Rickettsia Deregulates Genes Coding for the Neurotoxic Cell Response Pathways in Cerebrocortical Neurons In Vitro.

Rickettsial infections of the central nervous system (CNS) are manifested by severe neurological symptoms and represent a serious life-threatening condition. Despite the considerable health danger, only a few studies have been conducted focusing on the pathogenesis induced by Rickettsia sp. in CNS. To investigate the signaling pathways associated with the neurotoxic effects of rickettsiae, we employed an experimental model of cerebrocortical neurons combined with molecular profiling and comprehensive bioinformatic analysis. The cytopathic effect induced by Rickettsia akari and Rickettsia slovaca was demonstrated by decreased neuronal viability, structural changes in cell morphology, and extensive fragmentation of neurites in vitro. Targeted profiling revealed the deregulation of genes involved in the neuroinflammatory and neurotoxic cell response pathways. Although quantitative analysis showed differences in gene expression response, functional annotation revealed that the biological processes are largely shared between both Rickettsia species. The identified enriched pathways are associated with cytokine signaling, chemotaxis of immune cells, responses to infectious agents, interactions between neurons, endothelial and glial cells, and regulation of neuronal apoptotic processes. The findings of our study provide new insight into the etiopathogenesis of CNS infection and further expand the understanding of molecular signaling associated with neuroinvasive Rickettsia species.

Culture Isolate of Rickettsia felis from a Tick.

Although the cat flea, Ctenocephalides felis, has been identified as the primary vector of Rickettsia felis, additional flea, tick, mite, and louse species have also been associated with this bacterium by molecular means; however, the role of these arthropods in the transmission of R. felis has not been clarified. Here, we succeeded in culture isolation of R. felis from a host-seeking castor bean tick, Ixodes ricinus, the most common tick in Slovakia. The bacterial isolation was performed on XTC-2 cells at 28 °C using the shell-vial technique. An evaluation of the growth properties was performed for both the XTC-2 and Vero cell lines. We observed R. felis in the infected host cells microscopically by Gimenez staining and immunofluorescence assay. The R. felis isolate was purified by gradient ultracentrifugation and visualized by electron microscopy. Fragments of the genes gltA, ompA, ompB, htrA, rpoB, sca4, rffE, and rrs were amplified and compared with the corresponding sequences of the type strain URRWXCal2 and other R. felis culture -isolated strains. We did not detect any nucleotide polymorphisms; however, plasmid pRFδ, characteristic of the standard strain, was absent in our isolate. Herein, we describe the first successful isolation and characterization of a tick-derived R. felis strain "Danube", obtained from an I. ricinus nymph.

Hymenopteran Parasitoids of Hard Ticks in Western Africa and the Russian Far East.

Some parasitoids of the genus Ixodiphagus (Hymenoptera, Chalcidoidea: Encyrtidae) are well-known natural enemies of ticks. In this study, we investigate the occurrence of parasitoid wasps in adult hard ticks from Western Africa (Côte d'Ivoire and Senegal) and Far Eastern Europe (Russia) using molecular methods. The morphological identification allowed the classification of 785 collected specimens of six species of ticks: Rhipicephalus (Boophilus) microplus (41%), Ixodes persulcatus (33%), Dermacentor silvarum (11%), Haemaphysalis concinna (7%), Amblyomma variegatum (5%), and Haemaphysalis japonica (3%). The newly developed MALDI-TOF MS protocol identified tick species in spite of their different storage (dried or in 70% ethanol) conditions for a long period. Molecular screening of ticks by a new standard PCR system developed in this study revealed the presence of parasitoid wasp DNA in 3% (28/785) of analyzed ticks. Ixodiphagus hookeri was detected in 86% (24/28) of infested ticks, including 13 I. persulcatus, 9 R (B) microplus, and one H. concinna and D. silvarum. While an unidentified parasitoid wasp species from the subfamily Aphidiinae and Braconidae family was detected in the remaining 14% (4/28) infested ticks. These infested ticks were identified as I. persulcatus. Our findings highlight the need for further studies to clarify the species diversity of parasitoid infesting ticks by combining molecular and morphological features. The novel molecular and MALDI-TOF MS protocols could be effective tools for the surveillance and characterization of these potential bio-control agents of ticks.

Optimized fixation of actin filaments for improved indirect immunofluorescence staining of rickettsiae.

OBJECTIVE: The objective was to investigate fixative solutions: 3.7% formaldehyde, 4% paraformaldehyde, 4% paraformaldehyde in the cytoskeletal buffer and 4% paraformaldehyde in PHEM buffer (containing PIPES, HEPES, EGTA and MgCl2), applicable for immunofluorescence assay. RESULTS: Herein we optimized this serological technique, testing four fixative solutions, for the sensitive detection of rickettsial antigens, and preservation of intracellular structures of the host cells, particularly filamentous actin. Rickettsial antigens were presented equally well both with formaldehyde and all paraformaldehyde-based fixations, but only protocol with 4% paraformaldehyde in PHEM buffer allowed accurate imaging of actin filaments, and simultaneously allows monitoring of rickettsiae using actin-based motility during infection inside the host cells.

Rickettsial infections of the central nervous system.

As a result of migrations and globalization, people may face a possible increase in the incidence of central nervous system rickettsial infections (CNS R). These diseases, caused by Rickettsia species and transmitted to humans by arthropod bites, are putatively lethal. However, the diagnosis of CNS R is challenging and often delayed due to their nonspecific clinical presentation and the strict intracellular nature of rickettsiae. Furthermore, transfer of rickettsiae to the brain parenchyma is not yet understood. The aim of this review is to analyze and summarize the features and correlated findings of CNS R in order to focus attention on these intriguing but frequently neglected illnesses. We also incorporated data on CNS infections caused by Rickettsia-related microorganisms.

Synthetic Biology and the United Nations.

Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).

Arsenophonus nasoniae and Rickettsiae Infection of Ixodes ricinus Due to Parasitic Wasp Ixodiphagus hookeri.

Arsenophonus nasoniae, a male-killing endosymbiont of chalcid wasps, was recently detected in several hard tick species. Following the hypothesis that its presence in ticks may not be linked to the direct occurrence of bacteria in tick's organs, we identified A. nasoniae in wasps emerging from parasitised nymphs. We confirmed that 28.1% of Ixodiphagus hookeri wasps parasitizing Ixodes ricinus ticks were infected by A. nasoniae. Moreover, in examined I. ricinus nymphs, A. nasoniae was detected only in those, which were parasitized by the wasp. However, in part of the adult wasps as well as in some ticks that contained wasp's DNA, we did not confirm A. nasoniae. We also found, that in spite of reported male-killing, some newly emerged adult wasp males were also infected by A. nasoniae. Additionally, we amplified the DNA of Rickettsia helvetica and Rickettsia monacensis (known to be Ixodes ricinus-associated bacteria) in adult parasitoid wasps. This may be related either with the digested bacterial DNA in wasp body lumen or with a role of wasps in circulation of rickettsiae among tick vectors.

The natural infection of birds and ticks feeding on birds with Rickettsia spp. and Coxiella burnetii in Slovakia.

Ixodid ticks (Acari: Ixodidae) are known as primary vectors of many pathogens causing diseases in humans and animals. Ixodes ricinus is a common ectoparasite in Europe and birds are often hosts of subadult stages of the tick. From 2012 to 2013, 347 birds belonging to 43 species were caught and examined for ticks in three sites of Slovakia. Ticks and blood samples from birds were analysed individually for the presence of Rickettsia spp. and Coxiella burnetii by PCR-based methods. Only I. ricinus was found to infest birds. In total 594 specimens of bird-attached ticks were collected (451 larvae, 142 nymphs, 1 female). Altogether 37.2% (16/43) of bird species were infested by ticks and some birds carried more than one tick. The great tit, Parus major (83.8%, 31/37) was the most infested species. In total, 6.6 and 2.7% of bird-attached ticks were infected with Rickettsia spp. and C. burnetii, respectively. Rickettsia helvetica predominated (5.9%), whereas R. monacensis (0.5%) was only sporadically detected. Coxiella burnetii was detected in 0.9%, Rickettsia spp. in 8.9% and R. helvetica in 4.2% of bird blood samples. The great tit was the bird species most infested with I. ricinus, carried R. helvetica and C. burnetti positive tick larvae and nymphs and was found to be rickettsaemic in its blood. Further studies are necessary to define the role of birds in the circulation of rickettsiae and C. burnetii in natural foci.

Survival of rat cerebrocortical neurons after rickettsial infection.

Neuroinvasive microorganisms are suspected to play an important role in the etiopathogenesis of neurological diseases. However, direct evidence for the pathogenic function is still missing. The main aim of this study was to investigate biochemical and morphological changes that may occur as a result of an in vitro infection of rat cerebrocortical neurons by selected members of the genus Rickettsia. Our results showed that survival of the neurons is significantly reduced after the infection. Intracellular level of ATP is gradually decreased and inversely correlates with the load of rickettsiae. Immunofluorescence revealed that rickettsiae can enter the neurons and are localized in perinuclear space and also in neuronal processes. Data obtained in this study correspond to the idea of possible involvement of rickettsiae in the etiopathogenesis of various neuropathies.

Isolation of Arsenophonus nasoniae from Ixodes ricinus ticks in Slovakia.

The tick Ixodes ricinus is the most prevalent and widely distributed tick species in Central Europe, commonly found in woodlands, heaths, and forests and particularly abundant in the Alpine region. This tick readily bites humans and transmits a number of bacterial and viral pathogens. We collected 10 live nymphs of I. ricinus ticks from vegetation in the Rovinka forest, Slovakia, and isolated a strain of Arsenophonus nasoniae from one tick using the BME/CTVM2 cell line. A new isolate was then subcultured on axenic media (Columbia agar supplemented with 5% sheep blood). To the best of our knowledge, this bacterium was never previously isolated from hard ticks or identified in ticks in Europe. We amplified and sequenced the 16S rRNA, rpoB, and ftsY genes. Limited genetic characterization showed that the isolated strain is almost identical to a strain from the parasitic wasp Nasonia vitripennis. Electron microscopy revealed a typical morphology of a Gram-negative bacterium, without pili or flagellae. Its role in human and animal pathology remains to be evaluated.

Multiple tick-associated bacteria in Ixodes ricinus from Slovakia.

Eighty Ixodes ricinus ticks collected in the Rovinka forest in Slovakia were tested by real-time and routine single-step PCR for the presence of different pathogenic and endosymbiotic bacteria. No evidence of Coxiella burnetii, Diplorickettsia massiliensis, or Bartonella spp. was found. However, we identified, as the pathogenic bacteria, Anaplasma phagocytophilum in 8%, Rickettsia helvetica in 11%, Borrelia afzelii in 9%, Bo. garinii in 8%, Bo. burgdorferi sensu stricto in 5%, Bo. valaisiana in 3%, and Bo. miyamotoi in 1% of ticks. Other bacteria with unknown or suspected pathogenicity for humans were also identified: Arsenophonus nasoniae in 25%, Spiroplasma ixodetis in 3%, Candidatus Midichloria mitochondrii in 4%, Wolbachia aff. pipientis in 14%, and Ehrlichia muris in 3% of ticks. A. nasoniae was found almost exclusively in nymphs, and E. muris was only found in adult ticks. In total, 65% of ticks contained at least one species of bacteria, and 16% contained 2 or more species.

Identification of Rickettsia africae and Wolbachia sp. in Ceratophyllus garei fleas from Passerine birds migrated from Africa.

The aim of the study was to reveal new aspects of the role of flea vector taken from migratory birds by screening of specimens with molecular biological methods. A field study was done in fishponds in Slovakia. Actually, 47 fleas were collected from reed warblers (Acrocephalus scirpaceus) and their nests. DNA was extracted and analyzed for representatives of the orders Rickettsiales. A rickettsia that shares 99.7% of identity by gltA gene with Rickettsia africae was identified in Ceratophyllus garei collected from A. scirpaceus. Moreover, two Wolbachia sp. were also detected in fleas. This is the first record of R. africae and Wolbachia sp. identified so far in Central Europe in fleas collected from migratory bird returning from Africa. This molecular study extends the geographic range and vector spectrum of arthropod-borne agents.

Evaluation of clinical specimens for Rickettsia, Bartonella, Borrelia, Coxiella, Anaplasma, Franciscella and Diplorickettsia positivity using serological and molecular biology methods.

We monitored clinical samples from patients of different age groups from selected regions in Slovakia. Overall seroprevalence evaluated by immunofluorescence (IFA) using nine Bartonella, two Borrelia, six rickettsial (spotted fever and typhus group), two Coxiella, and one human granulocytic ehrlichiosis Anaplasma, Franciscella tularensis and Diplorickettsia massiliensis antigens, in rural and city populations of Slovak Republic, was found to be 32% positive for spotted fever group rickettsiae. Only five (10%) of the rickettsia-positive cases evaluated by IFA were confirmed by polymerase chain reaction. Rickettsia helvetica, Rickettsia slovaca, and Rickettsia raoultii infection appear to be prevalent in Slovakia. Furthermore, Coxiella burnetii, Borrelia and, for the first time, Bartonella elisabethae were confirmed in Slovakia.

Proteomics paves the way for Q fever diagnostics.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. The disease most frequently manifests clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness that presents mainly as infective endocarditis. The extreme infectivity of the bacterium results in large outbreaks, and the recent outbreak in the Netherlands underlines its impact on public health. Recent studies on the bacterium have included genome sequencing, the investigation of host-bacterium interactions, the development of cellular and animal models of infection, and the comprehensive analysis of different clinical isolates by whole genome and proteomic approaches. Current approaches for diagnosing Q fever are based on serological methods and PCR techniques, but the diagnosis of early stage disease lacks specificity and sensitivity. Consequently, different platforms have been created to explore Q fever biomarkers. Several studies using a combination of proteomics and recombinant protein screening approaches have been undertaken for the development of diagnostics and vaccines. In this review, we highlight advances in the field of C. burnetii proteomics, focusing mainly on the contribution of these technologies to the development and improvement of Q fever diagnostics.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever.

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.

A novel obligate intracellular gamma-proteobacterium associated with ixodid ticks, Diplorickettsia massiliensis, Gen. Nov., Sp. Nov.

BACKGROUND: Obligate intracellular bacteria of arthropods often exhibit a significant role in either human health or arthropod ecology. METHODOLOGY/PRINCIPAL FINDINGS: An obligate intracellular gamma-proteobacterium was isolated from the actively questing hard tick Ixodes ricinus using mammalian and amphibian cell lines. Transmission electron microscopy revealed a unique morphology of the bacterium, including intravacuolar localization of bacteria grouped predominantly in pairs and internal structures composed of electron-dense crystal-like structures and regular multilayer sheath-like structures. The isolate 20B was characterized to determine its taxonomic position using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that this strain belongs to the family Coxiellaceae, order Legionellales of Gamma-proteobacteria, and the closest relatives are different Rickettsiella spp. The level of 16S rRNA gene sequence similarity between strain 20B and other recognized species of the family was below 94.5%. Partial sequences of the rpoB, parC and ftsY genes confirmed the phylogenetic position of the new isolate. The G+C content estimated on the basis of whole genome analysis of strain 20B was 37.88%. On the basis of its phenotypic and genotypic properties, together with phylogenetic distinctiveness, we propose that strain 20B to be classified in the new genus Diplorickettsia as the type strain of a novel species named Diplorickettsia massiliensis sp. nov. CONCLUSIONS/SIGNIFICANCE: Considering the source of its isolation (hard tick, often biting humans) the role of this bacterium in the pathology of humans, animals and ticks should be further investigated.

Life cycle of Rickettsia slovaca in L929 cell line studied by quantitative real-time PCR and transmission electron microscopy.

Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.

Coxiella burnetii genotyping.

Coxiella burnetii is a strict intracellular bacterium with potential as a bioterrorism agent. To characterize different isolates of C. burnetii at the molecular level, we performed multispacer sequence typing (MST). MST is based on intergenic region sequencing. These regions are potentially variable since they are subject to lower selection pressure than the adjacent genes. We screened 68 spacers in 14 isolates and selected the 10 that exhibited the most variation. These spacers were then tested in 159 additional isolates obtained from different geographic areas or different hosts or were implicated in different manifestations of human disease caused by C. burnetii. The sequence analysis yielded 30 different allelic combinations. Phylogenic analysis showed 3 major clusters. MST allows easy comparison and exchange of results obtained in different laboratories and could be a useful tool for identifying bacterial strains.

Naming of Rickettsiae and rickettsial diseases.

Over the last 20 years, advances in molecular techniques have greatly facilitated the identification of the members of the Rickettsiales, and numerous new species and diseases have been described. In this paper, we review taxonomic rules and appropriate approaches to valid naming of rickettsial species and the diseases they cause.