Variations of plasmid content in Rickettsia felis.

BACKGROUND: Since its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. (PLoS One. 2007;2(3):e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal(2) (T), but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37 degrees C but generates plaques at 30 degrees C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group. SIGNIFICANCE: We demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.

Effective size of two feral domestic cat populations (Felis catus L): effect of the mating system.

A variety of behavioural traits have substantial effects on the gene dynamics and genetic structure of local populations. The mating system is a plastic trait that varies with environmental conditions in the domestic cat (Felis catus) allowing an intraspecific comparison of the impact of this feature on genetic characteristics of the population. To assess the potential effect of the heterogenity of males' contribution to the next generation on variance effective size, we applied the ecological approach of Nunney & Elam (1994) based upon a demographic and behavioural study, and the genetic 'temporal methods' of Waples (1989) and Berthier et al. (2002) using microsatellite markers. The two cat populations studied were nearly closed, similar in size and survival parameters, but differed in their mating system. Immigration appeared extremely restricted in both cases due to environmental and social constraints. As expected, the ratio of effective size to census number (Ne/N) was higher in the promiscuous cat population (harmonic mean = 42%) than in the polygynous one (33%), when Ne was calculated from the ecological method. Only the genetic results based on Waples' estimator were consistent with the ecological results, but failed to evidence an effect of the mating system. Results based on the estimation of Berthier et al. (2002) were extremely variable, with Ne sometimes exceeding census size. Such low reliability in the genetic results should retain attention for conservation purposes.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Role of Inflammatory Monocytes in Vaccine-Induced Reduction of Helicobacter felis Infection.

Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. In this study, we evaluated the role of inflammatory monocytes in the vaccine-induced reduction of Helicobacter felis infection. We first showed by using flow cytometric analysis that Ly6C(low) major histocompatibility complex class II-positive chemokine receptor type 2 (CCR2)-positive CD64(+) inflammatory monocytes accumulate in the stomach mucosa during the vaccine-induced reduction of H. felis infection. To determine whether inflammatory monocytes played a role in the protection, these cells were depleted with anti-CCR2 depleting antibodies. Indeed, depletion of inflammatory monocytes was associated with an impaired vaccine-induced reduction of H. felis infection on day 5 postinfection. To determine whether inflammatory monocytes had a direct or indirect role, we studied their antimicrobial activities. We observed that inflammatory monocytes produced tumor necrosis factor alpha and inducible nitric oxide synthase (iNOS), two major antimicrobial factors. Lastly, by using a Helicobacter in vitro killing assay, we showed that mouse inflammatory monocytes and activated human monocytes killed H. pylori in an iNOS-dependent manner. Collectively, these data show that inflammatory monocytes play a direct role in the immunization-induced reduction of H. felis infection from the gastric mucosa.

ESCCAR international congress on Rickettsia and other intracellular bacteria.

The European Society for the study of Chlamydia, Coxiella, Anaplasma and Rickettsia (ESCCAR) held his triennial international meeting in Lausanne. This meeting gathered 165 scientists from 28 countries and all 5 continents, allowing efficient networking and major scientific exchanges. Topics covered include molecular and cellular microbiology, genomics, as well as epidemiology, veterinary and human medicine. Several breakthroughs have been revealed at the meeting, such as (i) the presence of CRISPR (the "prokaryotic immune system") in chlamydiae, (ii) an Anaplasma effector involved in host chromatin remodelling, (iii) the polarity of the type III secretion system of chlamydiae during the entry process revealed by cryo-electron tomography. Moreover, the ESCCAR meeting was a unique opportunity to be exposed to cutting-edge science and to listen to comprehensive talks on current hot topics.

Evaluation of a new serological test for the detection of anti-Coxiella and anti-Rickettsia antibodies.

Coxiella burnetii and members of the genus Rickettsia are obligate intracellular bacteria. Since cultivation of these organisms requires dedicated techniques, their diagnosis usually relies on serological or molecular biology methods. Immunofluorescence is considered the gold standard to detect antibody-reactivity towards these organisms. Here, we assessed the performance of a new automated epifluorescence immunoassay (InoDiag) to detect IgM and IgG against C. burnetii, Rickettsia typhi and Rickettsia conorii. Samples were tested with the InoDiag assay. A total of 213 sera were tested, of which 63 samples from Q fever, 20 from spotted fever rickettsiosis, 6 from murine typhus and 124 controls. InoDiag results were compared to micro-immunofluorescence. For acute Q fever, the sensitivity of phase 2 IgG was only of 30% with a cutoff of 1 arbitrary unit (AU). In patients with acute Q fever with positive IF IgM, sensitivity reached 83% with the same cutoff. Sensitivity for chronic Q fever was 100% whereas sensitivity for past Q fever was 65%. Sensitivity for spotted Mediterranean fever and murine typhus were 91% and 100%, respectively. Both assays exhibited a good specificity in control groups, ranging from 79% in sera from patients with unrelated diseases or EBV positivity to 100% in sera from healthy patients. In conclusion, the InoDiag assay exhibits an excellent performance for the diagnosis of chronic Q fever but a very low IgG sensitivity for acute Q fever likely due to low reactivity of phase 2 antigens present on the glass slide. This defect is partially compensated by the detection of IgM. Because it exhibits a good negative predictive value, the InoDiag assay is valuable to rule out a chronic Q fever. For the diagnosis of rickettsial diseases, the sensitivity of the InoDiag method is similar to conventional immunofluorescence.

ESCMID postgraduate technical workshop on intracellular bacteria: from biology to clinic.

Infection by intracellular bacteria can lead to several diseases in both veterinary and human medicine. Unfortunately, the biology of these intracellular bacteria is highly complex due to their interactions with their host cells. Thus, it is very important to develop several tools in order to better understand the complex intracellular life of these pathogens, so allowing to improve the diagnosis options and the treatments of infectious diseases that they are causing. The workshop organised in Villars-sur-Ollon (Switzerland) by the ESCMID Study group on intracellular bacteria was a good opportunity to enhance our knowledge on these fastidious pathogens. During 5 days, 15 speakers gave 41 talks, covering all fields, from biology to clinic of different intracellular bacteria such as Bartonella, Chlamydia, Coxiella, Ehrlichia, Listeria, Parachlamydia, Rickettsia, and Waddlia. The format of this postgraduate course, which took place in the Swiss mountains, allowed interactive sessions and living discussions between the participants coming from all around the world. One of the major strength was to gather epidemiologists, clinical microbiologists, infectious diseases specialists, entomologists, veterinarians as well as bioinformaticians, biochemists and biologists to deliver a unique "one-health science" on intracellular bacteria. Here, we summarise the main take-home messages delivered during this meeting.

African tick-bite fever: a new entity in the differential diagnosis of multiple eschars in travelers. Description of five cases imported from South Africa to Switzerland.

African tick-bite fever (ATBF) is a newly described spotted fever rickettsiosis that frequently presents with multiple eschars in travelers returning from sub-Saharan Africa and, to a lesser extent, from the West Indies. It is caused by the bite of an infected Amblyomma tick, whose hunting habits explain the typical presence of multiple inoculation skin lesions and the occurrence of clustered cases. The etiological agent of ATBF is Rickettsia africae, an emerging tick-borne pathogenic bacterium. We describe herein a cluster of five cases of ATBF occurring in Swiss travelers returning from South Africa. The co-incidental infections in these five patients and the presence of multiple inoculation eschars, two features pathognomonic of this rickettsial disease, suggested the diagnosis of ATBF. Indeed, the presence of at least one inoculation eschar is observed in 53-100% of cases and multiple eschars in 21-54%. Two patients presented regional lymphadenitis and one a mild local lymphangitis. Though a cutaneous rash is described in 15-46% of cases, no rash was observed in our series. ATBF was confirmed by serology. Thus, ATBF has recently emerged as one of the most important causes of flu-like illness in travelers returning from Southern Africa. The presence of one or multiple eschars of inoculation is an important clinical clue to the diagnosis. It can be confirmed by serology or by PCR of a biopsy of the eschar. Culture can also be done in reference laboratories. Dermatologists and primary care physicians should know this clinical entity, since an inexpensive and efficient treatment is available.

Reduction of Helicobacter infection in IL-10-/- mice is dependent on CD4+ T cells but not on mast cells.

BACKGROUND: In contrast to wild type, interleukin-10-deficient (IL-10(-/-)) mice are able to clear Helicobacter infection. In this study, we investigated the immune response of IL-10(-/-) mice leading to the reduction of Helicobacter infection. MATERIALS AND METHODS: We characterized the immune responses of Helicobacter felis-infected IL-10(-/-) mice by studying the systemic antibody and cellular responses toward Helicobacter. We investigated the role of CD4(+) T cells in the Helicobacter clearance by injecting H. felis-infected IL-10(-/-) mice with anti-CD4 depleting antibodies. To examine the role of mast cells in Helicobacter clearance, we constructed and infected mast cells and IL-10 double-deficient mice. RESULTS: Reduction of Helicobacter infection in IL-10(-/-) mice is associated with strong humoral (fivefold higher serum antiurease antibody titers were measured in IL-10(-/-) in comparison to wild-type mice, p < .008) and cellular (urease-stimulated splenic CD4(+) T cells isolated from infected IL-10(-/-) mice produce 150-fold more interferon-gamma in comparison to wild-type counterparts, p < .008) immune responses directed toward Helicobacter. Depletion of CD4(+) cells from Helicobacter-infected IL-10(-/-) mice lead to the loss of bacterial clearance (rapid urease tests are threefold higher in CD4(+) depleted IL-10(-/-) in comparison to nondepleted IL-10(-/-) mice, p < .02). Mast cell IL-10(-/-) double-deficient mice clear H. felis infection, indicating that mast cells are unnecessary for the bacterial eradication in IL-10(-/-) mice. CONCLUSION: Taken together, these results suggest that CD4(+) cells are required for Helicobacter clearance in IL-10(-/-) mice. This reduction of Helicobacter infection is, however, not dependent on the mast cell population.

PAR2 Promotes Vaccine-Induced Protection Against Helicobacter Infection in Mice.

BACKGROUND & AIMS: Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS: Immune responses against Helicobacter infection were compared between vaccinated PAR2(-/-) and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS: Following vaccination, PAR2(-/-) mice did not have reductions in Helicobacter felis infection (RUT values were 0.01 ± 0.01 for WT mice and 0.11 ± 0.13 for PAR2(-/-) mice; P < .05). The vaccinated PAR2(-/-) mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83 ± 1.47 for WT mice and 4.86 ± 1.35 for PAR2(-/-) mice; P < .002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350 ± 436 pg/mL for PAR2(-/-) mice; P < .03) and reduced recruitment of CD4(+) IL-17(+) T cells into the gastric mucosa of PAR2(-/-) mice following bacterial challenge (3.7% ± 1.5% for WT mice and 2.6% ± 1.1% for PAR2(-/-) mice; P < .05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4(+) T cells compared with DCs from PAR2(-/-) mice (4298 ± 347 and 3230 ± 779; P < .04), indicating that PAR2(-/-) DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2(+/+) DCs into vaccinated PAR2(-/-) mice increased vaccine-induced protection (RUT values were 0.11 ± 0.10 and 0.26 ± 0.15 for injected and noninjected mice, respectively; P < .03). CONCLUSIONS: PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.

Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model.

BACKGROUND &amp; AIMS: Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS: In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS: Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P &lt; .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P &lt; .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P &lt; .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P &lt; .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P &lt; .01], respectively). CONCLUSIONS: IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Parachlamydia acanthamoebae in domestic cats with and without corneal disease.

Corneal samples of cats with and without corneal diseases were screened with a pan-Chlamydiales PCR and specific PCRs for Parachlamydia, Protochlamydia, Chlamydophila felis, Acanthamoeba and feline herpesviruses (FHV-1). Several corneal samples tested positive for Parachlamydia and related Chlamydiales, indicating cat exposure to these intracellular bacteria.