Protein composition of the phase I Coxiella burnetii soluble antigen prepared by extraction with trichloroacetic acid.

Q fever is a highly infectious, widespread airborne zoonosis caused by Coxiella burnetii bacterium. Humans usually acquire the disease by inhalation of contaminated aerosol produced by infected livestock. Vaccination is the most practical way for prevention and control of the disease in the exposed population. In this work, we reviewed the most important Q-fever outbreaks in Slovakia as well as the progress in vaccine development. One of them represents a soluble antigen complex produced by extraction with trichloroacetic acid from a highly purified C. burnetii phase I strain Nine Mile. It was developed at the Institute of Virology in Bratislava. The protein content of this vaccine was separated by gel electrophoresis and analyzed by mass spectrometry. The study has resulted in the identification of 39 bacterial proteins from which 12 were recognized as immunoreactive. Most of the proteins were involved in bacterium pathogenicity (41.6%) and cell wall maintenance (25%). Four of the immunoreactive proteins may possess the moonlighting activity. Definition of the vaccine components represents a prerequisite for vaccine standardization and approval by governmental authorities.

Two mice models for transferability of zoonotic bacteria via tick vector.

Spotted fever and typhus-related diseases caused by rickettsiae, Lyme borreliosis induced by spirochetes from Borrelia burgdorferii sensu lato complex, and Q fever evoked by Coxiella burnetii, are important zoonoses occurring worldwide. In order to study the pathogenesis of these infections, the efficacy of vaccines from the perspective of protection against the pathogens, pathogen - pathogen interactions during co-infections or pathogen-vector-host interrelationship, a suitable animal model should be established. In this study, we evaluated two mouse models - the C3H/N and Balb/c strains for susceptibility to infection and ability to transmit the pathogens via tick vector and to reveal the potential interactions between various bacterial tick-borne agents. Our results indicated that the C3H/N and Balb/c mice are well-accepted models of B. afzelii infection. However, they are not suitable for interaction studies with R. helvetica since the animals did not acquire rickettsiemia and do not transmit Rickettsia sp. to feeding ticks.

Update on rickettsioses in Slovakia.

The reported incidence of vector-borne diseases including various cases of Rickettsioses in humans is increasing due to a combination of climatic and social factors, escalating the opportunities for contact between people and ticks, fleas or lice. Many of the emerging infectious diseases currently challenging human health in Europe are transmitted by ticks which normally feed on domestic or wild animals. Each Rickettsia spp. has one or several tick vectors, and their geographical distribution varies according to geographical conditions; e.g.; altitude or temperature, which is gradually changing due to a global warming. Evidence of Rickettsia spp. particularly of a newly discovered species is a strong indication that a great number of diseases may be caused by so far undetected or unrecognized organisms. Their diagnosis relies mostly on rare "spot like" cooperation of clinicians with scientists, the members of the working groups that are devoted to the scientific studies of the corresponding research areas. The clinical picture of the disease caused by rickettsiae varies significantly from flu like symptoms to severe fatal outcomes, reflecting the various factors, e.g. a variability of virulence of rickettsial species due to cell invasion, dissemination of rickettsiae, genomics, immune response of an infected organism, or a tricky impact of a treatment. Several major reviews on rickettsioses have been previously published, e.g. in 1997 (Raoult and Roux, 1997a), in 2005 (Parola et al., 2005), and in 2011 (Botelho-Nevers and Raoult, 2011). In this work we intend to present a short historical overview and to describe new trends in research studies of rickettsiology. The main focus will be on rickettsioses affecting Europe΄s population.

Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever.

Q fever has emerged as an important human and veterinary public health problem in the Netherlands with major outbreaks in three consecutive years. Goat farms are probably the prime source from which Coxiella burnetii have spread throughout the environment, infecting people living in the vicinity. Coxiella burnetii infection not only spilled over from animal husbandry to humans but could also have spread to neighbouring wildlife and pets forming novel reservoirs and consequently posing another and lingering threat to humans, companion animals and livestock. In these cases, transmission routes other than airborne spread of contaminated aerosols may become significant. Therefore, the role of ticks in the transmission of Coxiella burnetii in the current situation was investigated. A total of 1891 questing Ixodes ricinus ticks and 1086 ticks feeding on pets, wildlife and livestock were tested by a recently developed multiplex Q-PCR. All ticks were negative, except for a few ticks feeding on a herd of recently vaccinated sheep. Coxiella-positive ticks were not detected after resampling this particular herd three months later. Based on these data we conclude that the current risk of acquiring Q fever from questing ticks in the Netherlands is negligible. However, for future risk assessments, it might be relevant to sample more ticks in the vicinity of previously C. burnetii infected goat farms and to assess whether C. burnetii can be transmitted transovarially and transstadially in I. ricinus ticks.

Static and dynamic systems in Rickettsia slovaca life cycle evaluated by quantitative real-time polymerase chain reaction.

Quantitative real-time polymerase chain reaction was used to characterize the growth of Rickettsia slovaca, a tick-borne pathogen transmitted by Dermacentor reticulatus and D. marginatus ticks, in static (L929 and Vero cells) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. The highest points of bacterial multiplication and the time-spans between the inoculum and the maximum of rickettsial copies were increased in consecutive order from eukaryotic cells, I. ricinus to D. marginatus systems. In dynamic system, multiplication maximum of R. slovaca was achieved 9 days earlier in I. ricinus; however, the number of rickettsial DNA copies was approximately 3.6 x 10(6) more in D. marginatus.

Ultrastructural study of the life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and Vero cell lines.

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Identification of protein candidates for the serodiagnosis of Q fever endocarditis by an immunoproteomic approach.

Q fever is a worldwide zoonosis caused by Coxiella burnetii bacterium. Two clinical forms are present: acute Q fever and chronic disease, including endocarditis. Currently, the diagnosis of Q fever endocarditis is based on the detection of anti-phase I antibodies. The objective of the study was to identify candidate proteins for the serological diagnosis of endocarditis due to C. burnetii. The immunoreactivities of sera from 12 patients with C. burnetii infections, including the sera from patients with endocarditis and with the acute clinical form of Q fever, were compared with those of three control subjects who did not have Q fever. We identified 29 candidate antigenic proteins by mass spectrometry. Two proteins, arginine repressor and OmpH, were recognised exclusively by the sera of patients with Q fever endocarditis. These proteins are promising candidates for the development of serodiagnostic assays for Q fever endocarditis.

Incidence of various tick-borne microorganisms in rodents and ticks of central Slovakia.

In this study, we detected Rickettsia helvetica, Candidatus Midichloria mitochondrii, Anaplasma phagocytophilum, Ehrlichia muris, Candidatus Neoehrlichia mikurensis, and Bartonella sp. infections in wild rodents and ticks collected from the vegetation of central Slovakia. The microorganisms were identified by PCR and sequencing. Yellow-necked mice (Apodemus flavicollis) were infected with E. muris and Bartonella sp., while ticks Ixodes ricinus collected from the vegetation were infected with R. helvetica, Candidatus M. mitochondrii, Candidatus N. mikurensis, A. phagocytophilum, and E. muris.

Rickettsial agents in Slovakian ticks (Acarina, Ixodidae) and their ability to grow in Vero and L929 cell lines.

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty-six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA-based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR-positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram-negative cell wall, and a cytoplasmic membrane.

Seasonal dynamics of Ceratophyllus sciurorum sciurorum (Schrank, 1803) (Siphonaptera) in Slovakia.

The authors pay attention to an occurrence of the taxon Ceratophyllus sciurorum sciurorum (Schrank, 1803) (Siphonaptera) in Slovakia during the whole season. The curve of occurrence this taxon of fleas has showed two high points. The first top has been achieved in April and second one followed in October. In December the authors have noticed a period and named it "cocoon block". This course of the curve has been found at the species from family Myoxidae, mostly. On the other hand, the course of curve of the birdboxes nesters has the opossite position, the high points have been recorded in autumn. A conspicuous differences has been probably caused by different biology of nesters of Myoxidae and birds in boxes. The ratio of males to females these ecological groups of hosts was different, too.