Annie Rodolakis

The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction

The polymerase chain reaction (PCR), targeting the repetitive transposon-like region of Coxiella burnetii (Trans-PCR), was evaluated for its ability to detect directly C. burnetii in genital swabs, milk and fecal specimens of ewes. By using a combination of centrifugation step, DNA purification using Qiamp Tissue kit followed by Trans-PCR assay, the efficiency for detection of coxiella in ewes milk samples was further improved and one C. burnetii-cell could be detected in 1ml of milk. In addition, an effective, simple and rapid method to remove PCR-inhibitory substances from fecal specimens by simply diluting the DNA template to 1:100 is described, which made the detection of one microorganism per mg of sample then possible. The results obtained from seropositive ewes proved that C. burnetii could also be detected in milk and fecal samples of naturally infected animals.

Coxiella burnetii Shedding Routes and Antibody Response after Outbreaks of Q Fever-Induced Abortion in Dairy Goat Herds

ABSTRACT 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.

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

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

Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep.

Two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes. The seroprevalence of C bumetii infection was studied by using an ELISA and the immunofluorescence (IF) assay was applied to the contents of vaginal swabs. In addition, a KCR assay, with primers based on a transposonlike repetitive region of the C bumetii genome (trans-PCR), was used for the highly sensitive and specific detection of C bumetii in vaginal swabs, milk and faeces. Of the 34 animals tested at parturition, eight (24 per cent) were positive by ELISA, 11 (32 per cent) were positive by IF, and 15 (44 per cent) were positive when the vaginal swab extract was subjected to the trans-PCR assay. C bumetii was therefore detected by PCR in the vaginal swabs of seven seronegative ewes. However, five weeks after lambing, 16 (47 per cent) of the animals tested were ELISA positive but only two animals (6 per cent) were positive by PCR. Among the ELISAand PCR-positive animals, eight (25 per cent) shed coxiella in their milk and six (18 per cent) did so in their faeces.

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.

Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.

Q Fever in Dairy Animals

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.

Zoonotic potential of Chlamydophila.

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.

Experimental Coxiella burnetii infection in pregnant goats: a histopathological and immunohistochemical study.

Pregnant goats were inoculated subcutaneosly with Coxiella burnetii and the course of infection was studied. Abortion in the last third of pregnancy occurred in all infected animals. Tissues from the placenta and other organs were studied before and after abortion by immunohistochemistry and PCR analysis. After infection, mild lesions were observed in several maternal organs, mainly the mammary gland but also the lung and the liver. The trophoblast cells of the choriallantoic membrane were the first target cells of the placenta; there was, however, a substantial delay between initial infection and placental colonization. In the last weeks of pregnancy, just before abortion, massive bacterial multiplication was detected in the placenta. In this stage of infection a necrotic and suppurative placentitis separated the fetal trophoblast cells from maternal syncytial epithelium. Vasculitis was observed in the fetal mesenchyme. A strong maternal T-cell response was detected in the inter-placentomal areas but not in the placentomes, where only neutrophils and smaller numbers of macrophages were associated with the lesions. Neither lesions nor C. burnetii DNA were found in maternal organs in animals maintained until day 120 post-abortion.

Improved sensitivity of PCR for Chlamydophila using pmp genes.

Primers targeting the conserved pmp gene family of Chlamydophila abortus were evaluated for their ability to improve the polymerase chain reaction (PCR) sensitivity. In purified DNA, specific pmp primers (named CpsiA and CpsiB) allowed at least a 10-fold increase of the PCR sensitivity compared to the specific ompA primers for C. abortus, but also for C. psittaci and C. caviae strains. No amplification was observed on C. felis, C. pecorum, C. pneumoniae and Chlamydia trachomatis strains. Tested on contaminated specimens such as genital swabs, the PCR sensitivity observed with CpsiA/CpsiB was also better than with the ompA primers. This study demonstrated that these specific pmp primers could serve as valuable, sensitive and common tools for a specific Chlamydophila diagnosis in ruminant, avian and human diseases. Digestion by AluI of the CpsiA/CpsiB fragments allowed a specific discrimination of the strains in function of their hosts and/or their serotypes.