Zoonotic pathogens in fluctuating common vole (Microtus arvalis) populations: occurrence and dynamics.

Diseases and host dynamics are linked, but their associations may vary in strength, be time-lagged, and depend on environmental influences. Where a vector is involved in disease transmission, its dynamics are an additional influence, and we often lack a general understanding on how diseases, hosts and vectors interact. We report on the occurrence of six zoonotic arthropod-borne pathogens (Anaplasma, Bartonella, Borrelia, Coxiella, Francisella and Rickettsia) in common voles (Microtus arvalis) throughout a population fluctuation and how their prevalence varies according to host density, seasonality and vector prevalence. We detected Francisella tularensis and four species of Bartonella, but not Anaplasma, Borrelia, Coxiella or Rickettsia. Bartonella taylorii and B. grahamii prevalence increased and decreased with current host (vole and mice) density, respectively, and increased with flea prevalence. Bartonella doshiae prevalence decreased with mice density. These three Bartonella species were also more prevalent during winter. Bartonella rochalimae prevalence varied with current and previous vole density (delayed-density dependence), but not with season. Coinfection with F. tularensis and Bartonella occurred as expected from the respective prevalence of each disease in voles. Our results highlight that simultaneously considering pathogen, vector and host dynamics provide a better understanding of the epidemiological dynamics of zoonoses in farmland rodents.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Rapid and Highly Sensitive DNA Flow Technology Platform to Detect Tick-Borne Bacterial Pathogens in Clinical Samples.

Zoonotic diseases represent a significant public health concern worldwide due to the emergence/re-emergence of vector-borne diseases in the last decade. Ticks are the most important vectors in the northern hemisphere and can transmit diseases such as Lyme disease, human granulocytic anaplasmosis, and spotted fever rickettsioses, among others. Therefore, there is a growing need to develop better and faster diagnostic tools that can detect zoonotic human pathogens in clinical samples. In this study, we present the results for a new kit tick-borne bacteria flow chip (TBFC), which allows the simultaneous screening of seven different bacterial pathogens in human samples using a DNA flow technology platform (hybriSpot system). The analytical sensitivity and specificity of the TBFC were calculated spiking bacterial DNA in human DNA samples, and the results were compared with an in-house single PCR-reverse line blot (RLB) routinely used for diagnosis at the National Center for Microbiology in Spain. The analytical sensitivity and specificity of the TBFC were almost identical to the PCR-RLBs used in diagnosis. In addition, samples from patients (n = 212) with a wide range of clinical signs/symptoms consistent with multisystem disorders suggestive of a tick-borne infection were tested using the TBFC, and the results were compared with those obtained by PCR-RLB. The concordance of both methods using patient samples was 97.2%. The TBFC kit is a rapid new and cost-efficient diagnostic molecular tool capable of detecting tick-borne pathogens in clinical samples.

Molecular Survey of Rickettsia spp., Anaplasma spp., Ehrlichia spp., Bartonella spp., and Borrelia spp. in Fleas and Lice in Ethiopia.

Bacterial arthropod-borne pathogens can often cause fever in Africa, but rural laboratories in these settings are usually too basic to provide a precise picture of their epidemiological impact. Our aim was to determine the prevalence of bacterial pathogens in fleas and lice in a rural area of southeast Ethiopia. Between July and November 2013, we extracted DNA from 91 fleas (Ctenocephalides felis [n = 50; 54.9%], Pulex irritans [n = 37; 40.1%], and C. canis [n = 4; 4.4%] and 30 lice (Pediculus humanus capitis [n = 16; 53.3%] and Pediculus humanus humanus [n = 14; 46.7%]), using two quantitative PCR (qPCR) analyses to look for bacteria from the genera: Anaplasma, Bartonella, Borrelia, Coxiella, Ehrlichia, Francisella, and Rickettsia. Of the 91 fleas analyzed, pathogens were present in 79 (86.8%), including Rickettsia felis (n = 41; 45%), Anaplasma platys (n = 40; 44.0%), Rickettsia monacensis (n = 2; 2.2%), Ehrlichia muris-like agent (n = 1; 1.1%), and Bartonella clarridgeiae (n = 1; 1.1%). P. irritans was the flea species most frequently infected with A. platys (67.7%), followed by C. felis (30.7%) (p < 0.001). Of the 30 lice identified, pathogens were present in 7 (23.3%): Bartonella quintana (n = 4; 16.7%), E. muris (n = 2, 6.7%), and Borrelia recurrentis (n = 1, 3.3%). Thus, in this rural area of Africa, fleas and lice can transmit parasitic pathogens to humans, causing febrile symptoms.

Molecular method for Bartonella species identification in clinical and environmental samples.

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.

Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

Genotypes of Coxiella burnetii in wildlife: disentangling the molecular epidemiology of a multi-host pathogen.

Evidences point to a relevant role of wildlife in the ecology of Coxiella burnetii worldwide. The lack of information on C. burnetii genotypes in wildlife prevents tracing-back clinical animal and human Q fever cases with potential wildlife origin. To compare C. burnetii genotypes circulating in wildlife, livestock and humans, 107 samples from red deer, European wild rabbit, racoon, small mammals, goat and sheep were genotyped by polymerase chain reaction and reverse line blot hybridization. Genomic groups I, II, VI and VII were found in wildlife and groups I, II, III and IV in domestic ruminants. Livestock genotypes clustered mainly with genotypes reported previously in livestock. Genotyping confirmed previous findings that suggest that C. burnetii may display host specificity since most genotypes of sympatric deer and rabbits clustered in separate groups. Wildlife genotypes clustered with genotypes from ticks and from acute hepatitis human Q fever cases, suggesting that particular C. burnetii genotypes circulating in a wildlife-tick cycle may occasionally jump into humans through tick bites or exposure to wildlife. This finding could be behind the reported geographic variation in the clinical presentation of acute Q fever in humans in Spain: atypical pneumonia in the north and hepatitis in the south.

Distribution of Bartonella henselae variants in patients, reservoir hosts and vectors in Spain.

We have studied the diversity of B. henselae circulating in patients, reservoir hosts and vectors in Spain. In total, we have fully characterized 53 clinical samples from 46 patients, as well as 78 B. henselae isolates obtained from 35 cats from La Rioja and Catalonia (northeastern Spain), four positive cat blood samples from which no isolates were obtained, and three positive fleas by Multiple Locus Sequence Typing and Multiple Locus Variable Number Tandem Repeats Analysis. This study represents the largest series of human cases characterized with these methods, with 10 different sequence types and 41 MLVA profiles. Two of the sequence types and 35 of the profiles were not described previously. Most of the B. henselae variants belonged to ST5. Also, we have identified a common profile (72) which is well distributed in Spain and was found to persist over time. Indeed, this profile seems to be the origin from which most of the variants identified in this study have been generated. In addition, ST5, ST6 and ST9 were found associated with felines, whereas ST1, ST5 and ST8 were the most frequent sequence types found infecting humans. Interestingly, some of the feline associated variants never found on patients were located in a separate clade, which could represent a group of strains less pathogenic for humans.

Tick-borne zoonotic bacteria in ticks collected from central Spain.

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.

Molecular method for identification of Rickettsia species in clinical and environmental samples.

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.