New way of purification of pathogenic rickettsiae reducing health risks.

In general, cultivation and purification of intracellular pathogenic rickettsiae represents a risk for laboratory personnel due to exposure to highly infectious aerosol or accidental inoculation during these procedures. In this study, we describe an alternative, effective and time saving technique for rickettsial purification using digitonin to release intracellular bacteria from host cell without physical disruption. No significant differences were noted in yield and infectivity between digitonin treated rickettsiae and rickettsiae purified by sonication. This is the first report of using digitonin in purification of pathogenic rickettsiae and this approach might be effective for other intracellular pathogenic bacteria.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

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.

Isolation of rickettsia helvetica from ticks in Slovakia.

UNLABELLED: To date, only three rickettsial species have been found in ticks in Slovakia by serological and/or molecular-biological techniques, namely Rickettsia slovaca, Candidatus rickettsia IRS, and Rickettsia raoultii. Recently, we succeeded in isolation of the forth species, Rickettsia helvetica from Ixodes ricinus, the most frequent tick in Slovakia. The isolation, positive for 10% of tested ticks, was performed on XTC cells by the shell-vial technique, Gimenez staining and light microscopy. The infected cell cultures contained rod-shaped particles morphologically identical to rickettsiae. The isolation was confirmed by direct detection of a fragment of the R. helvetica gene for citrate synthase in the positive ticks by PCR and its subsequent cloning, sequencing and comparison with the database. KEYWORDS: Rickettsia helvetica; isolation; Ixodes ricinus; Slovakia.

Characterization of antigens for Q fever serodiagnostics.

The aim of this study was to identify candidate proteins for serodiagnostics of Q fever by monoclonal antibodies (MAbs), and to clone, express, and purify the selected proteins for use as antigens in ELISA. The reactivity of three MAbs to Coxiella burnetii (C. b.) Nine Mile strain and one MAb to Priscilla strain was tested using SDS-PAGE, 2-D gel electrophoresis, immunoblot analysis, and mass spectrometry. Three immunoreactive Q fever-specific proteins discriminated by MAbs, namely the CBU_0937 protein, outer membrane Com1 (CBU_1910) protein, and elongation factor Tu (CBU_0236) were identified. Successful PCR-amplification, cloning, expression, and purification of the recombinant proteins Com1 and CBU_0937 allowed their use for the screening of sera from patients with Q fever endocarditis (18) or acute Q fever (16) in ELISA. The recombinant protein CBU_0937 with unknown biological function proved to be a more applicable diagnostic tool for Q fever ELISA as compared to the Com1 protein.

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.

Identification and characterization of Coxiella burnetii strains and isolates using monoclonal antibodies.

We evaluated 6 monoclonal antibodies (MAbs) for their usefulness in identifying and characterizing recognized laboratory strains as well as field isolates of Coxiella burnetii. Five had been generated in response to strain Nine Mile (3 IgM class, 1 IgG class, 1 light chain producers only) and were polypeptide-specific, and 1 was anti-Priscilla (IgG class) and was lipopolysaccharide (LPS)-specific. Initially, the MAbs were used in conjunction with a dot blot assay with which we could differentiate C. burnetii from rickettsiae or chlamydiae. Confirmation of the specificity of these MAbs was provided by demonstrating that only C. burnetii antigens were recognized by certain combinations of antibodies used for immunoblotting proteins of various C. burnetii strains. Subsequently, we characterized antigens of 11 C. burnetii field isolates and 3 reference strains by Western blotting with individual MAbs. MAb 921 and 922 (IgG class), MAb 241, 242, 384, 386, 614 (IgM class), and 7A5, 7A1 (light chain) consistently recognized a protein. Staining intensity differed, depending on the strain tested, and there was variability in the size of the antigen immunoreactive with MAb 14H (IgG class, LPS-specific). The most reactive region was at about 249 kD. Variability of reactivities with field isolates was seen in both the distribution of individual bands and their intensities. We conclude that an extensive immunoblotting technique may be useful for C. burnetii strain differentiation and routine identification of C. burnetii can be accomplished using this MAb-based dot blot assay.

Monitoring of humans and animals for the presence of various Rickettsiae and Coxiella burnetii by serological methods.

Serological examination of humans in Slovakia suspected of having rickettsial infections revealed the presence of antibodies to spotted fever group rickettsiae (R. conorii, R. slovaca, and R. typhi). Of interest is the finding of serological positivity to the newly recognized "IRS" agent. Antibodies to these rickettsiae and to C. burnetii were demonstrated also in domestic and hunting dogs and pet animals. These results confirm the occurrence and possible circulation of these rickettsiae and C. burnetii in the Slovak Republic.

Analysis of specificity of interaction between synthetic key saccharide components of lipopolysaccharides and monoclonal antibodies to Coxiella burnetii.

Two monoclonal antibodies (MAbs) against the lipopolysaccharides (LPSs) of Coxiella burnetii (C.b.) strains Priscilla and Nine Mile were prepared characterized by their interaction with synthetic glycoconjugates representing parts of LPSs of C.b. in virulent phase. Both MAbs were directed against immunodominant epitopes comprising core constituent of LPSs, Kdo (3-deoxy-alpha-D-manno-2-octulo-pyranosylonic acid). ELISA showed that the anti-Nine Mile MAb 4/11 bound preferably to disaccharides (alpha-Kdo (2 --> 4) alpha-Kdo and alpha-Kdo (2 --> 4) alpha-(5d) Kdo), while the anti-Priscilla MAb 1/4/H bound to all conjugates, though with various intensity. On the other hand, immunoelectron microscopy revealed a positive binding of only one glycoconjugate, namely the trisaccharide alpha-Kdo (2 --> 4) alpha-Kdo (2 --> 4) alpha-Kdo-BSA, to both MAbs. In competitive ELISA (cELISA), the anti-Priscilla MAb 1/4/H distinguished the strains Nine Mile and Priscilla, while the anti Nine Mile MAb 4/11 did not.

Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein.

'Gene D' is the PS120-protein-encoding gene, first described in Rickettsia conorii and Rickettsia japonica. Sequence analysis of a 3030 bp fragment of 'gene D' in 24 representatives of the genus Rickettsia was carried out to complete phylogenetic analyses previously inferred by comparison of gene sequences encoding citrate synthase, 17 kDa antigen and rOmpA and rOmpB. The phylogenetic relationships between rickettsiae were inferred from the comparison of both the gene and the derived protein sequences, using the parsimony, neighbour-joining and maximum-likelihood methods. Five distinct groups of rickettsiae were identified. These were: the Rickettsia massiliae group, including R. massiliae, Bar 29, Rickettsia rhipicephali and Rickettsia aeschlimannii; the Rickettsia rickettsii group containing Rickettsia sibirica, 'Rickettsia mongolotimonae', Rickettsia parkeri, strain S, Rickettsia africae, the R. conorii complex, Rickettsia slovaca, Rickettsia honei, R. rickettsii, R. japonica and Rickettsia montanensis; the group currently containing only Rickettsia helvetica; the Rickettsia akari group including Rickettsia australis, R. akari and the ELB agent; Rickettsia prowazekii and Rickettsia typhi clustered in the typhus group. As significant bootstrap values were obtained for most of the nodes, sequence comparison of 'gene D' should be considered as a complementary approach in phylogenetic studies of rickettsiae.

Characterization of a new spotted fever group rickettsia detected in Ixodes ricinus (Acari: Ixodidae) collected in Slovakia.

Two previously undescribed rickettsiae were detected in Ixodes ricinus Ricketts by polymerase chain reaction. Ixodes ricinus Slovakia (IRS) 3 and IRS4 were identified in ticks collected in northeastern and southwestern Slovakia, respectively. Sequences of the 16S rRNA citrate synthase (gltA) and outer membrane protein rOmpA (ompA) encoding genes of both strains were nearly identical but were distinct from those of all other known rickettsiae. Phylogenetic relationships inferred from the comparison of these sequences with those of other members of the genus Rickettsia indicate that IRS3 and IRS4 constitute a new rickettsial genotype and form a separate cluster among the spotted fever group rickettsiae.

Intraspecies diversity of Coxiella burnetii as revealed by com1 and mucZ sequence comparison.

Coxiella burnetii is classified within the gamma subgroup of the Proteobacteria. All strains tested to date have an identical 16S rRNA sequence but 20 different genotypes have been determined by pulsed field gel electrophoresis (PFGE). In this study, intraspecies genetic diversity was investigated by sequence comparison of 715 bp of the Com1 encoding gene (com1) and 774 bp of the MucZ encoding gene (mucZ) in 37 strains isolated from animals and humans with acute or chronic Q fever in Europe, North America and Africa. Five and four groups were established from sequence analysis of com1 and mucZ, respectively. Neither relation of the defined groups to geographical distribution of the isolates was noted nor relation to disease form (acute/chronic). The same isolates were grouped together regardless of the gene being investigated. Comparison of the five proposed groups to previous groups, yielded after digestion by NotI PFGE, allowed for an intermediate classification of C. burnetii isolates between those obtained by using 16S rDNA (one group) and PFGE (20 groups).

Rickettsia slovaca sp. nov., a member of the spotted fever group rickettsiae.

The name Rickettsia slovaca sp. nov. (type strain is strain B) is proposed for a member of the spotted fever group (SFG) rickettsiae which was isolated from Dermacentor marginatus ticks in Slovakia in 1968, and was recently implicated in human febrile illness. This rickettsia can be phenotypically distinguished from other SFG rickettsiae by microimmunofluorescence serotyping, SDS-PAGE, Western blotting and mAbs. Genotypic differences between R. slovaca and the other SFG representatives can be demonstrated by PCR-RFLP analysis, pulsed-field gel electrophoresis and sequencing of 16S rRNA, gltA and ompA genes.

Monoclonal antibody based differentiation of Coxiella burnetii isolates.

Isolates of Coxiella burnetii from different geographic regions in Europe, USA, Japan and Africa were compared in their binding properties to the monoclonal antibody (MoAb) 1/4/H directed against the lipopolysaccharide (LPS) of C. burnetii strain Priscilla. Immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) revealed different binding patterns of C. burnetii isolates under study. Most of the isolates tested did react with MoAb 1/4/H. Only four of 20 groups of isolates and one isolate of an otherwise positively reacting group did not react with MoAb 1/4/H. The results indicate a significant variation of LPS structure of the C. burnetii isolates studied.

Monoclonal antibodies to Coxiella burnetii that cross-react with strain Nine Mile.

Results are presented to show the binding properties of five monoclonal antibodies directed to Coxiella burnetii Priscilla with cross-reactions to the Nine Mile strain. The monoclonal antibodies preferentially recognize phase I epitopes by ELISA and recognize phase II epitopes by immunoblotting but do not allow differentiation between so-called chronic and acute strains of C. burnetii. The only difference in reactivity was in the staining pattern revealed after reactions with lipopolysaccharide I antigens.

Penetration of host cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin.

Internalization of obligate intracellular bacteria belonging to the genus Rickettsia by eukaryotic cells requires participation of both the parasitized host and the microorganism. The term "induced phagocytosis" has been used specifically to describe the entry of Rickettsia prowazekii, although a similar mechanism is likely for R. rickettsii. A role for a phospholipase in the internalization process has been proposed for both of these organisms, with the strongest supporting evidence provided for R. prowazekii. Despite general acceptance of the notion that phospholipase activity is involved in the internalization process of these bacteria, the origin of the enzyme is not known. The results of the study presented here, which used R. rickettsii and Vero cells, suggest that a rickettsial phospholipase, rather than a host cell phospholipase, mediates internalization of the organism. This conclusion is based upon results which show that pretreatment of R. rickettsii, but not of host cells, with a specific chemical inhibitor of phospholipase, and also antiserum to this enzyme, significantly reduces uptake of the organism and its ability to cause plaque formation.

Heparin protects human endothelial cells infected by Rickettsia rickettsii.

Routine culture of endothelial cells currently includes the use of heparin, which significantly reduces cell doubling time and increases cell population size. Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites produced by the action of xanthine and xanthine oxidase. Because of our hypothesis implicating free radicals in cell injury caused by Rickettsia rickettsii, we have carried out a series of experiments to examine the effects of heparin on injury to endothelial cells infected by this microorganism. These studies showed that heparin does not inhibit replication of R. rickettsii in the cytoplasm of endothelial cells. Furthermore, heparin appears to exhibit a protective effect on the infected host cell as measured by (i) reduced plaque size, (ii) increased longevity of the cell monolayer, (iii) reduction in the amount of lactic dehydrogenase released from infected cells, and (iv) reduction in the levels of intracellular peroxides formed in infected cells. Electron microscopic studies also show a significant reduction in dilatation of the rough-surfaced endoplasmic reticulum of the infected cells in the presence of heparin. These observations appear to lend additional support to involvement of an oxidative mechanism in human endothelial cell injury caused by R. rickettsii.

Extensive examination of different tick species for infestation with Coxiella burnetii in Slovakia.

Between 1987-1989 almost 7000 adult Ixodes ricinus, Dermacentor reticulatus, Dermacentor marginatus, Haemaphysalis concinna, Haemaphysalis punctata and Haemaphysalis inermis ticks collected in all 38 districts of Slovakia were screened for the presence of Coxiella burnettii. The proportion of ticks containing C. burnetii as indicated by the haemocyte test was less than 3%. Attempts to recover C. burnetii by inoculation of yolk sacs of embryonated hen eggs from pools of 1-6 specimens of haemocyte test positive ticks resulted in the isolation of 10 rickettsial strains. Six strains were recovered from I. ricinus, the remaining ones from single pools of D. reticulatus, D. marginatus. H. concinna and H. inermis ticks. In addition to the previous recovery of C. burnetii from H. punctata ticks, the agent was thus isolated from all important ticks living in Slovakia. The agent was found in tick habitats regardless of the latitude and altitude in the entire country. These results are not consistent with the negligible number of Q fever cases occurring in past years in Slovakia.