Karim Sidi-Boumedine

The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii

Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

The Importance of Ticks in Q Fever Transmission: What Has (and Has Not) Been Demonstrated?

Q fever is a widespread zoonotic disease caused by Coxiella burnetii, a ubiquitous intracellular bacterium infecting humans and a variety of animals. Transmission is primarily but not exclusively airborne, and ticks are usually thought to act as vectors. We argue that, although ticks may readily transmit C. burnetii in experimental systems, they only occasionally transmit the pathogen in the field. Furthermore, we underscore that many Coxiella-like bacteria are widespread in ticks and may have been misidentified as C. burnetii. Our recommendation is to improve the methods currently used to detect and characterize C. burnetii, and we propose that further knowledge of Coxiella-like bacteria will yield new insights into Q fever evolutionary ecology and C. burnetii virulence factors.

Circulation of Coxiella burnetii in a Naturally Infected Flock of Dairy Sheep: Shedding Dynamics, Environmental Contamination, and Genotype Diversity

ABSTRACT Q fever is a worldwide zoonosis caused by Coxiella burnetii. Domestic ruminants are considered to be the main reservoir. Sheep, in particular, may frequently cause outbreaks in humans. Because within-flock circulation data are essential to implementing optimal management strategies, we performed a follow-up study of a naturally infected flock of dairy sheep. We aimed to (i) describe C. burnetii shedding dynamics by sampling vaginal mucus, feces, and milk, (ii) assess circulating strain diversity, and (iii) quantify barn environmental contamination. For 8 months, we sampled vaginal mucus and feces every 3 weeks from aborting and nonaborting ewes (n = 11 and n = 26, respectively); for lactating females, milk was obtained as well. We also sampled vaginal mucus from nine ewe lambs. Dust and air samples were collected every 3 and 6 weeks, respectively. All samples were screened using real-time PCR, and strongly positive samples were further analyzed using quantitative PCR. Vaginal and fecal samples with sufficient bacterial burdens were then genotyped by multiple-locus variable-number tandem-repeat analysis (MLVA) using 17 markers. C. burnetii burdens were higher in vaginal mucus and feces than in milk, and they peaked in the first 3 weeks postabortion or postpartum. Primiparous females and aborting females tended to shed C. burnetii longer and have higher bacterial burdens than nonaborting and multiparous females. Six genotype clusters were identified; they were independent of abortion status, and within-individual genotype diversity was observed. C. burnetii was also detected in air and dust samples. Further studies should determine whether the within-flock circulation dynamics observed here are generalizable.

Serological evidence of exposure to Coxiella burnetii in sheep and goats in central Portugal.

The recent outbreak of Q fever in The Netherlands warned European health authorities of the need of studying Coxiella burnetii. In Portugal, little is known about C. burnetii infection in animals. A cross-sectional study was designed to investigate the exposure to C. burnetii in sheep and goats in the Central region of Portugal, estimating the herd and individual prevalence. A serosurvey was conducted in a two levels random sampling of 89 herds and 460 animals. Individual blood samples were collected from animals older than 6 months, and specific antibodies anti-C. burnetii were detected by ELISA testing. Results showed a global herd prevalence of 32.6% (95% CI: 23.1-42.1%). Herd prevalence was higher in mixed herds (38.5%; 95% CI: 12-65%) and in sheep herds (37.5%; 95% CI: 21-54%) than in goat herds (28.8%; 95% CI: 17-41%). Global individual prevalence was estimated at 9.6% (95% CI: 6.9-12.2%), and it was higher in goats (10.4%; 95% CI: 7.8-13%) than in sheep (8.6%; 95% CI: 5.8-11.4%). Sample positive percentages (S/P) ranged from 41.5% to 185.9%. S/P percent higher than 100 was found in 18.2% (8/44) of sera from distinct herds. Positive results were significantly associated with goats, older animals and larger herds. These results revealed the presence of C. burnetii in small ruminants evidencing their potential role in the infection cycle.

Prevalence and molecular typing of Coxiella burnetii in bulk tank milk in Belgian dairy goats, 2009–2013

Q fever, a worldwide zoonosis, is an arousing public health concern in many countries since the recent Dutch outbreak. An emerging C. burnetii clone, genotype CbNL01, was identified as responsible for the Dutch human Q fever cluster cases. Since 2009, Q fever surveillance in the goat industry was implemented by the Belgian authorities. The herd prevalence (December 2009-March 2013) ranged between 6.3 and 12.1%. Genotypic analysis highlighted the molecular diversity of the Belgian strains from goats and identified an emerging CbNL01-like genotype. This follow-up allowed the description of shedding profiles in positive farms which was either continuous (type I) and associated to the CbNL01-like genotype; or intermittent (type II) and linked to other genotypes. Despite the circulation of a CbNL01-like strain, the number of notified Belgian human cases was very low. The mandatory vaccination (in June 2011) on positive dairy goat farms in Belgium, contributed to a decrease in shedding.

Draft Genome Sequences of Six Ruminant Coxiella burnetii Isolates of European Origin

ABSTRACT Coxiella burnetii is responsible for Q fever, a worldwide zoonosis attributed to the inhalation of aerosols contaminated by livestock birth products. Six draft genome sequences of European C. burnetii isolates from ruminants are presented here. The availability of these genomes will help in understanding the potential host specificity and pathogenicity and in identifying pertinent markers for surveillance and tracing.

Evaluation of randomly amplified polymorphic DNA (RAPD) for discrimination of Coxiella burnetii ruminant strains isolated in France

Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Ruminant species represent the main reservoir of the bacterium, as high levels of the bacteria are shed in birth products and other excreta. Human infection generally occurs after inhalation of contaminated aerosols [1]. The availability of methods to study the distribution and the spread of a given C. burnetii strain, from different geographical areas or hosts, would promote a better understanding of the epidemiology of this pathogen. Several genetic typing methods for C. burnetii have been evaluated and appeared to be useful tools for epidemiological and phylogenetic purposes. However, RAPD [2,3] has never been evaluated for C. burnetii typing. In this work, a RAPD protocol was used to evaluate the genetic diversity among C. burnetii ruminant strains in comparison to published MLVA data [4]. Ten French C. burnetii isolates obtained from goats, sheep, cows and the reference strain Nine Mile, were used for RAPD typing (Table 1). Whole DNA was extracted using the Qiagen Dneasy kit (Courtaboeuf, France), following the manufacturer’s protocol. To assess optimal RAPD conditions several parameters were tested, for instance using a range of MgCl2 concentrations (from 1.5 to 5.5 mM), or different brands of Taq polymerase. RAPD PCRs were performed using a set of four 10-mer primers (P4M, 5¢-AAGACGCCGT-3¢; PR5, 5¢-AGTCGTCCCC-3¢; PR10A, 5¢AGGGCCGTCT3¢; and PR12A, 5¢-CAGCTCACGA-3¢). Unless specified, the optimised PCR conditions were, for a final volume of 25 lL in dH2O: 1X Taq polymerase buffer (without MgCl2); 3 mM MgCl2; 0.2 mM each dNTPs; 1U of Taq polymerase; 0.5 lM 10-mer primer; and 10 ng of DNA (all the reagents were purchased from Invitrogen, Cergy Pontoise, France). Negative controls consisting of dH2O only were included in each run. Amplifications were performed in an Eppendorf Mastercycler (Le Pecq, France), programmed for an initial denaturation step of 5 min at 94 C, 45 cycles of 96 C for 30 s, 37 C for 30 s, 72 C for 90 s and a final extension step at 72 C for 5 min. The PCR products were separated on 0.8% agarose gel containing ethidium bromide, visualised and photographed under UV light. Gel analysis was performed using the Quantity One 1-D Analysis software from Bio-Rad (Marnes la Coquette, France). An RAPD type was defined after the combination of the patterns obtained with the four primers used in this study (Table 1). The reproducibility of the method was evaluated by repeating the same assay several times, by different manipulators, as well as by using independent DNA preparations. Briefly, RAPD profiles were stable when varying MgCl2 concentration. However, a higher yield of amplification, estimated by the intensity of the bands observed on the gels, was observed using 2.5–3.5 mM MgCl2. This yield decreased when using higher concentrations of MgCl2 (data not shown). A MgCl2 concentration of 3 mM was therefore chosen for subsequent analyses. Different RAPD patterns were observed when different brands of Taq polymerases were used (data not shown). The Hot start platinum Taq polymerase (Invitrogen) produced the best patterns in our hands and was chosen for all the experiments. After optimisation of our RAPD protocol, the use of each primer generated distinct polymorphisms allowing differentiation of the studied strains. Strains from neighbouring flocks, that were indistinguishable using MLVA analysis, could be discriminated using RAPD typing (Table 1). For instance, CbB2 and CbB5 are two strains obtained in 2001 from neighbouring flocks, but were isolated from cows displaying different clinical signs (Table 1). CbB2 was isolated from a case of metritis and CbB5 from an aborted cow. Corresponding author and reprint requests: Dr V. Duquesne, AFSSA, Sophia Antipolis, Unite de Pathologie des Petits Ruminants, 105 Route des Chappes, BP111, 06902 SophiaAntipolis, France E-mail. v.duquesne@afssa.fr

Molecular methods routinely used to detect Coxiella burnetii in ticks cross-react with Coxiella-like bacteria

Background Q fever is a widespread zoonotic disease caused by Coxiella burnetii. Ticks may act as vectors, and many epidemiological studies aim to assess C. burnetii prevalence in ticks. Because ticks may also be infected with Coxiella-like bacteria, screening tools that differentiate between C. burnetii and Coxiella-like bacteria are essential. Methods In this study, we screened tick specimens from 10 species (Ornithodoros rostratus, O. peruvianus, O. capensis, Ixodes ricinus, Rhipicephalus annulatus, R. decoloratus, R. geigy, O. sonrai, O. occidentalis, and Amblyomma cajennense) known to harbor specific Coxiella-like bacteria, by using quantitative PCR primers usually considered to be specific for C. burnetii and targeting, respectively, the IS1111, icd, scvA, p1, and GroEL/htpB genes. Results We found that some Coxiella-like bacteria, belonging to clades A and C, yield positive PCR results when screened with primers initially believed to be C. burnetii-specific. Conclusions These results suggest that PCR-based surveys that aim to detect C. burnetii in ticks by using currently available methods must be interpreted with caution if the amplified products cannot be sequenced. Future molecular methods that aim at detecting C. burnetii need to take into account the possibility that cross-reactions may exist with Coxiella-like bacteria.

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats.

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.

Validation study for using lab-on-chip technology for Coxiella burnetii multi-locus-VNTR-analysis (MLVA) typing: application for studying genotypic diversity of strains from domestic ruminants in France.

Coxiella burnetii, the etiologic bacterium of Q fever zoonosis, is still difficult to control. Ruminants are often carriers and involved in human epidemics. MLVA is a promising genotyping method for molecular epidemiology. Different techniques are used to resolve the MLVA band profiles such as electrophoresis on agarose gels, capillary electrophoresis or using the microfluidic Lab-on-Chip system. In this study, system based on microfluidics electrophoresis with Lab-on-Chip technology was assessed and applied on DNA field samples to investigate the genotypic diversity of C. burnetii strains circulating in France. The Lab-on-Chip technology was first compared to agarose gel electrophoresis. Subsequently, the set-up Lab-on-Chip technology was applied on 97 samples collected from ruminants in France using the 17 markers previously described. A discordance rate of 27% was observed between Lab-on-Chip and agarose gel electrophoresis. These discrepancies were checked and resolved by sequencing. The cluster analysis revealed classification based on host species and/or geographic origin criteria. Moreover, the circulation of different genotypic strains within the same farm was also observed. In this study, MLVA with Lab-on-Chip technology was shown to be more accurate, reproducible, user friendly and safer than gel electrophoresis. It also provides an extended data set from French ruminant C. burnetii circulating strains useful for epidemiological investigations. Finally, it raises some questions regarding the standardization and harmonization of C. burnetii MLVA genotyping.