Francisella-like Endosymbionts Research Articles

Identification, Distribution and Population Dynamics of Francisella-like Endosymbiont in Haemaphysalis doenitzi (Acari: Ixodidae).

Francisella-like endosymbionts (FLEs) with significant homology to Francisella tularensis (γ-proteobacteria) have been characterized in several tick species, whereas knowledge on their distribution and population dynamics in ticks remains meager. Hence, in the current study, we identified a novel Francisella-like endosymbiont (FLEs-Hd) from the tick Haemaphysalis doenitzi and evaluated the putative functions of this symbiont. Results indicated that FLEs-Hd had 100% infection rate and a perfect vertical transmission in H. doenitzi, and that it is distributed in ovaries, malpighian tubules, salivary glands and midguts of the ticks, suggesting that FLEs-Hd presumably is a crucial symbiont of the host without specific tissue tropism. To further explore the function of the symbiont, the population dynamics of FLEs-Hd at each developmental stage of ticks and in tissues at different reproductive statuses were determined by real-time quantitative polymerase chain reaction (real-time qPCR). Results showed that the high density and regular population dynamics of FLEs-Hd appeared in female ovaries, suggesting that the symbiont may provide necessary nutrients or regulators to ensure normal ovary development of ticks.

A Francisella-like endosymbiont in the Gulf Coast tick evolved from a mammalian pathogen.

Ticks (order Ixodida) vector pathogenic bacteria that cause diseases in humans and other mammals. They also contain bacteria that are closely related to pathogens but function as endosymbionts that provide nutrients that are missing from mammalian blood—their sole food source. For instance, mammalian pathogens such as Coxiella burnetii and Francisella tularensis, as well as Coxiella-like and Francisella-like endosymbionts (CLEs and FLEs, respectively) occur in ticks worldwide. However, it is not clear whether the pathogens evolved from symbionts or symbionts from pathogens. Recent studies have indicated that C. burnetii likely originated from a tick-associated ancestor, but the origins of FLEs are not clear. In this study, we sequenced the genome of an FLE, termed FLE-Am, present in the Gulf Coast tick, Amblyomma maculatum. We show that FLE-Am likely evolved from a pathogenic strain of Francisella, indicating that tick endosymbionts can evolve from mammalian pathogens. Although the genome of FLE-Am is almost the same size as the genomes of pathogenic Francisella strains, about one-third of its protein-coding genes contain inactivating mutations. The relatively low coding capacity and extensive metabolic capabilities indicate that FLE-Am transitioned recently to its current endosymbiotic lifestyle and likely replaced an ancient endosymbiont with degraded functionality.

Tick species, tick-borne pathogens and symbionts in an insular environment off the coast of Western France

Insular environments provide ideal natural conditions to study disease ecology, especially emerging diseases, due to clear differentiation between local and long-distance transmission. Such environments are of particular interest regarding tick-borne pathogens (TBP), since animal exchange with the mainland (along with any ticks they carry) is limited, and because such locations could lie on migratory routes for birds carrying ticks. Therefore both tick species and TBP may display different prevalence than those observed on the continent. As such, an epidemiological survey was performed on Belle-Ile-en-Mer, an island off the coast of Western France, in order to estimate the prevalence of tick species and the microorganisms they carried. Three tick species, Dermacentor marginatus, D. reticulatus, and Haemaphysalis punctata were collected at five different sites in 2010 and 2011. All ticks were tested for pathogen’s and symbiont’s DNA by (i) PCR for Anaplasma spp., Borrelia spp., Rickettsia spp.; (ii) real-time PCR for Francisella tularensis, Francisella-like endosymbionts (FLE) and Coxiella spp. and (iii) PCR-RLB for Babesia-Theileria spp. Pathogen DNA detected in D. marginatus including Borrelia spp. (18%), Rickettsia spp. (13%) which was identified as R. slovaca, Babesia spp. (8%), and Theileria spp. (1%). Pathogens detected in D. reticulatus including Rickettsia spp. (31%) identified as R. raoulti, Francisella-like endosymbiont (86%), and Babesia spp (21%). Pathogens detected in H. punctata including Rickettsia spp. (1%) identified as R. aeschlimannii, FLE (0.4%), Babesia spp. (18%), and Theileria spp. (7%). Anaplasma spp., F. tularensis, or Coxiella spp. were not detected in any of the collected ticks. This study represents the first epidemiological survey of the insular Belle-Ile-en-Mer environment. It demonstrated the presence of expected pathogens, consistent with reports from island veterinarians or physicians, as well as unexpected pathogens, raising questions about their potential introduction through infected animals and/or their dispersion by migratory birds.

A snapshot of the microbiome of Amblyomma tuberculatum ticks infesting the gopher tortoise, an endangered species

The gopher tortoise tick, Amblyomma tuberculatum, has a unique relationship with the gopher tortoise, Gopherus polyphemus, found in sandy habitats across the southeastern United States. We aimed to understand the overall bacterial community associated with A. tuberculatum while also focusing on spotted fever group Rickettsia. These tortoises in the Southern Mississippi region are a federally threatened species; therefore, we have carefully trapped the tortoises and removed the species-specific ticks attached to them. Genomic DNA was extracted from individual ticks and used to explore overall bacterial load using pyrosequencing of bacterial 16S rRNA on 454-sequencing platform. The spotted fever group of Rickettsia was explored by amplifying rickettsial outer membrane protein A (rompA) gene by nested PCR. Sequencing results revealed 330 bacterial operational taxonomic units (OTUs) after all the necessary curation of sequences. Four whole A. tuberculatum ticks showed Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the most dominant phyla with a total of 74 different bacterial genera detected. Together Rickettsiae and Francisella showed >85% abundance, thus dominating the bacterial community structure. Partial sequences obtained from ompA amplicons revealed the presence of an uncharacterized Rickettsia similar to the Rickettsial endosymbiont of A. tuberculatum. This is the first preliminary profile of a complete bacterial community from gopher tortoise ticks and warrants further investigation regarding the functional role of Rickettsial and Francisella-like endosymbionts in tick physiology.

Molecular Investigation of Francisella-Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey.

This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission.

Francisella species in ticks and animals, Iberian Peninsula

The presence of Francisella species in 2134 ticks, 93 lagomorphs and 280 small mammals from the Iberian Peninsula was studied. Overall, 19 ticks and 6 lagomorphs were positive for Francisella tularensis subsp. holarctica, suggesting, as described for other regions, that lagomorphs may have an important role in the maintenance of F. tularensis in nature. Of the 6 positive lagomorphs, 4 were identified as the European rabbit, Oryctogalus cuniculus. Additionally, 353 ticks and 3 small mammals were PCR positive for Francisella-like endosymbionts (FLEs) and one small mammal was also positive for Francisella hispaniensis-like DNA sequences. Among FLE positive specimens, a variety of sequence types were detected: ticks were associated with 5 lpnA sequence types, with only one type identified per tick, in contrast to 2 lpnA sequence types detected in a single wood mouse (Apodemus sylvaticus). To our knowledge, this is the first report of FLEs in free-living small mammals as well as the first detection of F. hispaniensis-like sequences in a natural setting.

Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland

A total of 530 questing Dermacentor reticulatus ticks and 861 questing Ixodes ricinus ticks were collected from Lublin province (eastern Poland) and examined for the presence of Francisella by PCR for 16S rRNA (rrs) and tul4 genes. Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with Francisella tularensis subspecies holarctica, as determined by PCR of the rrs gene. None of 861 I. ricinus ticks were infected with F. tularensis. In contrast, the presence of Francisella-like endosymbionts (FLEs) was detected in more than half of the D. reticulatus ticks (50.4%) and 0.8% of the I. ricinus ticks. The nucleotide sequences of the FLEs detected in D. reticulatus exhibited 100% homology with the nucleotide sequence of the FLE strain FDrH detected in Hungary in D. reticulatus. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. This finding, however, needs to be confirmed by further studies in other areas. Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time, a low grade of infection of I. ricinus with FLEs.

TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations.

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.

Detection of Francisella-like endosymbiont in Hyalomma rufipes from Ethiopia

The expanding family of Francisellaceae includes the genus Francisella, where several pathogen bacteria, e.g. the zoonotic F. tularensis, and different Francisella-like agents belong to. Francisella-like endosymbionts (FLEs) are widespread in hard and soft ticks and their pathogenicity is unknown. The examination of 296 ticks collected in Ethiopia was performed for the detection of F. tularensis and FLEs using polymerase chain reaction (PCR) assays based on the amplification of 16S rRNA, sdhA and tul4 gene fragments. FLE was described in one Hyalomma rufipes tick based on the 16S rRNA and sdhA gene sequences. The 16S rRNA gene fragment was identical with the ones detected previously in Rhipicephalus sanguineus and Hyalomma marginatum marginatum in Bulgaria. The presence of endosymbionts with identical 16S rRNA gene sequence in both Rhipicephalus and Hyalomma species further supports the hypotheses, that certain FLEs had independent evolution from their tick hosts.

Phylogenetic relationships of Francisella-like endosymbionts detected in two species of Amblyomma from snakes in Thailand

Ticks are important vectors of several bacterial pathogens, including Francisella. In this study, a total of 24 adult ticks (Amblyomma varanense and Amblyomma helvolum) collected from 4 species of snake from 3 provinces of Thailand was screened for the presence of Francisella-like endosymbionts (FLEs) by PCR. FLEs were detected in 46% (11/24) of all ticks examined. A phylogenetic analysis of the 16S rDNA sequence indicated that the 11 distinct genotypes of FLEs amplified from A. varanense and A. helvolum (from Khon Kaen and Pichit provinces, respectively) are in the same group, along with other FLEs amplified from the other tick genera. Interestingly, these FLEs are closely related to, but distinct (different clade) from, the FLEs isolated from different tick species previously reported. This work represents the first report of Francisella spp. in snake ticks from Thailand.

Prevalence ofFrancisella tularensisandFrancisella-Like Endosymbionts in the Tick Population of Hungary and the Genetic Variability ofFrancisella-Like Agents

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.

Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany

The zoonotic disease tularaemia is caused by the bacterial pathogen Francisella tularensis. Although the causative agent is known for 100 years, knowledge of its enzootic cycles is still rudimentary. Apart from tabanids and mosquitoes, hard ticks have been described as important vectors and potential reservoirs for F. tularensis. Available data on the incidence of human tularaemia indicate an increase in cases in the federal state of Baden-Wuerttemberg. To determine whether ticks are involved in the reported increase in F. tularensis infections in humans and wildlife in this south-western part of Germany, 916 Ixodes ricinus and 211 adult Dermacentor marginatus and D. reticulatus ticks were collected in two different locations. Screening for the presence of F. tularensis was performed by real-time PCR of the 16S rRNA gene. Of the 95 pools of I. ricinus ticks (representing 916 individual ticks), 8 tick pools (8.4%) were positive in this PCR. 30-bp deletion PCR confirmed that the F. tularensis subspecies holarctica was present. FtM24 VNTR analysis revealed that they belong to the emerging Franco-Iberian subclone group of F. tularensis holarctica. Of the 211 ticks of the genus Dermacentor, 35 randomly chosen DNAs were subjected to 16S rRNA gene screening PCR; 20 of these (57%) gave positive signals. For cluster analysis, the lpnA gene region of all Francisella-positive I. ricinus pools and 6 Dermacentor ticks with a positive reaction in the screening PCR was amplified and sequenced. In the resulting neighbour-joining tree, all Francisella-positive I. ricinus samples clustered with sequences of F. tularensis, whilst all Dermacentor tick samples clustered with FLE (Francisella-like endosymbiont) sequences. This study shows that I. ricinus ticks may serve as vectors and/or reservoirs of F. tularensis in Germany and supports the hypothesis that the state of Baden-Wuerttemberg represents an emerging endemic focus of tularaemia.

Analysis of pathogen co-occurrence in host-seeking adult hard ticks from Serbia

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis. As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified (Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. concinna and I. ricinus) and analyzed for the presence of seven pathogens. Anaplasma ovis, A. phagocytophilum, Babesia canis, B. burgdorferi s.l., Coxiella burnetii, Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella-like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species (D. reticulatus, H. concinna and I. ricinus). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.

Association of different genetic types of Francisella-like organisms with the rocky mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis) in localities near their northern distributional limits.

Dermacentor andersoni and Dermacentor variabilis from allopatric and sympatric populations near their northern distributional limits were examined for the presence of Francisella species using molecular techniques that targeted 373 bp of the 16S rRNA gene. Although there was no evidence for the presence of Francisella tularensis in any tick, Francisella-like endosymbionts (FLEs) were common in D. andersoni and D. variabilis adults and immatures. A significantly greater proportion of female ticks contained FLEs compared to male ticks. In addition, significantly more D. variabilis adult individuals contained multiple FLE sequence types than did D. andersoni adults. Ten different types of FLEs were identified based on the sequence data, which has implications for diagnostic tests and epidemiological studies of F. tularensis in tick populations in Canada. The three most prevalent types of FLEs have been detected previously in D. andersoni or D. variabilis from other parts of their distributional ranges, whereas the other seven FLE types have not been reported previously. A comparison of the FLEs from both allopatric and sympatric populations of these two tick species provided insight into the relative host-specificity and the modes of transmission of these tick-borne bacteria. In general, each FLE type was specific for one tick species, suggesting vertical transmission of each bacterium. However, there were a few instances of potential cross-transfer of two FLE types to the other tick species at locations where D. andersoni and D. variabilis occurred in sympatry, suggesting that there may be occasional horizontal transmission of some FLEs.

Deep Mitochondrial DNA Lineage Divergences Within Alberta Populations of Dermacentor albipictus (Acari: Ixodidae) Do Not Indicate Distinct Species

The winter tick Dermacentor albipictus (Packard) has a single-host life cycle that allows it to reach severe infestation levels on ungulates, particularly moose. Genotypic variation within these and related ticks has been a source of taxonomic confusion, although the continuity in their morphology and life history has generally been interpreted as indicating the existence of a single species. To further investigate this variation, we sequenced regions of two mitochondrial DNA (mtDNA) genes (COI and 16S rDNA), two nuclear genes (lysozyme and ITS-2), and two bacterial markers from Francisella-like endosymbionts found in these ticks (eubacterial mtDNA 16S rRNA and a homolog of Francisella tularensis [Dorofe’ev] 17-kDa lipoprotein). We sampled 42 D. albipictus individuals from whitetail and mule deer culled from three populations in east-central Alberta, as well as four D. albipictus and two Dermacentor variabilis (Say) from other locations. We then compared DNA sequence variation between the genes and related this to variation in the morphology of spiracle plates. Both mtDNA regions indicated two deeply diverged lineages (mean difference of 7.1% for COI and 4.5% for 16S) that would normally be considered diagnostic of distinct species in DNA barcoding studies. However, very little divergence was revealed by nuclear gene sequences, bacterial endosymbionts, and morphometric analyses, and any variation that did occur in these markers was not congruent with mtDNA divergences. We conclude that the sampled populations in Alberta represent a single species, D. albipictus, and reiterate the importance of integrative approaches in species delimitation.

Deep Mitochondrial DNA Lineage Divergences Within Alberta Populations of <I>Dermacentor albipictus</I> (Acari: Ixodidae) Do Not Indicate Distinct Species

The winter tick Dermacentor albipictus (Packard) has a single-host life cycle that allows it to reach severe infestation levels on ungulates, particularly moose. Genotypic variation within these and related ticks has been a source of taxonomic confusion, although the continuity in their morphology and life history has generally been interpreted as indicating the existence of a single species. To further investigate this variation, we sequenced regions of two mitochondrial DNA (mtDNA) genes (COI and 16S rDNA),two nuclear genes (lysozyme and ITS-2), and two bacterial markers from Francisella-like endosymbionts found in these ticks (eubacterial mtDNA 16S rRNA and a homolog of Francisella tularensis [Dorofe'ev] 17-kDa lipoprotein). We sampled 42 D. albipictus individuals from whitetail and mule deer culled from three populations in east-central Alberta, as well as four D. albipictus and two Dermacentor variabilis (Say) from other locations. We then compared DNA sequence variation between the genes and related this to variation in the morphology of spiracle plates. Both mtDNA regions indicated two deeply diverged lineages (mean difference of 7.1% for COI and 4.5% for 16S) that would normally be considered diagnostic of distinct species in DNA barcoding studies. However, very little divergence was revealed by nuclear gene sequences, bacterial endosymbionts, and morphometric analyses, and any variation that did occur in these markers was not congruent with mtDNA divergences. We conclude that the sampled populations in Alberta represent a single species, D. albipictus, and reiterate the importance of integrative approaches in species delimitation.

Francisella-Like Endosymbiont inDermacentor reticulatusCollected in Portugal

In Portugal, recent studies have confirmed the presence of Francisella tularensis in Dermacentor reticulatus. Bacterial endosymbionts with significant homology to F. tularensis have been described in several species of ticks. In this work we identified Francisella-like endosymbionts in D. reticulatus ticks (39%), confirming the presence of these bacteria in Portugal. This finding should be considered in future studies using molecular approaches to detect Francisella prevalence in ticks and environmental samples.

Transovarial Transmission ofFrancisella-Like Endosymbionts andAnaplasma phagocytophilumVariants inDermacentor albipictus(Acari: Ixodidae)

Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F1 larvae by individual ticks at efficiencies of 10-40 and 95-100%, respectively. The FLEs were transovarially transmitted to F2 larvae obtained as progeny of adults from F1 larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission.

Francisella-Like Endosymbiont DNA andFrancisella tularensisVirulence-Related Genes in Brazilian Ticks (Acari: Ixodidae)

Ticks are vectors of a variety of pathogens, including Francisella tularensis. Bacteria in the genus Francisella have been identified mostly in the Northern Hemisphere and include tick endosymbionts. Francisella has never been described in Brazil, where Amblyomma spp. ticks are known as the vector of many bacterial zoonotic pathogens. In the present work, we have identified bacterial DNA sequences with identity to Francisella genes in Amblyomma dubitatum Neumann Dermacentor nitens (Neumann), and Rhipicephalus microplus (Canestrini) in Brazil. DNA fragments with homology to Francisella spp. 16S rDNA and the tul4 gene were polymerase chain reaction amplified from tick DNA samples collected in Minas Gerais and Mato Grosso states. These sequences were 96-99% identical to the reported sequences for Francisella-like tick endosymbionts (FLEs). Sequences similar to the tularemia agent F. tularensis pathogenicity island gene iglC and its regulatory gene mglA also were identified in FLEs.

Detection of a NovelFrancisellainDermacentor reticulatus: A Need for Careful Evaluation of PCR-Based Identification ofFrancisella tularensisin Eurasian Ticks

Francisella tularensis, the causative agent of tularemia, has been detected in ixodid ticks in some regions of North America, Europe, and Asia. In the present study, 245 Dermacentor reticulatus, 211 Ixodes ricinus, and 194 Haemaphysalis concinna adults from Hungary were tested for the presence of F. tularensis by polymerase chain reaction (PCR) assays based on 16S ribosomal RNA (16S rDNA) and T-cell epitope of a Francisella membrane protein (TUL4). No Francisella-specific amplification products were detected in I. ricinus and H. concinna ticks. Francisella DNA was identified using PCR assays based on 16S rDNA and TUL4 gene in D. reticulatus with similar prevalence (minimum 1.2%) as demonstrated in earlier European and Asian studies detecting F. tularensis in D. reticulatus. However, the 16S rDNA and TUL4 gene sequences of the Francisella-like agent occurring in D. reticulatus differed from the homologous sequences of Francisella spp. deposited in GenBank. Phylogenetic reconstructions showed that the new genotype detected in D. reticulatus was closely related to Francisella-like endosymbionts of North American Dermacentor ticks. Although further studies are needed on the relationship of this bacterium with ticks, the results highlight the need for careful evaluation of PCR-based identification in European and Asian laboratories that screen ixodid ticks for F. tularensis.