Armel Souriau

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.

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.

Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescence test with monoclonal antibodies

Monoclonal antibodies (mAbs) were produced to find strain markers essential to the epidemiological study of chlamydial abortion of ruminants. Their specificity was tested on 53 C. psittaci strains including 35 ruminant strains isolated mainly from abortion, belonging to serotype 1 and which are invasive in our mouse model (Rodolakis et al., 1989), and 14 ruminant strains mostly intestinal, belonging to serotype 2 and non-invasive for mouse. One strain specific mAb was obtained reacting only with the non-invasive strain iB1. Six sub serotype 2 mAbs were found. They reacted only with some non-invasive strains. They allowed the distinction of 9 patterns of response among the 14 non-invasive strains. No serotype 2 specific mAbs reacting with all non-invasive serotype 2 strains were selected. In return all the invasive strains reacted with all the 18 serotype 1 specific mAbs produced. No cross-reactivities between invasive and non-invasive strains were observed, whereas common epitopes were demonstrated between invasive strains and avian or feline strains.

Restriction endonuclease analysis of DNA from ruminant Chlamydia psittaci and its relation to mouse virulence

DNA from 20 pathogenic or non-pathogenic ruminant strains of Chlamydia psittaci was compared by restriction endonuclease analysis. The strains could be easily differentiated according to their invasiveness for mouse, whatever their pathological origin. DNA patterns of invasive strains were similar, whereas those of non-invasive strains were distributed in two groups.

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

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

Serotyping of Chlamydial Clinical Isolates from Birds with Monoclonal Antibodies

Forty-nine avian chlamydial strains, isolated mainly from various regions in France and from different species of birds, were analyzed and tested with a panel of nine monoclonal antibodies (MAbs) by the indirect microimmunofluorescence test (MIF). The MAbs included five serovar-specific MAbs, three MAbs raised against Chlamydia psittaci and Chlamydia pecorum ovine strains, and one genus-specific MAb. Of the 49 isolates, 41 came from parrots or budgerigars; the rest were from pigeons, a canary, a duck, and a dove. Two additional strains were from unknown hosts. Most of these avian strains were successfully serotyped according to their reactions with five serovar-specific MAbs by the MIF test. The serovars of 44 strains were determined: 39 were of serovar A, 3 of serovar B, and 2 of serovar E. The remaining five isolates were unclassified because they did not react with any of five serovar-specific MAbs but did react with genus MAb or the MAbs produced with ovine strains. The five unclassified isolates (two from budgerigars, two from Gabon gray parrots, and one from a duck) indicate that one or more additional serovars of C. psittaci exist in birds. The heterogeneity within each subgroup was evident because the 49 avian isolates gave 10 subgroups when the results of the five serovar-specific MAbs were combined with results from the three MAbs produced with ovine strains. This heterogeneity of the serovar isolates, as shown by the combination of MAbs, could provide strain markers very useful for epidemiologic studies.