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.

Strain FhSp1^T, 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. FhSp1^T shared similarity of more than 99 % with strains of Francisella tularensis subspecies and Francisella novicida U112^T, 98 % with Francisella piscicida GM2212^T and 98.4 % with Francisella philomiragia ATCC 25015^T. In the recA gene sequence, Francisella sp. FhSp1^T exhibited 91.6–91.7 % similarity to strains of F. tularensis subspecies, 91.2 % to F. novicida U112^T 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 C_10 : 0 (17.2 %), C_14 : 0 (11.2 %), C_16 : 0 (13.1 %), C_18 : 0 3-OH (14.2 %) and C_18 : 1 ω9c (7.1 %). DNA–DNA hybridization, which showed unambiguously that FhSp1^T 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 FhSp1^T 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 FhSp1^T (=FnSp1^T =FSC454^T =F62^T =DSM 22475^T =CCUG 58020^T). 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 15482^T =CCUG 33449^T =CIP 56.12^T). We also present an emended description of the genus Francisella.

The prevalence of tick-borne and related bacteria infecting adult ticks in central Spain was assessed by molecular methods. Six areas were sampled monthly during a 2-year longitudinal study. A total of 1,038 questing and 442 feeding ticks, belonging to eight different species, were tested. The most abundant species were Hyalomma lusitanicum (54% of captures), followed by Dermacentor marginatus (23%) and Rhipicephalus sanguineus (10%). Four human pathogens, including seven Rickettsia species, Anaplasma phagocytophilum, Borrelia burgdorferi, and Francisella tularensis, were detected at percentages of 19.0, 2.2, 1.7, and 0.5, respectively, whereas Bartonella spp. was never detected. In terms of infection and tick abundance, H. lusitanicum seems to be the most significant tick species in the area, carrying three of the five agents tested, and the anthropophilic tick, D. marginatum, infected with Rickettsia spp. and F. tularensis, is the most relevant in terms of public health. The significance of these data is discussed.

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

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.

Waterborne Outbreak of Tularemia Associated with Crayfish Fishing