaires. Peer review of the questionnaire by a panel of specialists and subsequent exploratory analysis was carried out by applying the questionnaire in children with confirmed food allergy. Test-retest analysis was performed by giving a face-to-face questionnaire to 159 children with suspected adverse food reactions, aged between three and 11 years. Temporal stability using Spearman Rho correlation test and reproducibility was studied using Cohen's Kappa index. Results: 115 children confirmed adverse food reactions that occurred with one or more foods. Retest was given about three weeks after the test, to 50 of these children who were randomly selected. The questionnaire showed good temporal stability (Spearman correlation coefficient of 0.834), and good reproducibility (only two of the 27 items had a Kappa index < 0.60). Conclusions. This questionnaire showed good temporal stability and reproducibility. Its validation for screening children with suspected food allergy will allow a standardised approach to diagnosis and comparison of results obtained in different centres

No disponible

Apoptosis induced by synthetic compounds containing a 3,4,5-trimethoxyphenyl fragment against lymphoid immature neoplasms.

T-cell acute lymphoblastic leukemia is an aggressive hematological malignancy originating from the malignant transformation of progenitor T cells at different stages of development. The treatment causes severe adverse effects and is associated with relapses and high morbidity and mortality rates. The present study aimed to evaluate the cytotoxic activity of 28 new compounds containing 3,4,5-trimethoxyphenyl analogues on hematological neoplastic cells lines. Cytotoxicity screening by the MTT method revealed that compound 1d was the most promising. Cell viability of neoplastic cells decreased in a concentration- and time-dependent manner, with compound 1d not causing hemolysis or reducing peripheral blood mononuclear cells viability, suggesting a selective cytotoxicity. We also suggested that compound 1d induced apoptotic-like cell death with mitochondrial involvement in Jurkat cells.

Development of a tool for screening adverse food reactions and food allergy in Portuguese children.

INTRODUCTION AND OBJECTIVES: A standardised questionnaire may be an excellent tool for epidemiological studies aiming at screening children with suspected food allergies. Thus, the aim of the present study was to develop a screening questionnaire for assessing children with suspected food allergy and to analyse its reproducibility. MATERIALS AND METHODS: A questionnaire of adverse food reactions was developed by literary review of similar questionnaires validated in other countries as well as less well defined, non-validated Portuguese questionnaires. Peer review of the questionnaire by a panel of specialists and subsequent exploratory analysis was carried out by applying the questionnaire in children with confirmed food allergy. Test-retest analysis was performed by giving a face-to-face questionnaire to 159 children with suspected adverse food reactions, aged between three and 11 years. Temporal stability using Spearman Rho correlation test and reproducibility was studied using Cohen's Kappa index. RESULTS: 115 children confirmed adverse food reactions that occurred with one or more foods. Retest was given about three weeks after the test, to 50 of these children who were randomly selected. The questionnaire showed good temporal stability (Spearman correlation coefficient of 0.834), and good reproducibility (only two of the 27 items had a Kappa index <0.60). CONCLUSIONS: This questionnaire showed good temporal stability and reproducibility. Its validation for screening children with suspected food allergy will allow a standardised approach to diagnosis and comparison of results obtained in different centres.

Background levels of trace elements in brown and red seaweeds from Trindade, a remote island in South Atlantic Ocean.

Trace elements in organisms are normally higher in well-developed coastal areas than on oceanic islands. Few studies have used seaweeds as their sentinels on islands. This study established background levels of trace elements (As, Cd, Pb, Zn, Cu and Hg) for four seaweed species (Dictyopteris delicatula and Canistrocarpus cervicornis, brown algae; Ceratodictyon variabile and Palisada perforata, red algae) from Trindade, an oceanic Brazilian island, and verified potential differences associated to distinct environmental conditions. Spatial differences were not detected for As, Hg and Cd in samples, although the highest concentrations of these elements were observed in brown seaweeds. The highest Zn, Pb and Cu concentrations in seaweeds from the only inhabited beach may be a signal of the onset of human footprints on this still pristine, remote island. By comparison with background described in the literature, concentrations of trace elements in seaweeds were low, thus, allowing them to be considered reference levels.

Evaluation of leucocytes from sputum samples of pulmonary tuberculosis patients using flow cytometry.

SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.

Rhipicephalus bursa Sialotranscriptomic Response to Blood Feeding and Babesia ovis Infection: Identification of Candidate Protective Antigens.

Ticks are among the most prevalent blood-feeding arthropods, and they act as vectors and reservoirs for numerous pathogens. Sialotranscriptomic characterizations of tick responses to blood feeding and pathogen infections can offer new insights into the molecular interplay occurring at the tick-host-pathogen interface. In the present study, we aimed to identify and characterize Rhipicephalus bursa salivary gland (SG) genes that were differentially expressed in response to blood feeding and Babesia ovis infection. Our experimental approach consisted of RNA sequencing of SG from three different tick samples, fed-infected, fed-uninfected, and unfed-uninfected, for characterization and inter-comparison. Overall, 7,272 expressed sequence tags (ESTs) were constructed from unfed-uninfected, 13,819 ESTs from fed-uninfected, and 15,292 ESTs from fed-infected ticks. Two catalogs of transcripts that were differentially expressed in response to blood feeding and B. ovis infection were produced. Four genes coding for a putative vitellogenin-3, lachesin, a glycine rich protein, and a secreted cement protein were selected for RNA interference functional studies. A reduction of 92, 65, and 51% was observed in vitellogenin-3, secreted cement, and lachesin mRNA levels in SG, respectively. The vitellogenin-3 knockdown led to increased tick mortality, with 77% of ticks dying post-infestation. The reduction of the secreted cement protein-mRNA levels resulted in 46% of ticks being incapable of correctly attaching to the host and significantly lower female weights post-feeding in comparison to the control group. The lachesin knockdown resulted in a 70% reduction of the levels associated with B. ovis infection in R. bursa SG and 70% mortality. These results improved our understanding of the role of tick SG genes in Babesia infection/proliferation and tick feeding. Moreover, lachesin, vitellogenin-3, and secreted cement proteins were validated as candidate protective antigens for the development of novel tick and tick-borne disease control measures.

Normal variation of bone marrow B-cell precursors according to age - reference ranges for studies in myelodysplastic syndromes in Brazil.

BACKGROUND: Normal B lymphoid maturation occurs in bone marrow (BM) throughout life, but immature B-cell progenitors (BCPs) are more numerous in children than in adults. To assess the normal values according to age became important as BCPs are decreased in myelodysplastic syndromes and have been considered an important diagnostic and prognostic feature in these clonal disorders. METHODS: in a multicenter retrospective study from the Brazilian Group of Flow Cytometry we analyzed the variation of BCPs in normal BM according to age and technical peculiarities of each laboratory. We analysed of 45 BM donors and 89 cases examined for elucidation of transitory reactive cytopenias presenting a normal BM immunophenotyping. BCPs were enumerated as CD19+ /CD34+ /CD45dim /CD10+ cells (panel 1) or CD19+ /CD34+ /CD45dim cells (panel 2) among the total nucleated non-erythroid cells and as percentage of CD34+ cells. RESULTS: we included 134 cases. Panel 1 was applied in 88 cases and panel 2 was used in 46. Age range: 10 months to 89 years. In a multiple regression, % BCPs/total nucleated cells was an exponential function of age. Age explained alone 49.4% of the variance, while 'panel used' explained 1.8% and 'laboratory' explained 0.7%. Age explained only 24.9% of the variance of BCPs/CD34+ cells. CONCLUSIONS: in normal individuals, BM B-cell precursors varied mainly according to age, but were also dependent on technical peculiarities of operators and equipments. Analysis by phenotype and as percentage of total nucleated cells was more accurate and less susceptible to variation than evaluating % BCPs/total CD34+ cells. © 2017 International Clinical Cytometry Society.

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: Anaplasma marginale, 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.

Screening for the SMIM1*64_80 del Allele in blood donors in a population from Southern Brazil.

BACKGROUND AND OBJECTIVES: Serological screening for the Vel- phenotype is complex given the large individual variation in the levels of expression of the Vel antigen, and the polyclonal anti-human sera of immunised persons, when available, show heterogeneous reactivity levels. Studies of the SMIM1 gene have enabled the development of several molecular methodologies that will be crucially important for the screening of different populations, including Brazilians. To evaluate the deletion of 17 bp in the SMIM1 gene in a population from the south of Brazil, 448 unrelated blood donors from 7 regions comprising the haemotherapy network in the state of Santa Catarina were evaluated between August 2011 and March 2014. MATERIALS AND METHODS: DNA samples from these donors were analysed employing a 5' nuclease real-time polymerase chain reaction (PCR) assay targeting the 17 bp deletion in the SMIM1 gene. RESULTS: Among the 448 samples analysed, 10 (2·23%) harboured the 17 bp deletion of the gene SMIM1, and all were heterozygote for the SMIM1*64_80 del allele. CONCLUSION: The allelic frequency found differed from those observed in other Caucasian populations. This difference can be explained by the ethnic make-up of each Caucasian population. The data obtained are important to characterise the correct phenotype of the donor as the serological assay results are not reliable due to variations in the expression intensity of the Vel antigen in heterozygote donors for the SMIM1*64_80 del allele. Moreover, the tool used in this study is of great value for identifying a donor Vel- phenotype and supplying a possible need for transfusion.

Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines.

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 µM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion.

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.

Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis.

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow.

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

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).

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 agent's 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.

PCR-based survey of Anaplasma phagocytophilum in Portuguese ticks (Acari: Ixodidae).

A total of two-thousand and six ticks, collected from 2002 to 2006 in areas belonging to seven districts of Mainland Portugal and also in Madeira Island, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Active infections were detected exclusively in Ixodes species, including six questing I. ricinus nymphs from Madeira Island, one questing I. ventalloi nymph from Setúbal District, and two I. ventalloi adults found parasitizing domestic cats in both Setúbal and Santarém District. These findings confirm prior observations and suggest the persistence of A. phagocytophilum on Madeira Island. Moreover, it adds I. ventalloi and domestic cats to the list of potential elements of the agent's enzootic cycles in Portugal. Molecular analysis of PCR amplicons suggests the existence of two A. phagocytophilum genotypes in Portugal, one of which is identical or very similar to North American strains implicated in human disease.

Ticks and tick-borne Rickettsiae surveillance in Montesinho Natural Park, Portugal.

This study constitutes the first contribution to the knowledge of tick dynamics and its implication in the epidemiology of rickettsial diseases in Montesinho Natural Park (MNP), Bragança district of Portugal. Of 76 ticks collected, 12 (15.8%) were Dermacentor (D.) marginatus, 36 (47.4%) D. reticulatus, and 28 (36.8%) Rhipicephalus (R.) sanguineus. Isolation assays were performed by shell-vial technique on 41 ticks. Israeli spotted fever strain was an isolate from R. sanguineus, and three isolates of Rickettsia slovaca were obtained from D. reticulatus. All 76 ticks were screened by PCR for Rickettsia sp., Ehrlichia (E.) chaffeensis, and Anaplasma (A.) phagocytophilum. Rickettsia RpA4 strain DNA was detected in 10 D. marginatus and 2 D. reticulatus, and Israeli spotted fever strain in 1 R. sanguineus. No E. chaffeensis or A. phagocytophilum infection was detected. New host records are provided for D. reticulatus. Also described for the first time in Portugal is the isolation of R. slovaca from D. reticulatus and the isolation of Israeli spotted fever strain from R. sanguineus. This confirms the association of the last rickettsiae strain with the same vector tick as previously described in Israel and Sicily.