Francisella sciaenopsi sp. nov. isolated from diseased red drum Sciaenops ocellatus in Florida, USA.

Piscine francisellosis is one of the most important bacterial diseases affecting various fish species worldwide. Francisella orientalis, F. noatunensis, and F. salimarina (F. marina) have been reported as etiological agents of disease in fish. A Francisella sp. was isolated from several diseased red drum Sciaenops ocellatus experiencing morbidity in Florida, USA, in 2008. In this study, molecular and phenotypic characterization of the recovered isolate was conducted. Phenotypically, the isolate showed a biochemical reaction profile distinct from that of F. orientalis and F. salimarina. Although the 16S rRNA sequence of this isolate shared 99.61% identity to the type strain of F. philomiragia O#319LT, whole genome analysis (average nucleotide identity <95%; digital DNA-DNA hybridization <70%) and a multilocus sequence analysis of 8 concatenated housekeeping genes in comparison with other Francisella spp. indicated that this isolate was a novel Francisella species, more closely related to F. orientalis. Immersion, intracoelomic injection, and co-habitation challenges using a Nile tilapia Oreochromis niloticus fingerling model of infection were done to investigate virulence in a piscine model. Variably pigmented granulomas and pigmented macrophage aggregates were observed in the kidneys and spleens of the challenged fish, but no mortality was recorded during the 15 d challenge period, suggesting that this novel Francisella sp. might be an opportunistic pathogen of fish. Based on the phenotypic and genotypic differences from other Francisella spp. observed in this study, we propose the name Francisella sciaenopsi sp. nov. for this novel isolate.

Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Francisella philomiragia bacteremia in an immunocompromised patient: a rare case report.

BACKGROUND: Francisella philomiragia is a very rare opportunistic pathogen of humans which causes protean diseases such as pneumonia and other systemic infections. Subsequent failure of prompt treatment may result in poor prognosis with mortality among infected patients. CASE PRESENTATION: The present report describes a case of F. philomiragia bacteraemia first reported in Malaysia and Asian in a 60-year-old patient with underlying end-stage renal disease (ESRF) and diabetes mellitus. He presented with Acute Pulmonary Oedema with Non-ST-Elevation Myocardial Infarction (NSTEMI) in our hospital. He was intubated in view of persistent type I respiratory failure and persistent desaturation despite post haemodialysis. Blood investigation indicated the presence of ongoing infection and inflammation. The aerobic blood culture growth of F. philomiragia was identified using the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Score value: 2.16) and confirmed by 16S Ribosomal DNA (16S rDNA) sequencing. He was discharged well on day 26 of admission, after completing one week of piperacillin/tazobactam and two weeks of doxycycline. CONCLUSION: Clinical suspicion should be raised if patients with known risk factors are presenting with pneumonia or pulmonary nodules especially as these are the most common manifestations of F. philomiragia infection. Early diagnosis via accurate laboratory identification of the organism through MALDI-TOF mass spectrometry and molecular technique such as 16S rDNA sequencing are vital for prompt treatment that results in better outcomes for the afflicted patients.

Absence of Francisella tularensis in Finnish Ixodes ricinus and Ixodes persulcatus ticks.

Francisella tularensis subsp. holarctica is the causative agent of tularaemia in Europe. Finland is a high-incidence region for tularaemia, with mosquito bites as the most common sources of infection. However, in Central and Western Europe, ticks (Acari: Ixodidae) have been suggested as the main vectors. Indeed, several studies have reported the pathogen from the locally most common human-biting tick species, Ixodes ricinus. In Finland, the occurrence of the pathogen in ticks has started receiving attention only recently. Here, we collate previous tick screening data from Finland regarding F. tularensis as well as present the results from a novel screening of roughly 15 000 I. ricinus and I. persulcatus collected from across the country. In total, 14 878 ticks collected between 2015 and 2020 were screened for F. tularensis using a TaqMan-based qPCR assay targeting the 23 KDa gene. The combined screening efforts of the current and previous studies, encompassing roughly 20 000 ticks, did not find any positive ticks. Given the negative results despite the considerable sample size, it appears that the pathogen is not circulating in local tick populations in Finland. We discuss some possible reasons for the lack of the bacterium in ticks in this high-incidence region of tularaemia.

Molecular characterization of bacterial communities of two neotropical tick species (Amblyomma aureolatum and Ornithodoros brasiliensis) using rDNA 16S sequencing.

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America.

Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan.

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.

Development of a semi-quantitative PCR assay for the detection of Francisella halioticida and its application to field samples.

The current study describes the development and application of a TaqMan® real-time PCR assay for the detection of the bacterium Francisella halioticida. Previously, detection of F. halioticida is relied on bacterial culture and conventional PCR; however, the real-time PCR provides many advantages because it is faster, less labour-intensive and reduces the risk of cross-contamination. DNA samples from mussels collected in April 2020 from seven sites in northern Brittany (France) were tested using the newly developed real-time PCR assay. The objective was to screen for the presence of F. halioticida during spring mortality events. The bacterium was detected in 71.4% of the samples tested and was present at all sites except for Saint-Brieuc and Mont-Saint-Michel, two sites which were not concerned by mortality at the time of sampling. Less than a month later, Saint-Brieuc was affected by unusual mortalities and F. halioticida was detected in almost all mussels (81.25%). The findings from this study provide further evidence indicating that F. halioticida may be contributing to mussel mortalities; however, a direct causal relationship has not yet been established. The real-time PCR assay developed in this study allows for rapid, specific and sensitive detection of F. halioticida which should prove useful for future studies concerning the involvement of this bacterium with shellfish mortalities.

First metagenomic report of Borrelia americana and Borrelia carolinensis in Poland - a preliminary study.

INTRODUCTION AND OBJECTIVE: Ixodes ricinus (I. ricinus) and Dermacentor reticulatus (D. reticulatus) are the most common ticks in Poland. These ticks contain many bacteria, which compose a microbiome with potential impact on humans. The aim of the study was to discover the microbiome of ticks in Poland. MATERIAL AND METHODS: Ticks were collected in The Protected Landscape Area of the Bug and Nurzec Valley, Poland, in 2016-2018 by flagging. They were cleaned in 70% ethanol and damaged in mortar with PBS (without Ca2+ and Mg2+ ions). DNA was extracted from the homogenates with spin columns kits, and used as a matrix in end-point PCR for bacterial 16S rRNA fragments amplifications, and further for next generation sequencing (NGS) by ILLUMINA. RESULTS: In 22 ticks (3 I. ricinus and 19 D. reticulatus) 38 microorganisms were detected. The most common were Francisella hispaniensis and Francisella novicida. In 17 ticks, Sphingomonas oligophenolica, and in 12 Rickettsia aeshlimanii were found. In 2, I. ricinus specific DNA of Borrelia americana and Borrelia carolinensis were found. In one female, D. reticulatus Anaplasma phagocytophilum and Anaplasma centrale were found. Pseudomonas lutea and Ps. moraviensis were detected in 9 and 8 ticks, respectively. CONCLUSIONS: Polish ticks microbiome contains not only well-known tick-borne pathogens, but also other pathogenic microorganisms. For the first time in Poland, Borrelia americana and Borrelia carolinensis in I. ricinus collected from the environment were detected. The dominant pathogenic microorganisms for humans were Francisella spp. and Rickettsia spp., and non-pathogenic - Sphingomonas oligophenolica. Knowledge of a tick microbiome might be useful in tick-borne biocontrol and tick-borne diseases prevention.

Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.

Behavioral characteristics and endosymbionts of two potential tularemia and Rocky Mountain spotted fever tick vectors.

Due to climate change-induced alterations of temperature and humidity, the distribution of pathogen-carrying organisms such as ticks may shift. Tick survival is often limited by environmental factors such as dryness, but a predicted hotter and wetter world may allow the expansion of tick ranges. Dermacentor andersoni and D. variabilis ticks are morphologically similar, co-occur throughout the Inland Northwest of Washington State, U.S.A., and both can be injected with pathogenic Rickettsia and Francisella bacteria. Differences in behavior and the potential role of endosymbiotic Rickettsia and Francisella in these ticks are poorly studied. We wanted to measure behavioral and ecological differences between the two species and determine which, if any, Rickettsia and Francisella bacteria - pathogenic or endosymbiotic - they carried. Additionally, we wanted to determine if either tick species may be selected for if the climate in eastern Washington becomes wetter or dryer. We found that D. andersoni is more resistant to desiccation, but both species share similar questing behaviors such as climbing and attraction to bright light. Both also avoid the odor of eucalyptus and DEET but not permethrin. Although both tick species are capable of transmitting pathogenic species of Francisella and Rickettsia, which cause tularemia and Rocky Mountain Spotted Fever, respectively, we found primarily non-pathogenic endosymbiotic strains of Francisella and Rickettsia, and only one tick infected with F. tularensis subspecies holarctica.

First European report of Francisella tularensis subsp. holarctica isolation from a domestic cat.

Francisella tularensis subsp. holarctica is a select agent causing life-threatening tularemia. It has been isolated from humans and animals, mainly lagomorphs and rodents, rarely other wild carnivore species. Increasing numbers of human tularemia cases have been reported during the last 5 years in Switzerland. Here we report the first isolation of Francisella tularensis subsp. holarctica from a domestic cat in Europe and compare its genome sequence with other Swiss isolates. The cat isolate shows a close phylogenetic relationship with a contemporary hare isolate from close geographic proximity, indicating a possible epidemiological link.

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection.

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 106  CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.

Francisella salimarina sp. nov., isolated from coastal seawater.

Four strains (SYSU SYW-1T, SYW-2, SYW-3 and XLW-1) were isolated from seawater near the shore in Guangdong Province, China. Cells were Gram-stain-negative, aerobic, non-motile and non-spore-forming. Growth was observed at a temperature range of 16-40 °C (optimum, 32 °C), a pH range of 4-8 (optimum, pH 7) and in the presence of up to 10 % (w/v) NaCl. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unidentified phospholipid. The respiratory quinone was ubiquinone 8 (UQ-8), and the predominant fatty acids were C18 : 0 3-OH, C10 : 0, C14 : 0 and C18 : 1ω9c. Comparison of 16S rRNA gene and genome sequences confirmed that these strains represented a novel member of the genus Francisella, with less than 98.8 % 16S rRNA gene sequence similarity and less than 95 % genomic average nucleotide identity to recognized Francisella species. The phylogenetic tree based on 16S rRNA gene sequences and the protein-concatamer tree based on a concatenation of 28 protein marker sequences both indicated that the strains clustered with 'Francisella salina' TX07-7308 and 'Francisella marina' E95-16, but formed a distinct lineage group among the other members of the genus Francisella. The DNA G+C contents of the four strains were determined to be 32.9, 32.7, 32.9 and 32.9 %, respectively (genome). On the basis of phenotypic and genotypic features, the strains are considered to represent a novel species of the genus Francisella, for which the name Francisella salimarina sp. nov. is proposed. The type strain is SYSU SYW-1T (=CGMCC 1.17031T=NBRC 113781T).

Tick-borne pathogens in ticks (Acari: Ixodidae) collected from various domestic and wild hosts in Corsica (France), a Mediterranean island environment.

Corsica is a mountainous French island in the north-west of the Mediterranean Sea presenting a large diversity of natural environments where many interactions between humans, domestic animals and wild fauna occur. Despite this favourable context, tick-borne pathogens (TBPs) have not systematically been investigated. In this study, a large number of TBPs were screened in ticks collected over a period of one year from domestic and wild hosts in Corsica. More than 1,500 ticks belonging to nine species and five genera (Rhipicephalus, Hyalomma, Dermacentor, Ixodes and Haemaphysalis) were analysed individually or pooled (by species, gender, host and locality). A real-time microfluidic PCR was used for high-throughput screening of TBP DNA. This advanced methodology enabled the simultaneous detection of 29 bacterial and 12 parasitic species (including Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia and Theileria). The Crimean-Congo haemorrhagic fever (CCHF) virus was investigated individually in tick species known to be vectors or carriers of this virus. In almost half of the tick pools (48%), DNA from at least one pathogen was detected and eleven species of TBPs from six genera were reported. TBPs were found in ticks from all collected hosts and were present in more than 80% of the investigated area. The detection of DNA of certain species confirmed the previous identification of these pathogens in Corsica, such as Rickettsia aeschlimannii (23% of pools), Rickettsia slovaca (5%), Anaplasma marginale (4%) and Theileria equi (0.4%), but most TBP DNA identified had not previously been reported in Corsican ticks. This included Anaplasma phagocytophilum (16%), Rickettsia helvetica (1%), Borrelia afzelii (0.7%), Borrelia miyamotoi (1%), Bartonella henselae (2%), Babesia bigemina (2%) and Babesia ovis (0.5%). The high tick infection rate and the diversity of TBPs reported in this study highlight the probable role of animals as reservoir hosts of zoonotic pathogens and human exposure to TBPs in Corsica.

Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks.

To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.com) relies on a two-step workflow: a genus-level search followed by a species-level search. This strategy enables the rapid identification of microorganisms based on the analyzed proteome. This method was successfully used to identify strains of Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, Yersinia pestis and closely related species from simulated blood culture flasks. This newly developed LC-MS/MS method is a safe and rapid method for accurately identifying bacteria directly from positive blood culture flasks.

Francisella opportunistica sp. nov., isolated from human blood and cerebrospinal fluid.

Two isolates of a Gram-negative, non-spore-forming coccobacillus cultured from the blood and cerebrospinal fluid of immunocompromised patients in the United States were described previously. Biochemical and phylogenetic analyses revealed that they belong to a novel species within the Francisella genus. Here we describe a third isolate of this species, recovered from blood of a febrile patient with renal failure, and formally name the Francisella species. Whole genome comparisons indicated the three isolates display greater than 99.9 % average nucleotide identity (ANI) to each other and are most closely related to the tick endosymbiont F. persica, with only 88.6-88.8 % ANI to the type strain of F. persica. Based on biochemical, metabolic and genomic comparisons, we propose that these three isolates should be recognized as Francisella opportunistica sp. nov, with the type strain of the species, PA05-1188T, available through the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 107100) and the American Type Culture Collection (ATCC BAA-2974).

First molecular detection of Francisella-like endosymbionts in Hyalomma and Rhipicephalus tick species collected from vertebrate hosts from Sardinia island, Italy.

Ticks are vectors of a wide variety of human and animal pathogens as well as non-pathogenic microorganisms acting as endosymbionts and whose role in ticks is still little known. Symbionts such as Francisella-like endosymbionts (FLEs) are members of Francisellaceae family with unknown pathogenicity, detected in both hard and soft ticks. A total of 236 ticks collected from several sites in Sardinia were screened for Francisella species by PCR using primers targeting a fragment of the 16S rRNA gene. DNA of Francisella was detected in 5.1% (12/236) of the ticks tested. Sequencing results revealed that seven Rhipicephalus sanguineus s.l., three Hyalomma marginatum, one Hy. lusitanicum, and one Rh. bursa ticks exhibited DNA with 99-100% similarity to Francisella-like endosymbionts isolated from different tick species all over the world. Further research is needed in order to better characterize FLE strains obtained in Sardinia and to better understand if their presence could be related to the infection with other zoonotic pathogens.

Transmission of Francisella noatuensis subsp. orientalis from subclinically infected hybrid red tilapia broodstock (Oreochromis sp.) to their offspring.

Francisella noatunensis subsp. orientalis (Fno) has been reported as an important bacterial pathogen causing significant mortality (30-95%) in farmed tilapia in broad geographic areas. However, we found that there was a proportion of broodfish in our laboratory that appeared to be healthy but which tested positive for Fno. We therefore hypothesized that Fno might be able to be transmitted from subclinically infected tilapia mouthbrooders to their offspring through the current practice of fry production in tilapia hatcheries. To prove this, experimentally infected hybrid red tilapia broodstock were mated and their offspring were examined for the presence of Fno. In this study, three pairs of infected broodfish were mated for natural spawning and fertilized eggs from each couple were then collected from the female mouths for artificial incubation. The newly hatched larvae were cultured for 30 days and sample collection was performed at different developmental stages i.e. yolk-sac larvae, 5 and 30-day old fry. The results showed that the ovary and testis of all 3 pairs of the broodstock, as well as their fertilized eggs and offspring were Fno positive by Fno-specific PCR and in situ DNA hybridization. In summary, this study revealed that with the current practice in tilapia hatcheries, Fno might be able to transmit from subclinically infected tilapia mouthbrooders to their offspring. Therefore, using Fno-free broodfish in tilapia hatcheries should be considered in order to produce Fno-free tilapia fry.

Francisella spp. detected in Dermacentor ticks in Malaysian forest reserve areas.

Limited information is available on tropical ticks and tick-borne bacteria affecting the health of humans and animals in the Southeast Asia region. Francisella tularensis is a tick-borne bacterium which causes a potentially life-threatening disease known as tularemia. This study was conducted to determine the occurrence of Francisella spp. in questing ticks collected from Malaysian forest reserve areas. A total of 106 ticks (mainly Dermacentor and Haemaphysalis spp.) were examined for Francisella DNA using a Polymerase chain reaction (PCR) assay targeting the bacterial 16S rDNA. Francisella DNA was detected from 12 Dermacentor ticks. Sequence analysis of the amplified 16S rDNA sequences (1035 bp) show >99% identity with that of Francisella endosymbiont reported in a tick from Thailand. A dendrogram constructed based on the bacterial 16S rDNA shows that the Francisella spp. were distantly related to the pathogenic strains of F. tularensis. Three Francisella-positive ticks were identified as Dermacentor atrosignatus, based on sequence analysis of the tick mitochondrial 16S rRNA gene. Further screening of cattle and sheep ticks (Haemaphysalis bispinosa and Rhipicephalus microplus) and animal samples (cattle, sheep, and goats) did not yield any positive findings. Our findings provide the first molecular data on the occurrence of a Francisella strain with unknown pathogenicity in Dermacentor questing ticks in Malaysia.

Detection of tick-borne pathogens in ticks collected in the suburban area of Monte Romano, Lazio Region, Central Italy.

BACKGROUND: A study on tick species characterization and tick borne pathogens detection was performed by a survey conducted during 2012 and 2013 in the Viterbo province (Lazio Region, Central Italy). Seven sites were selected for the study investigation, including two farms and a military zone. METHODS: A total of 255 ticks, Rhipicephalus (Boophilus) annulatus (n = 215), Rhipicephalus bursa (n = 28), and Hyalomma marginatum (n = 12) were screened individually by molecular methods for the tick borne bacterial agents: Borrelia burgdorferi sensu lato group, Bartonella spp., Coxiella burnetii, Ehrlichia spp., Francisella spp., and Rickettsia spp. RESULTS AND CONCLUSION: Overall, 182 ticks (71%) were infected with at least one pathogen; among these co-infections were found in 94 ticks. Tick borne pathogens identified were C. burnetii, B. burgdorferi s.l., Bartonella spp., Rickettsia spp., Francisella spp., and Ehrlichia spp. In R. bursa and H. marginatum, the presence of B. burgdorferi s.l. was positively correlated with that of C. burnetii, Rickettsia spp., and Bartonella spp. and their coinfection probabilities were 29.8%, 22.7% and 11.7%, respectively; the probability of coinfection for Francisella spp. and Rickettsia spp. and for Francisella spp. and Bartonella spp. was 14.9% and 17.9%, respectively. In R. (Boophilus) annulatus, the probability of coinfection between C. burnetii and B. burgdorferi s.l. was 11.3%, while those between C. burnetii and Bartonella spp. and between B. burgdorferi s.l. and Bartonella spp. were 0.8%. Further studies are needed in order to assess the risk associated with these unusual tick-borne pathogens in Central Italy.