Publications by authors named "Annie Rodolakis"

34 Publications

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos.

Theriogenology 2019 Mar 1;126:114-120. Epub 2018 Dec 1.

LUNAM University, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, Sanitary Security of Reproduction Biotechnology Unit, Nantes, France. Electronic address:

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 10Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ± 9 × 10 bacteria/mL) than for batches of ZP-intact embryos (0.47 ± 0.19 × 10 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.
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http://dx.doi.org/10.1016/j.theriogenology.2018.11.033DOI Listing
March 2019

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

PLoS One 2015 22;10(5):e0126433. Epub 2015 May 22.

Anses, Animal Health Laboratory, Bacterial Zoonoses Unit, University Paris-Est, Maisons-Alfort, France.

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126433PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441495PMC
April 2016

Host adaptation of Chlamydia pecorum towards low virulence evident in co-evolution of the ompA, incA, and ORF663 Loci.

PLoS One 2014 1;9(8):e103615. Epub 2014 Aug 1.

INRA, UR1282 Infectiologie Animale et Santé Publique, Nouzilly, France.

Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P<10-4), as did a high number of amino acids in all incA CTRs (P = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence (P = 0.0001). Correction for ompA phylogram position in ORF663 and incA abolished the correlation between genetic changes and virulence, demonstrating co-evolution of ompA, incA, and ORF663 towards low virulence. Pairwise divergence of ompA, incA, and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals (P≤10-5), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118914PMC
April 2015

Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification.

J Microbiol Methods 2013 Dec;95(3):368-72

This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard.
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December 2013

High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

J Microbiol Methods 2012 Sep 28;90(3):241-4. Epub 2012 May 28.

Bacterial Zoonoses Unit, French Agency for Food, Environmental & Occupational Health Safety (Anses), Maisons-Alfort, France.

We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.
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http://dx.doi.org/10.1016/j.mimet.2012.05.014DOI Listing
September 2012

Detection of Coxiella burnetii, the agent of Q fever, in oviducts and uterine flushing media and in genital tract tissues of the non pregnant goat.

Comp Immunol Microbiol Infect Dis 2011 Jul 16;34(4):355-60. Epub 2011 Jun 16.

LUNAM University, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, Department of Research into the Health Risk and Biotechnology of Reproduction, Nantes, France.

The aim of the present study was the detection and quantification of Coxiella burnetii DNA in the flushing media (oviducts and uterine horns) and genital tract tissues of non pregnant goats from 20 goats chosen at random from 86 goats originating from 56 different breeding herds in south-west France. The serological prevalence rate of C. burnetii in the study population was 70.3%. The DNA of C. burnetii was identified using conventional PCR in the flushing media from the oviducts and uterus in 8/20 goats (40%) and in genital tract tissues (oviduct, uterus and ovary) in 5/20 goats (25%). This study clearly shows for the first time that the media used to flush the oviducts or uterine horns, collected using the standard embryo harvesting technique in goats, are susceptible to infection with C. burnetii. The 16 conventional PCR-positive samples were also analyzed using real-time PCR. The bacterial load of the oviduct and uterine flushing media varied from 2.9×10(4) to 7.5×10(6) bacteria per flushing medium, while the bacterial load of the tissue samples varied from 1.0×10(2) to 1.5×10(5) bacteria per mg of tissue. The infection of genital tract flushing media and tissues is a risk factor for the transmission of C. burnetii from donor to recipient during embryo transfer or to the embryo and fetus when gestation is pursued to term.
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http://dx.doi.org/10.1016/j.cimid.2011.05.002DOI Listing
July 2011

Coxiella burnetii associated placental lesions and infection level in parturient cows.

Vet J 2011 Nov 2;190(2):e135-e139. Epub 2011 Feb 2.

Department of Veterinary Disease Biology, Ridebanevej 3, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark; Department of Large Animal Sciences, Dyrlaegevej 68, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark. Electronic address:

Cotyledons (n=170) from dairy cattle were analysed for Coxiella burnetii by real-time (rt) PCR targeting the IS1111a and icd genes. Positive cases (n=90) and a random selection of negative cases (n=20) were examined by histology, immunohistochemistry and, if infection level was high, by fluorescence in situ hybridisation. PCR results were compared to bulk tank milk (BTM) antibody levels. Placental infection was detected in cows from herds at all BTM antibody levels. However the likelihood of placental infection was generally higher in herds with intermediate or high BMT antibody levels than in herds with low antibody levels. Histological examination revealed a range of mostly mild cotyledonary changes; C. burnetii infection was only rarely associated with inflammation. This may explain why bovine Q fever is usually not clinically apparent. Nevertheless, infected cattle will shed C. burnetii at calving and this can occur even in herds without BTM antibodies.
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http://dx.doi.org/10.1016/j.tvjl.2010.12.021DOI Listing
November 2011

Differential identification of Chlamydophila abortus live vaccine strain 1B and C. abortus field isolates by PCR-RFLP.

Vaccine 2010 Aug 29;28(35):5653-6. Epub 2010 Jun 29.

Unité Zoonoses Bactériennes, Agence Française de Sécurité Sanitaire des Aliments (Lerpaz), 23 Avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex, France.

Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydophila abortus and its nitrosoguanidine-induced, temperature-sensitive and virulence-attenuated live vaccine derivative identified point mutations unique to the mutant (Burall et al. [1]). Here, we evaluate the capacity of some of these mutations to either create or eliminate restriction sites using the wild-type strain C. abortus S26/3 as a reference. Three of eight genomic sites with confirmed point mutations (CAB153, CAB636 and CAB648) were retained for analysis as each resulted in the loss of a restriction site in the genome sequence of the vaccine strain. PCR-restriction fragment length polymorphism analysis using restriction enzymes chosen to specifically target the three genomic sites was then applied to a large number of C. abortus field isolates and reference strains. Our results indicate that the three mutations are uniquely present in the vaccine strain, and as such provide easy-to-use markers for the differential identification of the vaccine strain and wild-type isolates.
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http://dx.doi.org/10.1016/j.vaccine.2010.06.064DOI Listing
August 2010

Recent advances in the understanding of Chlamydophila pecorum infections, sixteen years after it was named as the fourth species of the Chlamydiaceae family.

Vet Res 2010 May-Jun;41(3):27. Epub 2009 Dec 10.

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

Chlamydophila pecorum found in the intestine and vaginal mucus of asymptomatic ruminants has also been associated with different pathological conditions in ruminants, swine and koalas. Some endangered species such as water buffalos and bandicoots have also been found to be infected by C. pecorum. The persistence of C. pecorum strains in the intestine and vaginal mucus of ruminants could cause long-term sub-clinical infection affecting the animal's health. C. pecorum strains present many genetic and antigenic variations, but coding tandem repeats have recently been found in some C. pecorum genes, allowing C. pecorum strains isolated from sick animals to be differentiated from those isolated from asymptomatic animals. This review provides an update on C. pecorum infections in different animal hosts and the implications for animal health. The taxonomy, typing and genetic aspects of C. pecorum are also reviewed.
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http://dx.doi.org/10.1051/vetres/2009075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820232PMC
June 2010

Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.

Vet Microbiol 2010 Jul 6;143(2-4):424-8. Epub 2009 Dec 6.

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

Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection.
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http://dx.doi.org/10.1016/j.vetmic.2009.11.017DOI Listing
July 2010

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

Q Fever in dairy animals.

Authors:
Annie Rodolakis

Ann N Y Acad Sci 2009 May;1166:90-3

INRA, UR 1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly.

This review evaluates the threat to human health--with the shedding of C. burnetii in dairy animals with reproductive disorders or those without clinical signs. The review also discusses the diagnosis of Q fever in livestock and the possibility of Coxiella-free herds, and it reports the available methods for controlling Q fever. C. burnetii shedding seems to occur frequently in milk taken from asymptomatic dairy cows. The number of Coxiella shed in milk is generally low. The phase I vaccine prevented abortion and greatly decreased the shedding of C. burnetii in milk.
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http://dx.doi.org/10.1111/j.1749-6632.2009.04511.xDOI Listing
May 2009

Zoonotic potential of Chlamydophila.

Vet Microbiol 2010 Jan 13;140(3-4):382-91. Epub 2009 Mar 13.

INRA, UR1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly, France.

The purpose of this article is to present the diseases induced in humans and animals by the different species of Chlamydophila, after providing an overview on the history of these infectious agents and their taxonomy. The route of transmission and the available methods for prevention and control in the different animal species are reviewed.
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http://dx.doi.org/10.1016/j.vetmic.2009.03.014DOI Listing
January 2010

Genotyping of Chlamydophila abortus strains by multilocus VNTR analysis.

Vet Microbiol 2009 Jun 24;137(3-4):335-44. Epub 2009 Jan 24.

Unité Zoonoses Bactériennes, Agence Française de Sécurité Sanitaire des Aliments (Lerpaz), 94706 Maisons-Alfort cedex, France.

Chlamydophila (C.) abortus is the causative agent of ovine enzootic abortion with zoonotic potential whose epidemiology has been held back because of the obligate intracellular habitat of the bacterium. In the present study, we report on a molecular typing method termed multiple loci variable number of tandem repeats (VNTR) Analysis (MLVA) for exploring the diversity of C. abortus. An initial analysis performed with 34 selected genetic loci on 34 ruminant strains including the variant Greek strains LLG and POS resulted in the identification of five polymorphic loci, confirming the widely held notion that C. abortus is a very homogeneous species. Analysis of additional 111 samples with the selected five loci resulted in the classification of all strains into six genotypes with distinct molecular patterns termed genotypes [1] through [6]. Interestingly, the classification of the isolates in the six genotypes was partly related to their geographical origin. Direct examination of clinical samples proved the MLVA to be suitable for direct typing. Analysis of the genomic sequences in six C. abortus prototypes of amplicons generated with each of the five selected VNTR primers revealed that variation between genotypes was caused by the presence or absence of coding tandem repeats in three loci. Amplification of Chlamydophila psittaci reference strains with the five selected VNTR primers and of the six C. abortus prototype strains with the eight VNTR primers established for the typing of C. psittaci [Laroucau, K., Thierry, S., Vorimore, F., Blanco, K., Kaleta, E., Hoop, R., Magnino, S., Vanrompay, D., Sachse, K., Myers, G.S., Bavoil, P.M., Vergnaud, G., Pourcel, C., 2008. High resolution typing of Chlamydophila psittaci by multilocus VNTR analysis (MLVA). Infect. Genet. Evol. 8(2), 171-181] showed that both MLVA typing systems were species-specific when all respective VNTR primer sets were used. In conclusion, the newly developed MLVA system provides a highly sensitive, high-resolution and easy-to-perform tool for the differentiation of C. abortus isolates of different origin, which is suitable for molecular epidemiological studies.
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http://dx.doi.org/10.1016/j.vetmic.2009.01.029DOI Listing
June 2009

Coxiella burnetii shedding routes and antibody response after outbreaks of Q fever-induced abortion in dairy goat herds.

Appl Environ Microbiol 2009 Jan 14;75(2):428-33. Epub 2008 Nov 14.

Agence Française de Sécurité Sanitaire des Aliments (French Food Safety Agency) (AFSSA), Unité Pathologie des Ruminants (Laboratory of Pathologies in Ruminants), Sophia Antipolis, France.

Q fever is a zoonosis caused by Coxiella burnetii, a bacterium largely carried by ruminants and shed into milk, vaginal mucus, and feces. The main potential hazard to humans and animals is due to shedding of bacteria that can then persist in the environment and be aerosolized. The purpose of this study was to evaluate shedding after an outbreak of Q fever abortion in goat herds and to assess the relationship with the occurrence of abortions and antibody responses. Aborting and nonaborting goats were monitored by PCR for C. burnetii shedding 15 and 30 days after the abortion episodes. PCR analysis of all samples showed that 70% (n = 50) of the aborting and 53% (n = 70) of the nonaborting goats were positive. C. burnetii was shed into vaginal mucus, feces, and milk of 44%, 21%, and 38%, respectively, of goats that aborted and 27%, 20%, and 31%, respectively, of goats that delivered normally. Statistical comparison of these shedding results did not reveal any difference between these two groups. PCR results obtained for the vaginal and fecal routes were concordant in 81% of cases, whereas those for milk correlated with only 49% of cases with either vaginal or fecal shedding status. Serological analysis, using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and complement fixation tests, showed that at least 24% of the seronegative goats shed bacteria. Positive vaginal and fecal shedding, unlike positive milk shedding, was observed more often in animals that were weakly positive or negative by ELISA or IFA. Two opposite shedding trends were thus apparent for the milk and vaginal-fecal routes. Moreover, this study showed that a nonnegligible proportion of seronegative animals that delivered normally could excrete C. burnetii.
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http://dx.doi.org/10.1128/AEM.00690-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620711PMC
January 2009

Preliminary phylogenetic identification of virulent Chlamydophila pecorum strains.

Infect Genet Evol 2008 Dec 29;8(6):764-71. Epub 2008 Jul 29.

INRA, UR1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly, France.

Chlamydophila pecorum is an obligate intracellular bacterium associated with different pathological conditions in ruminants, swine and koala, which is also found in the intestine of asymptomatic animals. A multi-virulence locus sequence typing (MVLST) system was developed using 19 C. pecorum strains (8 pathogenic and 11 non-pathogenic intestinal strains) isolated from ruminants of different geographical origins. To evaluate the ability of MVLST to distinguish the pathogenic from the non-pathogenic strains of C. pecorum, the sequences of 12 genes were analysed: 6 potential virulence genes (ompA, incA, incB, incC, mip and copN), 5 housekeeping genes (recA, hemD, aroC, efp, gap), and the ORF663 gene encoding a hypothetical protein (HP) that includes a variant 15-nucleotides coding tandem repeat (CTR). MVLST provided high discriminatory power (100%) in allowing to distinguish 6 of 8 pathogenic strains in a single group, and overall more discriminatory than MLST targeting housekeeping genes. ompA was the most polymorphic gene and the phylogenetic tree based only on its sequence differentiated 4 groups with high bootstrap values. The number of CTRs (rich in serine, proline and lysine) in ORF663 detected in the pathogenic strains was generally lower than that found in the intestinal strains. MVLST appears to be a promising method for the differential identification of virulent C. pecorum strains, and the ompA, incA and ORF663 genes appear to be good molecular markers for further epidemiological investigation of C. pecorum.
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http://dx.doi.org/10.1016/j.meegid.2008.06.009DOI Listing
December 2008

Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum.

Vet Res 2008 Nov-Dec;39(6):56. Epub 2008 Jul 25.

INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de recherche de Tours, F-37380 Nouzilly, France.

Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.
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http://dx.doi.org/10.1051/vetres:2008032DOI Listing
January 2009

Comparative diagnostic potential of three serological tests for abortive Q fever in goat herds.

Vet Microbiol 2007 Oct 27;124(3-4):286-97. Epub 2007 Apr 27.

AFSSA, Agence Française de Sécurité Sanitaire des Aliments (French Agency for Food Sanitary Safety), Unité Pathologie des Petits Ruminants (Laboratory of Pathologies in Small Ruminants), Sophia Antipolis, France.

Performances of an ELISA, an immunofluorescence assay (IFA) and a complement fixation test (CFT) were assessed for detecting antibodies against Coxiella burnetii after Q fever abortions in naturally infected goats. The goal of the study was to provide information useful for veterinary serodiagnosis in regard to categories of goats either experiencing Q fever abortion or not, blood sampling times and recommended cut-offs. The study was conducted on eight goat herds with evidence of C. burnetii abortions. In each herd, at least 5 goats that had aborted and 10 goats prior to parturition or at term were monitored 15, 30 and 60 days (D15, D30, D60) after the onset of Q fever abortion. The overall CFT results distribution did not differ between the two groups of goats and showed poor agreement with the ELISA results. In contrast, the ELISA and IFA results revealed comparable significant differences, but overall the ELISA test was slightly more sensitive than the IFA test. Seroprevalence, according to ELISA and IFA respectively, was higher in the aborting (88% and 82%) than in the non-aborting group (60% and 50%). High levels of serum antibodies were detected in goats post-abortion with an average of 114 %OD using ELISA and a log10(titer) of 2.4 using IFA. Strongly positive ELISA (%OD>80) and positive IFA results (log10(titers)>1.9) were significantly associated with abortion. Sampling on D15 gave the best association with ORs of 10 for ELISA and 6 for IFA. The practical interest of these results is discussed.
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http://dx.doi.org/10.1016/j.vetmic.2007.04.033DOI Listing
October 2007

Shedding routes of Coxiella burnetii in dairy cows: implications for detection and control.

Vet Res 2006 Nov-Dec;37(6):827-33. Epub 2006 Sep 15.

Unit of Animal Health Management, Veterinary School and INRA, BP 40706, 44307 Nantes Cedex 03, France.

Reliable detection of Coxiella burnetii shedders is a critical point for the control of the spread of this bacterium among animals and from animals to humans. Coxiella burnetii is shed by ruminants mainly by birth products (placenta, birth fluids), but may also be shed by vaginal mucus, milk, and faeces, urine and semen. However, the informative value of these types of samples to identify shedders under field conditions is unknown. Our aim was then to describe the responses obtained using a real-time PCR technique applied to milk, vaginal mucus and faeces samples taken from 242 dairy cows in commercial dairy herds known to be naturally infected with Coxiella burnetii, and to assess their putative associations. Positive results were found in all types of tested samples even in faeces. No predominant shedding route was identified. Among the shedder cows, 65.4% were detected as shedders by only one route. By contrast, cows with positive results for all three samples were scarce (less than 7%). Testing a cow based on only one type of biological sample may lead to misclassify it with regards to its shedding of Coxiella burnetii and thereby underestimate the risk of bacterial spread within a herd.
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http://dx.doi.org/10.1051/vetres:2006038DOI Listing
December 2006

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

BMC Microbiol 2006 Apr 26;6:38. Epub 2006 Apr 26.

INRA, Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France.

Background: Coxiella 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).

Results: By 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.

Conclusion: Our 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.
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http://dx.doi.org/10.1186/1471-2180-6-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1488860PMC
April 2006

Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats.

Vaccine 2005 Aug;23(35):4392-402

INRA, Pathologie Infectieuse et Immunologie, F-37380 Nouzilly, France.

Livestock is considered to be the major "source" of human Q fever. The efficacy of two currently available vaccines (Coxevac, phase I, CEVA Santé Animale and Chlamyvax FQ, phase II, MERIAL) against Coxiella excretion was investigated in terms of risks to human health. Two months before mating, 17 goats were vaccinated subcutaneously against Coxiella burnetii with an inactivated phase I vaccine and 16 goats were vaccinated with an inactivated phase II Coxiella mixed with Chlamydophila abortus vaccine. Fourteen goats were left unvaccinated. At 84 days of gestation, the goats were subcutaneously challenged with 10(4) bacteria of C. burnetii strain CbC1. Phase I vaccine was effective and dramatically reduced both abortion and excretion of bacteria in the milk, vaginal mucus and feces. In contrast, the phase II vaccine did not affect the course of the disease or excretion.
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http://dx.doi.org/10.1016/j.vaccine.2005.04.010DOI Listing
August 2005

Is Q fever an emerging or re-emerging zoonosis?

Vet Res 2005 May-Jun;36(3):327-49

Pathologie Infectieuse et Immunologie, INRA, Centre de Tours-Nouzilly, 37380 Nouzilly, France.

Q fever is a zoonotic disease considered as emerging or re-emerging in many countries. It is caused by Coxiella burnetii, a bacterium developing spore-like forms that are highly resistant to the environment. The most common animal reservoirs are livestock and the main source of infection is by inhalation of contaminated aerosols. Although the culture process for Coxiella is laborious, advances on the knowledge of the life cycle of the bacterium have been made. New tools have been developed to (i) improve the diagnosis of Q fever in humans and animals, and especially animal shedders, (ii) perform epidemiological studies, and (iii) prevent the disease through the use of vaccines. This review summarizes the state of the knowledge on the bacteriology and clinical manifestations of Q fever as well as its diagnosis, epidemiology, treatment and prevention in order to understand what factors are responsible for its emergence or re-emergence.
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http://dx.doi.org/10.1051/vetres:2005010DOI Listing
July 2005

Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model.

Can J Vet Res 2004 Jul;68(3):226-8

Unité de Pathologie Infectieuse et Immunologie, INRA-Centre de Tours, Nouzilly 37380 France.

Q fever and chlamydiosis often affect ovine and caprine flocks simultaneously or successively. Combination vaccines effective against these 2 diseases would be of great value in veterinary medicine. Unfortunately, the current effective vaccines are a live vaccine for chlamydiosis and killed vaccine for Q fever. Vaccination of mice with live chlamydiosis vaccine 1B and killed phase I vaccine against Q fever at 2 points on the back at the same time produced good protection against chlamydial abortion. This suggests that it may be possible to vaccinate ewes and goats against chlamydiosis and Q fever simultaneously.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142145PMC
July 2004

Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination.

J Med Microbiol 2004 Sep;53(Pt 9):861-868

Unité de Pathologie Infectieuse et Immunologie, INRA-Centre de Tours, 37380 Nouzilly, France.

The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models. The C. abortus groEL gene was cloned by screening a genomic library constructed in lambdaFIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C. abortus. Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %). As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES. OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection. In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice. Nevertheless, partial protection of fetuses was obtained. Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response. The anti-GroEL antibodies showed no neutralizing effect in vitro on C. abortus infectivity. Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.
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http://dx.doi.org/10.1099/jmm.0.05442-0DOI Listing
September 2004

Abortive potency of Chlamydophila abortus in pregnant mice is not directly correlated with placental and fetal colonization levels.

Infect Immun 2003 Dec;71(12):7219-22

Unité de Recherche Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Centre de Tours-Nouzilly, 37380 Nouzilly, France.

Abortion, placental and fetal colonization, and levels of gamma interferon were analyzed for four Chlamydophila abortus strains presenting antigenic variations in a mouse model. Expression of virulence of these strains varied and indicated that abortion was not directly related to the number of bacteria in the placenta, and thus, other factors may have an important role in activating the abortion process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC308942PMC
http://dx.doi.org/10.1128/IAI.71.12.7219-7222.2003DOI Listing
December 2003

Experimental Coxiella burnetii infection in pregnant goats: excretion routes.

Vet Res 2003 Jul-Aug;34(4):423-33

Pathologie Infectieuse et Immunologie, INRA, Tours-Nouzilly, 37380 Nouzilly, France.

Q fever is a widespread zoonosis caused by Coxiella burnetii. Infected animals, shedding bacteria by different routes, constitute contamination sources for humans and the environment. To study Coxiella excretion, pregnant goats were inoculated by the subcutaneous route in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria (10(8), 10(6) or 10(4) i.d.). All the goats aborted whatever the dose used. Coxiella were found by PCR and immunofluorescence tests in all placentas and in several organs of at least one fetus per goat. At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samples up to 52 days. A few goats excreted Coxiella in their feces before abortion, and all goats, excreted bacteria in their feces after abortion. Antibody titers against Coxiella increased from 21 days post inoculation to the end of the experiment. For a Q fever diagnostic, detection by PCR and immunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortion should be preferred to serological tests.
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http://dx.doi.org/10.1051/vetres:2003017DOI Listing
January 2004

Comparison of the efficacy of Q fever vaccines against Coxiella burnetii experimental challenge in pregnant goats.

Ann N Y Acad Sci 2003 Jun;990:521-3

Unité de Pathologie Infectieuse et Immunologie, INRA, 37380 Nouzilly, France.

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http://dx.doi.org/10.1111/j.1749-6632.2003.tb07421.xDOI Listing
June 2003

Proteic boost enhances humoral response induced by DNA vaccination with the dnaK gene of Chlamydophila abortus but fails to protect pregnant mice against a virulence challenge.

Vet Res 2003 Jan-Feb;34(1):119-25

Unité de Pathologie Infectieuse et Immunologie, INRA-Centre de Tours, 37380 Nouzilly, France.

In order to enhance the quantity and the protective properties of the antibodies induced by DNA vaccination with the heat shock protein dnaK gene of Chlamydophila abortus AB7 as well as to elicit an efficient cellular immune response, we vaccinated mice with a DNA prime followed by a boost with the recombinant DnaK protein. In non-pregnant mice, this strategy induced the same predominance of the IgG2a isotype as DNA immunization alone with a substantial increased antibody level. The induced antibodies had no in vitro neutralizing properties on C. abortus infectivity. Moreover, the proteic boost probably failed to elicit an efficient cellular immune response since the pregnant or non-pregnant mice were not protected against the bacterial challenge.
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http://dx.doi.org/10.1051/vetres:2002052DOI Listing
May 2003

PCR-based detection of Coxiella burnetii from clinical samples.

Methods Mol Biol 2003 ;216:153-61

INRA Tours-Nouzilly, Nouzilly, France.

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http://dx.doi.org/10.1385/1-59259-344-5:153DOI Listing
July 2003