Publications by authors named "Karim Sidi-Boumedine"

23 Publications

  • Page 1 of 1

Coxiella burnetii in slaughterhouses in Brazil: A public health concern.

PLoS One 2020 30;15(10):e0241246. Epub 2020 Oct 30.

Departamento de Higiene Veterinária e Saúde Pública, Universidade Estadual Paulista "Júlio de Mesquita Filho", Botucatu, São Paulo, Brazil.

Q fever is an important zoonosis, yet it is often neglected and can present large outbreaks, as observed in the Netherlands. In the past few years, cases of Q fever have been described in Brazil; however, the epidemiological situation of Q fever in ruminants, the main reservoir of the pathogen, is unknown in this country. Our study aimed to estimate the prevalence of C. burnetii in cattle sent to slaughterhouses using an immunofluorescence assay (IFA) and quantitative real-time PCR (qPCR). From 1515 cattle serum samples collected from nine slaughterhouses, 23.8% (360/1515) were serologically positive by IFA (cutoff titer>1:64), indicating past or recent exposure to C. burnetii infection. Among the 54 cities sampled during the study, 83.3% (45/54) had at least one seropositive animal. Subsequently, all seropositive samples were submitted to qPCR for C. burnetii DNA, and 12.2% (44/360) of the sera were qPCR positive, which indicates bacteremia and suggests active or recent infection. The results highlight the risk for abattoir workers that results from exposure to contaminated aerosols produced during slaughter procedures. Moreover, the heat maps that were construction from the positive samples demonstrate the widespread distribution of C. burnetii in the State of São Paulo, Brazil and denotes the need for surveillance and preventive measures to reduce the prevalence in cattle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241246PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598456PMC
December 2020

New Genotypes of Circulating in Brazil and Argentina.

Pathogens 2019 Dec 28;9(1). Epub 2019 Dec 28.

Departamento de Higiene Veterinária e Saúde Pública, Universidade Estadual Paulista "Júlio de Mesquita Filho", Botucatu, 18610-000 São Paulo, Brazil.

, the zoonotic agent of Q fever, has a worldwide distribution. Despite the vast information about the circulating genotypes in Europe and North America, there is a lack of data regarding strains in South America. Here, we show the presence of novel multispacer sequence typing (MST) genotypes of in two clusters detected in Brazil and Argentina that seem to be distant in parenthood. Argentinian strains isolated from a tick belongs to a new phylogenetic branch of , and the Brazilians strains may be related to MST 20 and 61. Multilocus variable number tandem repeats analysis (MLVA) typing provided a deeper resolution that may be related to host clusters of bovines, caprine, ovine, and ticks. Our results corroborate with the reports of geotypes of Thus, we highlight the need for more genotyping studies to understand the genetic diversity of in South America and to confirm the hypothesis of host-related genotypes. We also emphasize the importance of virulence studies for a better understanding of Q fever in the region, which may help in surveillance and disease prevention programs.
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http://dx.doi.org/10.3390/pathogens9010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168634PMC
December 2019

Molecular typing of Coxiella burnetii from sheep in Egypt.

Comp Immunol Microbiol Infect Dis 2019 Dec 26;67:101353. Epub 2019 Sep 26.

Anses, Sophia Antipolis Laboratory, Animal Q fever Unit, Sophia Antipolis, France.

Coxiella burnetii, the etiological agent of Q fever, is a globally distributed zoonotic disease. The disease was reported serologically in different animal species and humans in Egypt but the genetic information about circulating Coxiella strains is limited. The present study aimed to genetically characterize Coxiella positive samples, identified in abortive sheep, based on a 17-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST). Four MLVA types were found among six examined samples. While all three samples examined by MST were identified as novel sequence type (ST) closely related to human heart valve isolates from France, Saudi Arabia, USA and United Kingdom. This study provides the first genetic information about circulating Coxiella strains in Egypt and improves epidemiological data of Q fever in the country.
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http://dx.doi.org/10.1016/j.cimid.2019.101353DOI Listing
December 2019

Real-time quantitative PCR-based detection of Coxiella burnetii in unpasteurized cow's milk sold for human consumption.

Zoonoses Public Health 2019 09 7;66(6):695-700. Epub 2019 Jun 7.

Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista "Júlio de Mesquita Filho", Botucatu, Brazil.

Coxiella burnetii is a zoonotic pathogen with a worldwide distribution that is responsible for Q fever in humans. It is a highly infectious bacterium that can be transmitted from cattle to humans through the consumption of unpasteurized milk. We report the molecular identification of C. burnetii in raw cow's milk being sold directly for human consumption in Brazil without official inspection or pasteurization. One hundred and twelve samples of raw milk were analysed by real-time quantitative PCR (qPCR), and C. burnetii was detected in 3.57% (4/112) of the samples at a concentration ranging from 125 to 404 bacteria per millilitre. The identification of this zoonotic pathogen in raw milk sold directly for human consumption is a public health concern since C. burnetii can be transmitted through the oral route. This result indicates that health education and other preventive measures should be officially implemented in Brazil to prevent the spread of infection. To our knowledge, this is the first qPCR-based detection of C. burnetii in raw milk samples from cows sold in Brazil that do not undergo official inspection or pasteurization.
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http://dx.doi.org/10.1111/zph.12609DOI Listing
September 2019

Seroprevalence of horses to Coxiella burnetii in an Q fever endemic area.

Vet Microbiol 2018 Feb 13;215:49-56. Epub 2017 Nov 13.

University of Lyon, VetAgroSup, Marcy L'Etoile, France; EPIA, UMR 0346, Epidemiologie des maladies animales et zoonotiques, INRA, VetAgroSup, 63122 Saint-Genès Champanelle, France. Electronic address:

Coxiella burnetii can infect many animal species, but its circulation dynamics in and through horses is still unclear. This study evaluated horse exposure in an area known to be endemic for ruminants and humans. We assessed antibody prevalence in horse serum by ELISA, and screened by qPCR horse blood, ticks found on horses and dust from stables. Horse seroprevalence was 4% (n = 335, 37 stables) in 2015 and 12% (n = 294, 39 stables) in 2016. Of 199 horses sampled both years, 13 seroconverted, eight remained seropositive, and one seroreverted. Seropositive horses were located close to reported human cases, yet none displayed Q fever-compatible syndromes. Coxiella DNA was detected in almost 40% of collected ticks (n = 59/148 in 2015; n = 103/305 in 2016), occasionally in dust (n = 3/46 in 2015; n = 1/14 in 2016) but never in horse blood. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.
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http://dx.doi.org/10.1016/j.vetmic.2017.11.012DOI Listing
February 2018

Molecular epidemiology of Coxiella burnetii in French livestock reveals the existence of three main genotype clusters and suggests species-specific associations as well as regional stability.

Infect Genet Evol 2017 03 20;48:142-149. Epub 2016 Dec 20.

EPIA, INRA, 63122 Saint-Genès Champanelle, France. Electronic address:

Q fever is a worldwide zoonosis caused by the bacterium Coxiella burnetii. In domestic ruminants, Q fever main clinical manifestations are abortions. Although the clinical signs may differ between ruminant species, C. burnetii's genetic diversity remains understudied in enzootic areas. Here, we focused on France, where Q fever is enzootic, with the aims to (a) identify potential associations between C. burnetii genotypes and ruminant host species; (b) assess the distribution of C. burnetii genotypes both within French farms and across France's major livestock-farming regions; and (c) suggest a subset of markers for future genotypic studies. We used DNA samples collected between 2006 and 2015 from 301 females (160 cows, 76 ewes, 65 goats) aborted of Q fever within 7 different farming regions. C. burnetii diversity was determined using a multiple-locus variable-number of tandem repeat analysis (MLVA) considering 17 markers. Using a phylogenetic approach, we identified 3 main genotypic clusters divided into 12 sub-clusters. These clusters were significantly associated with ruminant species: almost all the cattle genotypes were found in a "cattle-specific" cluster whereas small ruminants genotypes essentially grouped into the two other clusters. The clusters also proved stable over space and time, some genotypes being more specifically observed in certain farming regions. We also observed some within-farm diversity but this diversity was restricted to a same genotypic cluster. Finally, we identified 6 MLVA markers that maximized the representativeness of the diversity described. Overall, we highlighted that molecular epidemiology is a relevant approach to assess C. burnetii's genetic diversity and to reveal the existence of species-specific associations and regional stability. These results will be valuable in the field to trace genotype circulation among ruminants and from ruminants to humans. Ultimately, the potential links between genotypes and virulence traits need to be investigated to adapt control measures in livestock farms.
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http://dx.doi.org/10.1016/j.meegid.2016.12.015DOI Listing
March 2017

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

Infect Ecol Epidemiol 2015 24;5:29230. Epub 2015 Nov 24.

Anses, Sophia-Antipolis Laboratory, Animal Q fever Unit, Sophia-Antipolis, France.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660934PMC
http://dx.doi.org/10.3402/iee.v5.29230DOI Listing
November 2015

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.

Microbes Infect 2015 Nov-Dec;17(11-12):782-8. Epub 2015 Oct 19.

Anses, Sophia-Antipolis Laboratory, Animal Q Fever Unit, Sophia-Antipolis, 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.
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http://dx.doi.org/10.1016/j.micinf.2015.09.026DOI Listing
October 2016

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

Trends Parasitol 2015 Nov 11;31(11):536-552. Epub 2015 Oct 11.

Unité d'Epidémiologie Animale, UR 0346 INRA, Saint Genès Champanelle, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pt.2015.06.014DOI Listing
November 2015

Impact of IS1111 insertion on the MLVA genotyping of Coxiella burnetii.

Microbes Infect 2015 Nov-Dec;17(11-12):789-94. Epub 2015 Sep 3.

Anses, Sophia-Antipolis Laboratory, Animal Q Fever Unit, Sophia-Antipolis, France.

Q fever epidemiological investigations of the likely sources of contamination may involve Coxiella burnetii MLVA for direct and rapid typing from clinical samples. However, little information is available with regards to PCR amplification failures in C. burnetii MLVA typing. This paper focuses on difficulties encountered with MLVA loci that may impact the interpretation of MLVA data and shows that some loci may constitute hotspots for mutational events. MLVA genotyping, using 17 different loci, was used on vaginal swabs (VS) from clinically infected animals as described elsewhere (Chmielewski et al., 2009). Amplicons of interest were sequenced and identified using the BLAST software by comparison with sequences available in GenBank. All VS samples produced MLVA patterns. However, amplification failures or unexpected sizes amplicons (>to 1.5 kbp), making the interpretation of MLVA complicated, were also observed. Sequencing of these amplicons revealed the presence of IS1111 element insertion. In this C. burnetii MLVA study some difficulties encountered with genotyping are highlighted and the role of IS1111 element in genome plasticity is confirmed. Finally, the need for the selection of a set of VNTRs for an efficient MLVA scheme and the question of standardization and harmonization for comparable MLVA typing data are raised again.
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http://dx.doi.org/10.1016/j.micinf.2015.08.009DOI Listing
October 2016

Whole genome PCR scanning (WGPS) of Coxiella burnetii strains from ruminants.

Microbes Infect 2015 Nov-Dec;17(11-12):772-5. Epub 2015 Aug 24.

Anses, Sophia-Antipolis Laboratory, Animal Q Fever Unit, Sophia-Antipolis, France.

Coxiella burnetii is the causative agent of Q fever, a zoonosis that spreads from ruminants to humans via the inhalation of aerosols contaminated by livestock's birth products. This study aimed to compare the genomes of strains isolated from ruminants by "Whole Genome PCR Scanning (WGPS)" in order to identify genomic differences. C. burnetii isolated from different ruminant hosts were compared to the Nine Mile reference strain using WGPS. The identified genomic regions of differences (RDs) were confirmed by sequencing. A set of 219 primers for amplification of 10 kbp segments covering the entire genome was obtained. The analyses revealed the presence of: i) conserved genomic regions, ii) genomic polymorphism including insertions and deletions and iii) amplification failures in some cases as well. WGPS, a descriptive approach, allowed the identification and localization of divergent genetic loci from various strains of C. burnetii which consisted of deletions, insertions and maybe genomic rearrangements. It also substantiates the role played by the IS1111 element in the genomic plasticity of C. burnetii. We believe that this approach could be combined with new sequencing technologies, as a selective/directed sequencing approach, particularly when repeated sequences are present in the analysed genomes.
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http://dx.doi.org/10.1016/j.micinf.2015.08.003DOI Listing
October 2016

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

PLoS Pathog 2015 May 15;11(5):e1004892. Epub 2015 May 15.

Laboratoire MIVEGEC (Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle), Centre National de la Recherche Scientifique (UMR5290)-Université de Montpellier-Institut pour la Recherche et le Développement (UR 224), Montpellier, France.

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.
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http://dx.doi.org/10.1371/journal.ppat.1004892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433120PMC
May 2015

Prevalence of Coxiella burnetii antibodies in Portuguese dairy cattle herds.

Trop Anim Health Prod 2015 Jan 23;47(1):227-30. Epub 2014 Oct 23.

CECAV, Department of Veterinary Science, Agricultural and Veterinary Sciences School, University of Trás-os-Montes e Alto Douro, Quinta de Prados, 5000-811, Vila Real, Portugal.

Q fever is an important zoonotic disease which has been recently diagnosed, mainly in sheep and goats, in Portugal. The aim of the present study was to determine the prevalence of bovine Coxiella burnetii antibodies in dairy farms from the northwest of Portugal. Bulk tank milk samples were randomly obtained, on November 2013, from 90 dairy farms and assayed using an ELISA kit. The apparent prevalence was 61.1% (95% C.I. from 50.8 to 70.5%). The proportion of negative and intermediate (inconclusive) herds was 34.5% (25.5 to 44.7%) and 4.4% (1.7 to 10.9%), respectively. In conclusion, a high level of exposure to Coxiella burnetii was observed in Portuguese dairy cattle herds, highlighting the needs to better understand the epidemiology of Q fever in Portugal by the implementation of a monitoring program based on harmonized serologic and molecular methodologies and elucidation of the infection status of the herds.
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http://dx.doi.org/10.1007/s11250-014-0679-1DOI Listing
January 2015

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

Genome Announc 2014 May 15;2(3). Epub 2014 May 15.

ANSES Sophia-Antipolis Laboratory, Sophia-Antipolis, France.

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.
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http://dx.doi.org/10.1128/genomeA.00285-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022799PMC
May 2014

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

Vet Microbiol 2014 May 3;170(1-2):117-24. Epub 2014 Feb 3.

Veterinary and Agrochemical Research Center, VAR-CODA-CERVA, Brussels, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vetmic.2014.01.025DOI Listing
May 2014

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR.

BMC Microbiol 2009 Jul 1;9:130. Epub 2009 Jul 1.

Institut National de la Recherche Agronomique (INRA), UR1282, Infectiologie Animale et Santé Publique (IASP), F-37380 Nouzilly, France.

Background: Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals.

Results: Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas) were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta) and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta) were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens.

Conclusion: We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and the pathogenetic significance of Q fever and chlamydiosis.
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http://dx.doi.org/10.1186/1471-2180-9-130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725139PMC
July 2009

Molecular epidemiology of Q fever in Poland.

Pol J Microbiol 2009 ;58(1):9-13

National Institute of Public Health-National Institute of Hygiene, Warsaw, Poland.

Coxiella burnetii is the etiologic agent of Q fever, a worldwide distributed zoonosis, accountable for serious health problem both for humans and animals. The exposure to C. burnetii infected animals and their products is the main risk factor for Q fever in humans. Several outbreaks of Q fever have been described in Poland which sources were recognized to be related to imported animals and their products or to wildlife using serological methods. Moreover, some of them have been confirmed by isolation of C. burnetii strains. In this study, multispacer sequence typing (MST) and multiple loci variable number tandem repeats (VNTR) analysis (MLVA) have been used to characterize C. burnetii strains isolated in Poland. A total of two sequence types (MST) and four MLVA types were identified among 6 C. burnetii isolates examined. This study highlighted the usefulness of these methods in the improvement of epidemiological investigations of Q fever loci on the Polish territory.
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June 2009

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

Vet Microbiol 2009 Mar 29;134(3-4):300-4. Epub 2008 Aug 29.

Agence Française de Sécurité Sanitaire des Aliments, Unité Pathologie des Petits Ruminants, 06902 Sophia Antipolis Cedex, France.

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.
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http://dx.doi.org/10.1016/j.vetmic.2008.08.024DOI Listing
March 2009

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

PLoS One 2007 Nov 28;2(11):e1229. Epub 2007 Nov 28.

Department of Cardiovascular Sciences-Surgery, St George's University of London, London, United Kingdom.

Background: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms.

Methods: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed.

Principal Findings: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice.

Conclusions/significance: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2082073PMC
November 2007

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga.

Appl Environ Microbiol 2006 Jan;72(1):854-9

Department of Cardiac and Vascular Sciences (Surgery), St. George's University of London, London SW17 0RE, United Kingdom.

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.
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http://dx.doi.org/10.1128/AEM.72.1.854-859.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1352277PMC
January 2006

Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease.

J Clin Microbiol 2003 Jul;41(7):2915-23

Department of Surgery, St. George's Hospital Medical School, London SW17 0RE, United Kingdom.

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165291PMC
http://dx.doi.org/10.1128/JCM.41.7.2915-2923.2003DOI Listing
July 2003

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests.

Microbes Infect 2003 Jun;5(7):593-602

Unité BioAgresseurs, Santé, Environnement, Institut National de la Recherche Agronomique, 37380, Nouzilly, France.

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.
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http://dx.doi.org/10.1016/s1286-4579(03)00091-1DOI Listing
June 2003

Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia.

Vet Res 2002 Mar-Apr;33(2):215-22

Institut de la Recherche Vétérinaire de Tunisie, La Rabta.

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunosorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.
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http://dx.doi.org/10.1051/vetres:2002009DOI Listing
July 2002