Nathalie Arricau-Bouvery

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

BackgroundCoxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).ResultsBy applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power.ConclusionOur analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.

Ovine manure used as a garden fertiliser as a suspected source of human Q fever

Correspondence to Dr Rodolakis Q FEVER is an emergent infectious disease caused by Coxiella burnetii, an intracellular bacterium. Domestic ruminants such as cattle, sheep and goats appear to be the most important source of human infections (Marrie and Raoult 1997, Maurin and Raoult 1999). Human beings contract the disease by exposure to infected animals, mostly by the inhalation of infected aerosol particles shed in the faeces, birth products and placenta of mammals. People living close to areas where ruminants graze may become infected. The organism persists for long periods in the environment and can be disseminated in air currents. This short communication describes two cases of Q fever among residents of a small town in the south of France. Ovine manure used as a garden fertiliser may have contained Coxiella species and might have been the source of contamination. In the second week of March 2000, a 58-year-old man and a 66-year-old woman visited their general practitioner with flu-like symptoms: a fever (temperature of 39 to 40°C), malaise, febrile illness, severe headaches and clinical signs of hepatitis. They were treated with a painkiller and penicillin antibiotic. However, they did not recover and were admitted to hospital for further clinical examinations. Numerous tests were performed on the basis of clinical suspicions. Blood cultures and serological examination for antibodies to pathogens were negative and C burnetii infection was suspected. By indirect immunofluorescence assay (IFA), the man and the woman had, respectively, an immunoglobulin G (IgG) anti-phase II antibody titre of 1280 and 2560, and an immunoglobulin M (IgM) anti-phase II antibody titre of 320 and 1280 (Tissot Dupont and others 1992). The presence of both an IgG antibody titre of more than 1:200 and an IgM antibody titre of

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions.

Spiroplamas are helical, cell wall‐less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram‐positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin‐less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface‐exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild‐type but not of the spiralin‐less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.

Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas.

Spiroplasma citri strain GII3 contains seven plasmids, pSciA and pSci1-6, that share extensive regions of sequence homology and display a mosaic gene organization. Plasmid pSci2 comprises 12 coding sequences (CDS), three of which encode polypeptides homologous to proteins Soj/ParA, involved in chromosome partitioning, and TrsE and Mob/TraG, implicated in the type IV secretion pathway. One CDS encodes the adhesin-like protein ScARP3d whereas the other eight encode polypeptides with no homology to known proteins. The pSci2 CDS pE and soj have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of S. citri 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS (pE) and its flanking intergenic regions. Shuttle (S. citri/Escherichia coli) plasmids, in which CDS pE was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein of the S. citri plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact soj gene were stably maintained, indicating that the soj-encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (S. citri/E. coli) plasmids, were shown to replicate in all S. citri strains tested regardless of whether the strain possesses endogenous plasmids, such as strain GII3, or not, such as strain R8A2. In addition, the pSci replicons were introduced efficiently into the plant-pathogenic spiroplasmas Spiroplasma kunkelii and Spiroplasma phoeniceum, the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of S. citri, the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plant-pathogenic spiroplasmas. As an example, a HA-tagged S. citri protein was expressed in S. kunkelii. Detection of pE-hybridizing sequences in various group I spiroplasma species indicated that pE replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas.

The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

ABSTRACT Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His6-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

Excretion of Coxiella burnetii during an experimental infection of pregnant goats with an abortive goat strain CbC1.

Coxiella burnetii is the agent of Q fever, a zoonosis responsible for mammalian reproductive disorders. Contaminated aerosols or airborne dust constitute the main source of contamination of humans and animals. They arise from placenta, feces, urine, milk, or environment because bacteria are very resistant to both drying and atmospheric agents. 1–3 Consumption of contaminated raw milk and dairy-produce could also be a source of infection. Cows, goats and sheep are more frequently involved in the disease cycle than are other animal species. Study of excretion is so important that experimental models have been made with sheep, 4 but only a few data are available about naturally infected goats. To study the pathogeny of the disease and the shedding of the bacteria in a goat model, three groups of five, six, or seven pregnant goats were subcutaneously inoculated with 10 8 (group 1), 10 6 (group 2) or 10 4 (group 3), C. burnetii strain CbC1 at 90 days of gestation. C. burnetii strain CbC1 was isolated from the placenta of an aborted goat in a French caprine flock (Allier, France). After goat inoculation, the animals were kept in a level 3 security building until about 6 weeks after delivery. Immunofluorescence, DNA extraction and Trans-PCR were used for bacterial detection as described previously 5 on cotyledon of placenta, organs of fetuses (spleen, liver, lung, stomach, and peritoneal fluids), fecal samples, vaginal swabs and milk. All pregnant goats aborted between days 25 and 48 post-inoculation, i.e., between 115 and 138 days of the gestation. Whatever the dose used, none of the kids survived more than 24 h. The cotyledons of all placentas were positive for C. burnetii by immunofluorescence and PCR tests. All the aborted kids presented a positive PCR in one organ or more, except for one kid of groups 1 and 3. At the time of abortion and the 2 subsequent days, C. burnetii organisms were shed profusely in vaginal fluid of all aborted goats. This excretion decreases with time and Coxiella was not detected 3, 14, and 10 days after abortion for group 1, group 2 or group 3, respectively. At the day of abortion and the following days, all the milk samples were

Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion.

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

Comparison of the Efficacy of Q Fever Vaccines against Coxiella burnetii Experimental Challenge in Pregnant Goats

Q fever is a zoonosis due to Coxiella burnetii, small Gram-negative intracellular bacteria. C. burnetii can be caught orally, by drinking raw milk or infected dairy products, 1 or parenterally from infected tick bites, 2 but inhalation of contaminated dust and spray is the most common route of contamination. 3,4 Although C. burnetii infects arthropods, pets, domestic and wild animals, the major source of contamination for humans is considered to be cattle, sheep, and goats . Indeed, infected female animals shed large amounts of C. burnetii through the environment in feces and vaginal discharges particularly during a normal or abortive delivery, which can contaminate other animals or humans. 5,6 Vaccination is the best way to control Q fever in ruminants, but a good vaccine must also prevent the shedding of C burnetii , in order to reduce the prevalence of Q fever and avoid risks for human health. In this work, the efficacy of inactivated vaccines against infection and Coxiella excretion, experimentally induced in goats, was evaluated.

Involvement of a Minimal Actin-Binding Region of Spiroplasma citri Phosphoglycerate Kinase in Spiroplasma Transmission by Its Leafhopper Vector

Background Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells. Methodology/Principal Findings In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49–154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His6-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission. Conclusion These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.