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Background: The ability of tick-borne agents to survive in stored blood bags is a key factor for their transmissibility by blood transfusion. The aim of this study was to evaluate the survival and potential infectivity of Rickettsia conorii (RC) in artificially contaminated canine whole blood (WB) and in leukoreduced whole blood (LR-WB) during the storage period., Methods: RC was cultured on L929 cells. We used a one-week 25-cm 2 flask with 70-80% of L929 infected cells to prepare the bacterial inoculum by pelleting cells and suspending the pellet in the donors' serum. We infected five 100 ml WB units with RC within 2 h from the collection and maintained it at room temperature for 4 h prior to refrigeration. We filtered 50 ml of each WB bag to obtain leukoreduced WB (LR-WB) at day 1 post-infection (dpi). We checked WB and LR-WB bags at 1, 4, 7, 14, 21, 28, 35 dpi for RC presence and viability through real-time PCR (rPCR) for DNA and mRNA, respectively, and by isolation. Identification of isolates was confirmed by indirect immunofluorescence and rPCRs., Results: RC survived for the entire storage period in both whole and leukoreduced blood. All bags contained viable bacteria until 7 dpi; RC viability generally decreased over time, particularly in LR-WB bags where the isolation time was longer than in WB. Viable bacteria were still isolated at 35 dpi in 3 WB and 3 LR-WB., Conclusions: Leukoreduction reduced but did not eliminate RC in infected units. The survival and infectivity of RC in canine blood during the storage period may represent a threat for recipients.

A number of spotted fever group (SFG) rickettsiae cause serious infections in humans. Several antigenically related rickettsial agents may coexist within the same geographical area, and humans or vertebrate hosts may be sequentially exposed to multiple SFG agents. We assessed whether exposure of a vertebrate reservoir to one SFG Rickettsia will affect the host's immune response to a related pathogen and the efficiency of transmission to uninfected ticks. Two pairs of dogs were each infected with either Rickettsia massiliae or Rickettsia conorii israelensis, and their immune response was monitored twice weekly by IFA. The four immunized dogs and a pair of naïve dogs were each challenged with R. conorii israelensis-infected Rhipicephalus sanguineus nymphs. Uninfected Rh. sanguineus larvae were acquisition-fed on the dogs on days 1, 7, and 14 post-challenge. These ticks were tested for the presence of rickettsial DNA after molting to the nymphal stage. The naive dogs became infected with R. conorii israelensis and were infectious to ticks for at least 3 weeks, whereas reservoir competence of dogs previously infected with either R. massiliae or R. conorii was significantly diminished. This opens an opportunity for decreasing the efficiency of transmission and propagation of pathogenic Rickettsia in natural foci by immunizing the primary hosts with closely related nonpathogenic SFG bacteria. However, neither homologous immunization nor cross-immunization significantly affected the efficiency of R. conorii transmission between cofeeding infected nymphs and uninfected larvae. At high densities of ticks, the efficiency of cofeeding transmission may be sufficient for yearly amplification and persistent circulation of a rickettsial pathogen in the vector population., (Published by Elsevier GmbH.)

The role played by dendritic cells (DCs), initiators and orchestrators of the immune response, remains unclear in rickettsial infections. To investigate their importance in rickettsioses, we analyzed the responses of murine bone marrow-derived DCs (BMDCs) after rickettsial stimulation in vitro and their protective role in vivo. Rickettsia conorii stimulation of BMDCs caused significant maturation and production of proinflammatory cytokines. Transfer of rickettsiae-stimulated DCs protected mice from lethal rickettsial challenge by limiting rickettsial proliferation in vivo, whereas partial protection was observed in mice receiving lipopolysaccharide (LPS)-stimulated DCs. Immunity to R. conorii after transfer of DCs was associated with up-regulation of CD40, CD80, CD86, and major histocompatibility complex class II; with production of IL-2, IL-12, and IL-23; and with production of antigen-specific interferon- gamma in T cells. Taken together, our data suggest that a vigorous proinflammatory response in DCs is associated with protective immunity to rickettsiae and that generation of antigen-specific immunity is crucial to complete protection.

The efficacy of several anti-tick treatments to prevent the transmission of Rickettsia was evaluated under natural conditions of tick pressure in a kennel. Only Amitraz (Preventic) provided total control on transmission (no dogs were infected), whereas with Frontline, Advantix, or Scalibor, the rate of infection varied among the dogs.

In the past century vaccine development for prevention of rickettsial diseases has been prolific. However, in the past 20 years no new rickettsial vaccine has been manufactured and there are currently no new or old rickettsial vaccines licensed. Early rickettsial vaccines were difficult, expensive and very hazardous to produce. Molecular biology techniques of today are currently being used to develop new rickettsial vaccines that are standardized, inexpensive, nonhazardous and efficacious.

Until very recently, Mediterranean spotted fever caused by Rickettsia conorii was the only spotted fever group (SFG) rickettsioses recognized in southern Africa. However, increasing medical awareness of tick-borne infections, together with the introduction of improved isolation methods and the availability of molecular techniques, have led to the identification of several new SFG rickettsioses in the region. African tick bite fever, caused by Rickettsia africae, is currently the most important of these new rickettsioses, affecting large numbers of international travellers each year, but infections due to Rickettsia aeschlimannii and Rickettsia mongolotimonae have also been recently encountered. In this review, we describe the current status of the epidemiology, microbiology, clinical presentation, diagnosis, treatment, and prevention of SFG rickettsioses in southern Africa.

An emphasis on cellular immunity against Rickettsia has led to neglect of analysis of the role of antibody. The availability of an excellent mouse model of spotted fever rickettsiosis enabled investigation of a potential role of antibody in immunity to Rickettsia conorii. C3H severe combined immunodeficiency (SCID) mice were passively transfused with monoclonal antibodies against rickettsial outer membrane protein A (OmpA), OmpB, or lipopolysaccharide (LPS), polyclonal anti-R. conorii serum, Fab fragments of polyclonal antiserum, or no antibodies and then challenged 48 h later with 10 50% lethal doses (LD(50)) of R. conorii. All mice that received monoclonal antibodies against OmpA and two of four mice that received monoclonal antibodies against OmpB or polyclonal antisera were completely protected, but the recipients of anti-LPS antibodies or the Fab fragments were not protected. Polyclonal antibody treatment of C3H SCID mice that had been infected with 10 LD(50) of R. conorii 4 or 5 days earlier prolonged the life of the infected mice from 10.4 to 22.5 days and resulted in decreased levels of infectious rickettsiae in the spleen and liver 24 and 48 h later. Treatment with protective antibodies resulted in the development of large aggregates of R. conorii antigens in splenic macrophages and intraphagolysosomal rickettsial death and digestion. The kinetics of development of antibodies to R. conorii determined by immunoblotting revealed antibodies to LPS on day 6 and antibodies to OmpA and OmpB on day 12, when recovery from the infection had already occurred. Antibodies to particular epitopes of OmpA and OmpB may protect against reinfection, but they may not play a key role in immunity against primary infection. Antibodies might be useful for treating infections with antibiotic-resistant organisms, and some B-cell epitopes should be included in a subunit vaccine.

A randomized clinical trial was undertaken to compare clinical and serologic parameters of the efficacy of one-dose azithromycin in the prophylaxis of Mediterranean spotted fever (MSF) in 122 persons with a tick bite history. Antibodies to Rickettsia conorii (R. conorii) were detected by indirect immunofluorescence (IFA) assay in 55 (45.1%) of 122 subjects. Positive result was obtained in 19 (31.1%) of 61 subjects with azithromycin prophylaxis and in 36 (59.0%) of 61 subjects without prophylaxis. Clinical signs and symptoms of MSF were recorded in 6 (9.8%) and asymptomatic infection in 30 (49.2%) of 61 subjects without prophylaxis. In subjects with prophylaxis, clinical signs of the disease were not recorded at all, whereas asymptomatic infection was detected in 19 (31.1%) of 61 subjects. Based on the results obtained in our study, we assume that a single dose of azithromycin is promising in MSF prevention.

Fragments representing the genes of the two major outer membrane proteins of spotted fever group rickettsiae (rOmpA and rOmpB) were tested as DNA vaccines. Immunizations with each of three fragments (rompA4999-6710, rompB1550-2738, and rompB2459-4123) conferred a degree of protection on vaccinated mice against virulent rickettsial challenge. Protection was achieved when DNA immunizations were followed by booster immunizations with the homologous recombinant protein. Proliferation and gamma-interferon secretion were detected after in vitro stimulation of lymphocytes from immunized animals with whole Rickettsia conorii antigen. The data validate particular segments of rOmpA and rOmpB as potent immunogens and hence as sources of immunostimulatory elements with specificity for T lymphocytes, which are the key effectors of protective immunity against rickettsial infections.

Background: Epidemiologic features of tick typhus among German travelers has not been surveyed recently., Methods: Clinical features, travel and medical histories in 78 patients with tick typhus who presented to a German outpatient clinic for Infectious and Tropical Diseases were investigated, in order to identify common epidemiological factors and potential strategies of prevention. Diagnosis was confirmed by serological detection of IgG- and IgM-antibodies to Rickettsia conorii by indirect immunofluorescence., Results: The majority of patients (71.8%) had visited southern Africa prior to presentation. All patients presented with fever as the main symptom. An eschar was still present in 68 patients (87.2%) with regional lymphadenitis in 19.2%. However, only a minority of patients (17.9%) remembered a tick bite at the location of the eschar., Conclusion: Efforts to reduce the incidence of tick typhus in travelers should focus on preventive measures targeting behavioral changes. Avoiding tick bites during travel to endemic areas appears to be the single most important prophylactic action. Taking this into consideration, it should be possible to decrease the number of travelers returning with tick typhus significantly by adequate pretravel counseling.

Antibodies to Rickettsia conorii were detected by the indirect immunofluorescence (IFA) assay in 64 (51.6%) out of 124 examinees living in North Dalmatia (Croatia) who had a history of recent tick bites during 1994 and 1995. Positive titers of IFA antibodies to R. conorii were detected in 12 (31.5%) out of 38 examinees with carried out prophylaxis by azithromycin. The usual clinical signs of Mediterranean spotted fever were registered in 22 (25.6%) and asymptomatic infection in 30 (34.8%) out of 86 examinees without prophylaxis. Clinical signs of the disease were not registered in examinees with prophylaxis.

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.