Genome sequence of Rickettsia conorii subsp. indica, the agent of Indian tick typhus

Rickettsia conorii subsp. indica is the agent of Indian tick typhus. The present study reports the draft genome of Rickettsia conorii subsp. indica strain ITTR (ATCC VR-597).     

Genome Sequence of Rickettsia conorii subsp. caspia, the Agent of Astrakhan Fever

Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.     

Spotted fever rickettsioses in southern and eastern Europe

Mediterranean spotted fever due to Rickettsia conorii conorii was thought, for many years, to be the only tick-borne rickettsial disease prevalent in southern and eastern Europe. However, in recent years, six more species or subspecies within the spotted fever group of the genus Rickettsia have been described as emerging pathogens in this part of the world. Tick-borne agents include Rickettsia conorii israelensis, Rickettsia conorii caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. Many Rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore, a new spotted fever rickettsia, Rickettsia felis, was found to be associated with cat fleas and is an emerging human pathogen. Finally, the mite-transmitted Rickettsia akari, the agent of rickettsialpox, is also known to be prevalent in Europe. We present here an overview of these rickettsioses, focusing on emerging diseases.     

Distribution of Bacillus thuringiensis subsp. israelensis in Soil of a Swiss Wetland reserve after 22 years of mosquito control

Recurrent treatments with Bacillus thuringiensis subsp. israelensis are required to control the floodwater mosquito Aedes vexans that breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of resting B. thuringiensis subsp. israelensis spores in the soil was measured. The B. thuringiensis subsp. israelensis concentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for the B. thuringiensis subsp. israelensis cry4Aa and cry4Ba genes. B. thuringiensis subsp. israelensis spores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number of B. thuringiensis subsp. israelensis treatments, the elevation of the sampling point, and the duration of the flooding periods. The number of B. thuringiensis subsp. israelensis treatments was the major factor influencing the distribution of spores in the different topographic zones (P < 0.0001). These findings indicated that B. thuringiensis subsp. israelensis spores are rather immobile after their introduction into the environment.     

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.     

Transmission electron microscopy as a tool for exploring bacterial proteins: model of RickA in Rickettsia conorii

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever, belongs to the spotted fever group of Rickettsia. It is an obligate intracellular bacterium that grows within the cytoplasm of its eukaryotic host cells. It is motile in the cytoplasm of infected cells and RickA is reported as critical protein in this aspect. However, the subcellular localization of RickA remains uncertain. We describe a simple method allowing RickA protein to be localized by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). By using IFA we showed the global expression of surface protein RickA in R. conorii organisms. The TEM results showed that RickA is widely expressed over the entire bacterial surface of R. conorii.     

Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis.

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.     

Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.     

Widespread use of real-time PCR for rickettsial diagnosis

We report 2?years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.?conorii, five Rickettsia sibirica mongolitimonae, four R.?slovaca, two R.?australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.     

Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter

Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.     

Protein candidates for the serodiagnosis of rickettsioses

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R.?typhi to be discriminated from those due to R.?conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.     

Tetracycline enhances Tn916-mediated conjugal transfer.

Pregrowth of the donor on medium containing tetracycline increased conjugative transposition of Tn916 and the transposon-dependent mobilization of pC194 19- to 119-fold in matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis. Tn916 and pC194 transferred independently under these conditions. When Enterococcus faecalis was the donor and B. thuringiensis subsp. israelensis the recipient, pregrowth in tetracycline increased the conjugative transposition frequency by approximately 15-fold. Tetracycline-enhanced conjugation appeared during matings as short as 3 h in length. Pregrowth in tetracycline did not enhance conjugation in Bacillus sphaericus x B. thuringiensis subsp. israelensis or B. thuringiensis subsp. israelensis x B. subtilis matings. Incorporation of tetracycline into the mating medium, at concentrations that did not inhibit growth of the B. thuringiensis subsp. israelensis recipient, resulted in conjugation frequencies similar to those obtained by pregrowth of the B. subtilis donors in antibiotic-containing medium. The data suggest stimulation of donor function by tetracycline.     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae.

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Continous alert for rickettsiosis in Sicily: molecular characterization of Rickettsia sp. obtained from ticks and human beings (1986-2001)

Several strains of Rickettsia sp. were isolated from patients in western Sicily with MSF (Mediterranean Spotted Fever) as well as ticks. Strains isolated were examined by PCR and identified as belonging to R. conorii sp. Importantly a strain of Israeli Spotted Fever Rickettsiae, obtained from a tick, was also identified. Our data prove that strains other than the classical R. conorii also circulate in Sicily.     

Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.     

Dog tick-borne diseases in Sicily

In Sicily many tick borne diseases are endemic, in particular way those that see like main carrier ticks that prefer, for their vital cycle, climatic conditions characterized by high temperatures and a warmth-humid atmosphere. The more important pathologies transmitted by ticks causing diseases in dogs are babesiosis and ehrlichiosis. Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia conorii, Coxiella burnetii and tick transmitted encephalitis virus assume particular relevance because they are agents of zoonosis. Our centre, C.R.A.Ba.R.T, have conducted many researches and carried out many tests for diagnostic aim in order to estimate the spread of the main tick borne diseases in Sicilians' dogs. A study lead on 342 dogs has evidenced seroprevalence for Babesia canis, Ehrlichia canis and Rickettsia respective of 5.17%, 21.70% and 53.43%. A study on zoonotic agent seroprevalences in dogs gave the following percentages: C. burnetii 31.50%, R. conorii 73.60% and A. phagocytophilum 32.80%. The data carried out from IZS Sicily diagnostic service on 5,634 tests done in 2004-2005, confirm the experimental results on the presence of B. canis, E. canis, R. conorii, A. phagocytophilum, C. burnetii, Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in all the Sicilian areas.