Detection of Rickettsia conorii strains in Portuguese dogs (Canis familiaris).

This study reports a serological and molecular survey on the spotted fever group Rickettsia found in a group of 51 dogs with suspected tick-borne illness from the south of Portugal. Additionally, a prevalence of IgG antibodies to R. conorii was also estimated in a group of 400 healthy dogs from the same region. In the group of healthy dogs, the immunofluorescence test revealed that 154 (38.5%) of the 400 dogs had IgG antibodies reactive with R. conorii. The highest proportion of dogs (45%) with R. conorii antibodies was found in blood samples collected from October to December. Among the group suspected with tick-borne illness, 35 (62%) dogs showed to be seroreactive (IgG≥128) for antibodies against R. conorii, and the analysis of PCR-positive amplicons revealed that 5 dogs were infected with R. conorii Malish and 2 dogs were infected with R. conorii Israeli tick typhus strain. There was a higher prevalence of antibodies to R. conorii in the group of sick dogs, and the detection of R. conorii DNA in blood samples from this group points to their potential role as a reservoir and sentinel host helping to evaluate and characterize the distribution of circulating rickettsial strains.

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.

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal.

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.

Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients.

BACKGROUND: The pathophysiologic mechanisms that determine the severity of Mediterranean spotted fever (MSF) and the host-related and microbe-related risk factors for a fatal outcome are incompletely understood. METHODS: This prospective study used univariate and multivariate analyses to determine the risk factors for a fatal outcome for 140 patients with Rickettsia conorii infection admitted to 13 Portuguese hospitals during 1994-2006 with documented identification of the rickettsial strain causing their infection. RESULTS: A total of 71 patients (51%) were infected with the Malish strain of Rickettsia conorii, and 69 (49%) were infected with the Israeli spotted fever (ISF) strain. Patients were admitted to the intensive care unit (40 [29%]), hospitalized as routine inpatients (95[67%]), or managed as outpatients (5[4%]). Death occurred in 29 adults (21%). A fatal outcome was significantly more likely for patients infected with the ISF strain, and alcoholism was a risk factor. The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, obtundation and/or confusion, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated levels of hepatic enzymes and creatine kinase. Some, but not all, of these findings were observed more often in ISF strain-infected patients. CONCLUSIONS: Although fatalities and similar clinical manifestations occurred among both groups of patients, the ISF strain was more virulent than the Malish strain. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strongly associated with a fatal outcome.

Rickettsia conorii Israeli tick typhus strain isolated from Rhipicephalus sanguineus ticks in Portugal.

This report describes the first establishment of five isolates of R. conorii Israeli tick typhus (ITT) strain from Rhipicephalus sanguineus ticks in different regions of Portugal. Ticks were collected on two separate expeditions that took place in Bragança, Montesinho Natural Park, and Portalegre City.

Intralesional expression of mRNA of interferon- gamma , tumor necrosis factor- alpha , interleukin-10, nitric oxide synthase, indoleamine-2,3-dioxygenase, and RANTES is a major immune effector in Mediterranean spotted fever rickettsiosis.

BACKGROUND: The mechanisms of immunity to Rickettsia conorii that have been elucidated in mouse models have not been evaluated in human tissues. METHODS: In this study, quantitative real-time polymerase chain reaction was used to determine the levels of expression of inflammatory and immune mediators in skin-biopsy samples collected from 23 untreated patients with Mediterranean spotted fever (MSF). RESULTS: In all 23 patients, the levels of intralesional expression of mRNA of tumor necrosis factor (TNF)- alpha , interferon (IFN)- gamma , interleukin (IL)-10, RANTES, and indoleamine-2,3-dioxygenase (IDO), an enzyme involved in limiting rickettsial growth by tryptophan degradation, were higher than those in control subjects; 6 of the 23 patients had high levels of inducible nitric oxide synthase (iNOS), a source of microbicidal nitric oxide. Positive correlations between TNF- alpha , IFN- gamma , iNOS, IDO, and mild/moderate MSF suggest that type 1 polarization plays a protective role. Significantly higher levels of intralesional expression of IL-10 mRNA were inversely correlated with levels of intralesional expression of IFN- gamma mRNA and TNF- alpha mRNA. The mRNA-expression level of the chemokine RANTES was significantly higher in patients with severe MSF. CONCLUSION: Mild/moderate MSF is associated with a strong and balanced intralesional proinflammatory and anti-inflammatory response, with a dominant type 1 immunity, whereas severe MSF is associated with increased expression of chemokine mRNA. Whether these factors are simply correlates of mild and severe MSF or contribute to antirickettsial immunity and pathogenesis remains to be determined.

Rickettsia sp. strain RpA4 detected in Portuguese Dermacentor marginatus ticks.

Dermacentor marginatus species is the common vector of Rickettsia slovaca. Recently, a new rickettsia genotype, RpA4, was detected in several Dermacentor sp. ticks in several countries of the former Soviet Union and in Spain. In an epidemiological surveillance study, ninety-eight D. marginatus, collected in the South of Portugal in spring of 2003 and 2004, were screened by polymerase chain reaction (PCR) to detect Rickettsiae. Overall, 59.2% of the ticks were PCR-positive, among them 34.5% were proved to be infected with R. slovaca and 65.5% with Rickettsia sp. RpA4. This study reports for the first time a high prevalence of Rickettsia sp. RpA4 in D. marginatus in Portugal, pointing out for a wider geographic distribution of this genotype than previously reported.

Rickettsiae phylogeny: a multigenic approach.

The development of molecular taxonomic methods has provided a large amount of data in the reorganization of Rickettsiae taxonomy. Nevertheless, phylogenetic relationships among some groups and species delimitation remain unclear. To clarify rickettsial phylogeny, a multigenic approach was used for the first time for the genus Rickettsia, based on simultaneous analyses of eight loci: atpA, recA, virB4, dnaA, dnaK, rrl-rrf internal transcribed spacer, ompA and gltA. Concatenation of different nucleotide sequences resulted in an improvement in phylogenetic resolution when compared to single gene data. This multigenic approach has enabled the differentiation of many groups, including the spotted fever group which includes a great number of closely related species. The reliability of some previously recognized groups was evaluated.

Boutonneuse fever and climate variability.

Researchers have long appreciated the role of climate in vector-borne diseases, including the resurgence of boutonneuse fever (BF). Portugal usually is classified as having temperate Mediterranean climate. In this new century, in analyzing the data from the Meteorology Institute, this pattern has changed and an accentuated variability in climate is being observed. BF (febre escaro nodular) is endemic and high season is from late spring and summer. The brown dog tick Rhipicephalus sanguineus. is the vector and reservoir of Rickettsia conorii complex strains: R. conorii Malish and Israeli spotted fever strain. To assess the influence of climate change in BF seasonality our aim was to compare the human sera samples received at CEVDI-INSA for laboratory diagnosis of MSF for 5 months per year from October to February, ("off-season") from 2000 to 2005. Of 1,299 sera samples in persons with suspected clinical diagnosis of MSF, 45 (3.4%) were considered positive cases and the number of positive cases has doubled in the last 2 years. BF epidemiology clearly appears to be associated with climate change, especially with low precipitation values. Physicians should be aware of increasing off-season BF cases.

Automated method based in VNTR analysis for Rickettsiae genotyping.

A genetic locus named Rc-65, which is 5' adjacent to gene dksA and 3' adjacent to xerC gene, has previously been demonstrated to contain a VNTR with high discriminatory power in several rickettsial strains and thus, potentially useful for genetically similar strains identification. In this work, we present an automated molecular identification method based on capillary electrophoresis separation of VNTRs amplicons. The resulting electropherograms were in agreement with the sequence data obtained in a previous work. The presented genotyping method is fast and suitable for full automation, being a powerful tool for epidemiological surveillance in a large number of samples and enables the detection co-infected samples. The combination of other VNTR loci should improve the discriminatory capacity of this typing system, providing greater resolution and contributing to a more accurate VNTR-based assay. To our knowledge, this is the first automated assay for rickettsial strains identification.

Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae.

A total of 56 fleas were collected from mice, rats, and one hedgehog in national parks of mainland Portugal and the Madeira Island. All fleas were tested for the presence of bacteria of the genera Rickettsia and Bartonella using PCR assays. In fleas from mainland Portugal, we detected Rickettsia felis in one Archaeopsylla erinacei maura flea and in one Ctenophtalmus sp. In five Leptopsylla segnis fleas taken from rats in the Madeira Island, we identified Rickettsia typhi. In addition, in four fleas from the genera Ornithophaga and Stenoponia collect from mice and a rat in mainland Portugal, we detected the presence of two new Bartonella genotypes closely related to Bartonella elizabethae. Our findings emphasize the potential risk of flea-transmitted infections in mainland Portugal and the Madeira archipelago, and extend our knowledge of the potential flea vectors of human pathogens.

[Ticks associated to infectious pathologies in Portugal]. / Carraças associadas a patologias infecciosas em Portugal.

Ticks are hematophagos arthropods that parasitize terrestrial vertebrates. They are world wide, living in almost all terrestrial regions. Although mainly associated to animals, there are several tick species that bite humans and transmit tick-borne agents causing important infectious disease. In this paper the authors revise the most outstanding aspects of those arthropods as vectors of infectious pathogens in Portugal, focusing biologic, ecologic and epidemiologic features with Public Health interest.

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.

Rickettsia sibirica isolation from a patient and detection in ticks, Portugal.

We report the first isolation of Rickettsia sibirica (strain mongolotimonae) from the blood of a patient and detection by polymerase chain reaction (PCR) of the rickettsia in a Rhipicephalus pusillus tick collected from a dead mongoose (Herpestes ichneumon) in the Alentejo region, Portugal. We describe also the first PCR detection of a new Rickettsia strain that is related to R. sibirica.

[Rickettsial diseases in Brazil and Portugal: occurrence, distribution and diagnosis]. / Riquetsioses no Brasil e Portugal: ocorrência, distribuição e diagnóstico.

The present study is an update review on the occurrence and diagnosis of rickettsial diseases in Brazil and Portugal, aiming at promoting their epidemiological surveillance in both countries. A literature review was carried out and unpublished data of laboratories and surveillance systems were presented. The results described the occurrence of rickettsial diseases and infections in Brazil and Portugal, including other new and still poorly understood rickettsial infections. Current diagnostic methods were discussed. As in many other countries, rickettsial diseases and infections seem to be an emerging public health problem. Treated as a minor problem for many decades, the interest in these infections has increased in both countries but further studies are needed to establish their role as a public health problem.

Riquetsioses no Brasil e Portugal: ocorrência, distribuição e diagnóstico / Rickettsial diseases in Brazil and Portugal: occurrence, distribution and diagnosis

O presente artigo é uma atualização sobre a ocorrência e diagnóstico das riquetsioses existentes no Brasil e Portugal, com o objetivo de incentivar e incrementar a vigilância epidemiológica dessas doenças nos dois países. Realizou-se levantamento bibliográfico e foram apresentados dados não publicados de laboratórios e serviços de epidemiologia. Os resultados descreveram a ocorrência das riquetsioses no Brasil e Portugal, inclusive aquelas recém-descritas, advindas de riquétsias de potencial patogênico ainda incerto. Os métodos diagnósticos atualmente empregados foram discutidos. Como em outros países, as riquetsioses parecem assumir crescente importância em saúde pública. Relegadas a um plano secundário por muitas décadas, o interesse por essas infecções tem aumentado nos dois países, mas ainda carece de investigação para esclarecer seu real significado em saúde pública.

Characterization of a tandem repeat polymorphism in Rickettsia strains.

Mediterranean spotted fever (MSF) is a tick-borne rickettsiosis caused by 'Rickettsia conorii complex' strains. In Portugal, R. conorii and Israeli tick typhus (ITT) are the aetiological agents of this disease. A novel 65 bp tandem repeat was identified by the analysis of the R. conorii Malish 7 whole genome sequence with an appropriate algorithm for searching for repeated sequences. The variable number tandem repeat (VNTR) was named VNTR Rc-65 and this locus was amplified by PCR and sequenced in order to characterize the repeat diversity within different rickettsial strains including Portuguese strains isolated from clinical and vector samples. The VNTR Rc-65 has seven alleles within the rickettsial strains studied and a diversity index value of 0.71, meaning that this locus has a great discriminatory capacity and therefore can be used for identification of closely related strains. PCR amplification of the Rc-65 locus can be used to differentiate between the Portuguese R. conorii Malish-like and Israeli tick typhus strains, enabling a more accurate and rapid identification of these rickettsial isolates.

The presence of eschars, but not greater severity, in Portuguese patients infected with Israeli spotted fever.

In Portugal, Mediterranean spotted fever (MSF) is caused by R. conorii Malish and Israeli spotted fever (ISF) strains. It has been suggested that the ISF strain isolated from patients with MSF causes different clinical manifestations compared to those caused by Malish strain, namely the absence of eschar and greater severity. The aim of this study was to analyze the presence or absence of eschar and of fatality in Portuguese patients infected with either Malish or ISF strain. Of 94 patients with a clinical diagnosis of MSF between 1994 to 2004, 47 were infected with Malish strain and 47 with ISF strain. Eschars were reported in 20 patients (49%) infected with Malish strain, and in 17 (39%) with ISF strain. The presence of eschar is not statistically associated to a greater extent with either R. conorii strain (P=0.346). A total of 22 patients died, 9 infected with Malish strain and 13 infected with ISF strain, and no statistically significant difference was found (P=0.330). This study showed that the concepts of absence of the eschar and greater severity in Israeli spotted fever infection should be revised.

Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal.

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.