Raquel Escudero

Rickettsia monacensis and Human Disease, Spain

We identified Rickettsia monacensis as a cause of acute tickborne rickettsiosis in 2 humans. Its pathogenic role was assessed by culture and detection of the organism in patients’ blood samples. This finding increases the number of recognized human rickettsial pathogens and expands the known geographic distribution of Mediterranean spotted fever–like cases.

Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

ABSTRACT Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

Description of Francisella hispaniensis sp. nov., isolated from human blood, reclassification of Francisella novicida (Larson et al. 1955) Olsufiev et al. 1959 as Francisella tularensis subsp. novicida comb. nov. and emended description of the genus Francisella.

Strain FhSp1T, isolated from human blood in Spain in 2003, was studied for its taxonomic allocation. By 16S rRNA and recA gene sequencing, the strain was shown to belong to the genus Francisella. In the 16S rRNA gene sequence, Francisella sp. FhSp1T shared similarity of more than 99% with strains of Francisella tularensis subspecies and Francisella novicida U112T, 98% with Francisella piscicida GM2212T and 98.4% with Francisella philomiragia ATCC 25015T. In the recA gene sequence, Francisella sp. FhSp1T exhibited 91.6-91.7% similarity to strains of F. tularensis subspecies, 91.2% to F. novicida U112T and 84% to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C10:0 (17.2%), C14:0 (11.2%), C16:0 (13.1%), C18:0 3-OH (14.2%) and C18:1omega9c (7.1%). DNA-DNA hybridization, which showed unambiguously that FhSp1T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hitherto-described Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1T (=FnSp1T =FSC454T =F62T =DSM 22475T =CCUG 58020T). Furthermore, we formally propose the transfer of the species Francisella novicida to the species Francisella tularensis as Francisella tularensis subsp. novicida comb. nov. (type strain ATCC 15482T =CCUG 33449T =CIP 56.12T). We also present an emended description of the genus Francisella.

Tick-Borne Zoonotic Bacteria in Wild and Domestic Small Mammals in Northern Spain

ABSTRACT The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.

Molecular Method for Identification of Rickettsia Species in Clinical and Environmental Samples

ABSTRACT We present a PCR method targeting the 23S-5S internal transcribed spacer coupled with reverse line blotting that allows Rickettsia species detection and identification in a single step. The method is highly sensitive and specific in identifying Rickettsia species from both patient and environmental samples. The generic approach used allowed us to identify new pathogens.

Molecular Method for Bartonella Species Identification in Clinical and Environmental Samples

ABSTRACT A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.

Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus (Acari: Ixodidae) ticks from the Basque Country, Spain.

Abstract Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA was detected in ≤12.5% of adults and ≥0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in ≤14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where I. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. burgdorferi appears to have an increasing occurrence in ticks in the Basque Country.

Variability of Bartonella Genotypes among Small Mammals in Spain

ABSTRACT In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas—247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)—by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.

Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

BackgroundBorreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH) and factor H like-1 (FHL1)], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diversity. Here we show the differential binding of borrelial proteins to fH from human and animal sera.FindingsAffinity ligand binding experiments, 2-D electrophoresis, and protein identification and peptide de novo sequencing based on mass spectrometry, revealed novel fH putative binding proteins of Lyme- and relapsing fever Borrelia. An OspA serotype-associated differential human and animal fH binding by B. garinii was also observed, which could be related with the ability of some strains from serotypes 4 and 7 to invade non-nervous system tissues. Also, the variable affinity of binding proteins expressed by different Borrelia to animal fH correlated with their host selectivity.ConclusionThe novel animal and human putative fH binding proteins (FHBPs) in this study underscore the importance of evasion of complement in the pathogenesis of Borrelia infections.

Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic

ABSTRACT The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.