Burnetii DNA Research Articles

Coxiella burnetii (Q fever) in Rattus norvegicus and Rattus rattus at livestock farms and urban locations in the Netherlands; could Rattus spp. represent reservoirs for (re)introduction?

The Q fever outbreak in the Netherlands in 2007–2010 prompted government interventions to reduce the human incidence by reduction of Q fever shedding at dairy goat farms. Mandatory hygiene measures were taken, including the control of animal reservoirs. It has been postulated that brown rats, through their commensal nature, form an important factor in the persistent dissemination of endemic circulating Coxiella burnetii in nature to domestic animals, livestock and humans. Here, the occurrence of C. burnetii in rats captured at different types of location during the Q fever outbreak in the Netherlands, viz. urban areas, nature areas and various types of farm has been determined. This is a first step towards the elucidation of the reservoir status of rats in veterinary and human Q fever epidemiology. C. burnetii DNA was detected in the spleen of 4.9% of the brown rats ( Rattus norvegicus) and 3.0% of the black rats ( Rattus rattus). Evidence for C. burnetii infection was also found in liver, kidney, lung and intestinal tissue but not in heart, brain and pancreas. C. burnetii IgGs were detected in 15.8% of the brown rats. Positive rats were collected at goat, pig, cattle and poultry farms, and urban locations; including locations outside the designated 5 km “increased-risk” zones around bulk milk positive goat farms. The percentage of rat-positive locations was the highest for goat farms (50%) and cattle farms (14.3%). The presence of actively infected rats outside the lambing season and at multiple environmental settings including urban locations might suggest that rats are not merely a spill-over host due to infection by a contaminated environment but might represent true reservoirs, capable of independent maintenance of C. burnetii infection cycles and thereby contributing to spread and transmission of the pathogen. If frequent (re)introduction of C. burnetii to small ruminant farms can be caused by rats as maintenance reservoirs, mandatory wildlife control and lifelong vaccination of herds will be necessary.

Study and Management of a Q Fever Outbreak among Machine Tool Workers in the Basque Country (Spain)

The aim of this study is to describe a Q fever outbreak that affected the staff of a machine-tool factory in the Basque Country between 2009/12/20 and 2010/02/23. Study subjects were interviewed using a Q fever specific questionnaire and tested for Q fever serology (immunofluorescence assay with phase II antigen) and detecting Coxiella burnetii DNA using real-time PCR. We interviewed and tested 40 employees (90% of the staff). 33 employees, all of them men, had positive serology (attack rate 82.5%, 95% CI: 70.2–94.8). Mean age was 43.7 years (95% CI: 38.7–48.7) in positive men, 33.7 years (95% CI: −16.6–83.9) in negative men, and 36.25 (95% CI: 27.5–45.0) in women (all negatives). 15 cases (45.5%) were asymptomatic, 9 (27.3%) had flu-like symptoms, and the other 9 (27.3%) had developed radiologically confirmed pneumonia. We obtained 28 blood samples, 22 faeces samples, 11 milk samples, and one vaginal swab from 28 goats resting in a stable near the factory. Serology was positive in 18 goats (64.3%). All environmental samples were negative.

A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small-cell variant (SCV) by real-time PCR (qPCR) in clinical samples. A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. The silica column extraction method was more efficient at recovering C. burnetii DNA, from large-cell and small-cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.

Coxiella burnetii DNA in goat milk after vaccination with Coxevac ®

Q fever is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally. A large Q fever epidemic is currently spreading throughout the Netherlands with more than 3500 human cases notified from 2007 to 2009. Governmental measures to prevent further spread of the disease imposed in December 2009 included vaccination of all dairy goats and sheep and, in parallel, bulk tank milk testing to identify contaminated goat and sheep farms. When bulk tank milk was found to contain C. burnetii DNA, pregnant ruminants were culled. An important, but unsolved issue in this policy was whether vaccine-derived C. burnetii DNA is excreted in milk after vaccination. Using real time PCR and single nucleotide polymorphism (SNP) genotyping techniques, we show here that within hours and up to 9 days after vaccination with Coxevac ®, vaccine-derived C. burnetii DNA can be detected in the milk of dairy goats. This is the first report describing DNAlactia of vaccine-derived DNA after vaccination with a completely inactivated vaccine. This finding had implications for the Dutch policy to combat the Q fever epidemic. A 2-week interval was introduced between vaccination and bulk tank milk testing to identify infected farms.

Q Fever in Woolsorters, Belgium

Q Fever in Woolsorters, Belgium

Coxiella burnetii shedding and environmental contamination at lambing in two highly naturally-infected dairy sheep flocks after vaccination

Abortion due to Coxiella burnetii was confirmed in the 2007/08 season in two naturally-infected dairy sheep flocks. Proportion of C. burnetii shedders and bacterial loads in vaginal mucus were high among aborted or lambed ewes, as was within-flock seroprevalence. Before the next reproductive season (2008/09) 75% of ewes and 50% of replacement lambs were vaccinated (Coxevac, CEVA Santé Animale) keeping the remaining as untreated controls. Compared with the previous year results when abortion outbreak started, a great reduction in the percentage of abortions, in the number of shedders and in the bacterial burden excreted by the ewes was found in both flocks. However, seroconversion in non-vaccinated yearlings from both flocks and the presence of C. burnetii DNA in bioaerosols taken at sheep premises at lambing indicated that infection was still active. No differences were observed between vaccinated and control groups in terms of proportion of C. burnetii shedders. These results suggest that optimal results of vaccination in heavily infected flocks may not be obtained in a short-term period.

A Large Q Fever Outbreak in an Urban School in Central Israel

BACKGROUND. On 28 June 2005, numerous cases of febrile illness were reported among 322 students and employees of a boarding high school located in an urban area in central Israel. Subsequent investigation identified a large outbreak of Q fever which started 2 weeks earlier. We describe the investigation of this outbreak and its possible implications. METHODS. We conducted a case-control study to identify risk factors for Q fever disease. Environmental sampling was conducted to identify the source and the mode of transmission of Coxiella burnetii, the infectious agent. RESULTS. Of 303 individuals, 187 (62%) reported being ill between 15 June and 13 July 2005. Serological evidence for C. burnetii infection was evident in 144 (88%) of the 164 tested individuals. Being a student, dining regularly at the school dining room, and boarding at school during a June religious holiday and the preceding weekend were all significant risk factors for contracting Q fever. C. burnetii DNA was detected using polymerase chain reaction on samples from the school dining room's air conditioning system, supporting contribution of the air conditioning system to the aerosol transmission of the infectious agent. CONCLUSIONS. We report a large outbreak of Q fever in an urban school, possibly transmitted through an air conditioning system. A high level of suspicion for C. burnetii infection should be maintained when investigating point source outbreaks of influenza-like disease, especially outside the influenza season.

A survey of Western Australian sheep, cattle and kangaroos to determine the prevalence of Coxiella burnetii

The objective of this study was to investigate the prevalence of Coxiella burnetii in two domestic ruminant species (cattle and sheep) and the western grey kangaroo ( Macropus fuliginosus) in Western Australia (WA). The IDEXX CHEKiT Q Fever ELISA and CFT were used to test sera from 50 sheep and 329 head of cattle for anti- C. burnetii antibodies and 343 kangaroo sera were tested using an indirect ELISA developed specifically for this study. Faecal or urine samples collected from the same animals were tested with two PCR assays to identify active shedding of C. burnetii in excreta. Only two of the 379 ruminant sera had detectable levels of anti- C. burnetii antibodies according to the ELISA while the CFT did not detect any positive samples. In contrast 115 of the 343 western grey kangaroo serum samples were positive when tested with the antibody-ELISA. The first qPCR assay, targeting the IS1111a element, identified 41 of 379 ruminant and 42 of 343 kangaroo DNA samples as positive for C. burnetii DNA. The second qPCR, targeting the JB153-3 gene, identified nine C. burnetii DNA-positive ruminant samples and six positive kangaroo samples. Sequence comparisons showed high degrees of identity with C. burnetii. Isolation of C. burnetii from faeces was also attempted but was not successful. From the results presented here it appears that domestic ruminants may not be the most significant reservoir of C. burnetii in WA and that kangaroos may pose a significant threat for zoonotic transfer of this pathogen.

Coinfection with Coxiella burnetii in infectious endocarditis

Currently, most cases of Q fever endocarditis are diagnosed serologically, when levels of anti-phase I antibodies are ‡1:800 [1]. In Marseille, France, we proposed a systematic serological testing for all pathogens that could cause negative blood culture IE for all patients suspected to have IE. We demonstrated that this approach is useful in establishing the aetiological diagnosis of IE [2]. Thanks to this systematic serological testing, we could diagnose four patients with IE due to a typical microorganism and high antibody titres to C. burnetii. In a retrospective study, we enrolled patients, hospitalised in Marseille, between April 1994 and January 2007, with C. burnetti phase I IgG ‡1:800 and IE caused by another microorganism. The modified Duke criteria were used to define cases of endocarditis [3]. Each patient underwent testing that was guided by a diagnostic kit as described previously [2]. Titres of IgG, IgM and IgA antibodies in serum samples were determined by the indirect immunofluorescence assay [2]. Valvular surgical samples or heparinised blood samples were inoculated onto human embryonic lung fibroblasts grown on a coverslip in a shell vial. After a 7-day incubation period at 37 C, cultivated bacteria were detected by a direct immunofluorescence assay incorporating rabbit monoclonal antibodies to C. burnetii. A DNA extract was prepared from the valvular surgical samples with the QIAmp tissue kit (Qiagen, Hilden, Germany) and the QIAamp Blood Kit (Qiagen) for blood samples according to the manufacturer’s instructions. Bacterial DNA was detected by PCR amplification with universal primers and sequencing of the amplified fragment, as previously described [4]. For C. burnetii, the PCR was performed using a LightCycler apparatus (Roche), using two different target genes: the IS1111 repeated intervening sequence and the IS30a intervening sequence. Valvular surgical samples, when available, were embedded in paraffin, and sections were stained with hematoxylin-eosin-safran for histology. For detection of C. burnetii in tissues, immunochemistry was done on deparaffinised sections by use of an anti-C. burnetii rabbit polyclonal antibody, as previously described [5]. Four patients (6.5& of patients with IE tested) had IE according to Duke’s criteria due to common endocarditis pathogens (Streptococcus gallolyticus for patients 1 and 2, Streptococcus mitis for patient 3, and Enterococcus faecalis for patient 4) and anti-phase I C. burnetii antibodies ‡1:800. Characteristics of the patients and microbiological results are presented in Table 1. Only patient 1 had a past history of previous fever and asthenia suggestive of Q fever. All these patients underwent valvular surgery in the course of the disease. The valvular sample of patient 1 was positive for C. burnetii for the two targeted genes. Patient 3 had Q fever serology very suggestive of chronic infection with high titres of IgG1 1:102 400 and IgA1 1:12 800. Patients 2 and 4 had serology IgG1 of 1:800. For these two patients there was no other evidence of chronic Q fever. All the patients were treated for bacterial IE according to local guidelines and for Q fever endocarditis. All the patients improved and remain healthy at present. We report here four patients with IE due to typical microorganisms and Q fever serology with IgG1 ‡800. We could recover C. burnetii DNA in valvular samples in patient 1. Patient 3 had very high titres (1:102 400). We concluded that these two patients surely had an ongoing chronic Q fever. The two other patients with IgG1 1:800 had no evidence of ongoing illness. Patient 1 and patient 3 were included in a prospective study evaluating the contribution of systematic serological testing in the diagnosis of IE [2]. As witnessed in these four cases, the diagnosis of Q fever may Corresponding author and reprint requests: Prof. Didier Raoult, Unite des Rickettsies, CNRS UMR6020, IFR48, Faculte de Medecine, 27, Boulevard Jean Moulin, 13385 Marseille, France E-mail: didier.raoult@univmed.fr

Role of specific antibodies in Coxiella burnetii infection of macrophages

Q fever is a zoonosis with worldwide distribution caused by Coxiella burnetii, an obligate intracellular bacterium. Q fever is commonly divided into acute and chronic forms. Acute Q fever manifestations consist of self-limited febrile illness, pneumonia and granulomatous hepatitis as well as neurological disorders and miscellaneous manifestations [1]. The manifestations of chronic Q fever are endocarditis and, less frequently, vascular aneurysm and prosthesis infections. These usually occur in patients with previous vascular or valvular disease or in a context of immunosuppression. The chronic disease is characterised by impaired immune response, defective microbicidal activity of monocytes, and production of antibodies (Abs) directed against C. burnetii. Here, we examined the role of specific antibodies in the infection of human macrophages by C. burnetii. Immunoglobulins G (IgG) from three patients with chronic Q fever and three healthy controls were purified using protein A-sepharose columns (Pharmacia, Uppsala, Sweden). Their ability to opsonise C. burnetii was investigated by flow cytometry. About 85% of phase I organisms were opsonised by specific Abs (1 ⁄ 200 dilution) whereas irrelevant IgG were unable to opsonise bacteria. The intracellular fate of opsonised organisms within human monocyte-derived macrophages (MDM) was studied by real-time PCR as previously described [2]. Opsonised organisms intensively replicated within MDM whereas unopsonised organisms survived but poorly replicated: after 9 days, the bacterial load was 10 times higher in MDM infected with opsonised bacteria (98 400 vs. 10 200 C. burnetii DNA copies for 10 MDM). In addition, electron microscopy revealed that the replication of opsonised organisms was associated with the formation of spacious parasitophore vacuoles containing large amounts of bacteria (Fig. 1a), which were absent in MDM infected with unopsonised bacteria (Fig. 1b). At day 9 post-infection, about 30% of MDM infected with opsonised bacteria presented large vacuoles (Fig. 1c) whereas only 3% of MDM displayed large vacuoles when infected with unopsonised bacteria (Fig. 1c). Note that the large parasitophore vacuoles presented an electron-dense membrane and seemed unable to fuse with lysosomes (Fig. 1a). This prompted us to characterise the nature of the large vacuoles by scanning fluorescence confocal microscopy using cathepsin D as a marker of phagolysosomes [3]. It has been previously shown that C. burnetii prevents the phagosome conversion and survives in monocytes ⁄macrophages within late phagosomes, in contrast to avirulent and inactivated organisms, which are eliminated within phagolysosomes [3,4]. At day 9 post-infection, opsonised organisms did not co-localise with cathepsin D (Fig. 1d), demonstrating that the replicative large vacuoles are not phagolysosomes. Classically, it is believed that Abs provide protection against pathogens through different mechanisms, including opsonisation. We demonstrated here that the high levels of specific Abs found in chronic Q fever, which are likely to be related to the host inability to cure the infection, favoured C. burnetii replication within human MDM. In patients with C. burnetii endocarditis, the major manifestation of chronic Q fever, C. burnetii was found in infected cardiac valves as voluminous intracytoplasmic masses within infected mononuclear cells, and not extracellularly as for other aetiological agents of infective endocarditis [1]. We can hypothesise that C. burnetii-specific Abs produced during chronic Q fever are responsible for C. burnetii infection of cardiac valves. We also demonstrated that C. burnetii prevented the phagosome conversion because large parasitophore vacuoles containing organisms did not express cathepsin D, a specific Corresponding author and reprint requests: E. Ghigo, URMITE, Faculte de Medecine, 27 Bld. Jean Moulin, 13385 Marseille Cedex 05, France E-mail: eric.ghigo@univmed.fr

Role of the cytoplasmic pattern recognition receptor Nod2 in Coxiella burnetii infection

Q fever is caused by Coxiella burnetii, an obligate intracellular bacterium, which survives in an acidic vacuole and escapes killing by preventing phagosome maturation [1]. In acute Q fever, granuloma formation is dependent on migration of leucocytes through the vascular endothelium. During chronic Q fever, granulomas are lacking and monocytes do not migrate across the endothelium [1], suggesting that host resistance against C. burnetii requires innate immune components. Toll-like receptors (TLRs) are pattern recognition receptors important in C. burnetii recognition. Although TLR4 ⁄) and control mice have a similar pattern of tissue infection and clearance, granuloma formation and cytokine response are controlled, in part, by TLR4 [1]. TLR2 is also involved in granuloma formation but seems dispensable for bacterial clearance [2]. The obligate intracellular lifestyle of C. burnetii drew our attention to elucidate the contribution of another family of proteins implicated in microbial detection, the cytoplasmic nucleotide-binding oligomerization domain (Nod) proteins, and more specifically Nod2, in innate immunity to C. burnetii. Nod2 contains leucine-rich repeats similar to those found in TLRs and is thought to be responsible for recognition of peptidoglycans from Gram positive and Gram negative bacteria through direct or indirect interaction with muramyl dipeptide. Nod2 signalling contributes to inflammatory response via activation of the transcription factor NF-jB, which is necessary for clearing pathogens. The intracellular localisation of Nod2 and C. burnetii makes Nod2 a potential candidate for C. burnetii recognition through leaking into the cytoplasm of muramyl dipeptide-containing cell wall fragments. Bone marrow-derived macrophages (BMDM) from control and Nod2 ⁄) mice [3] were incubated with C. burnetii for 4 h and cultured for 12 days. Infection was measured by determining the number of C. burnetii DNA copies as previously described [2]. Uptake and survival of C. burnetii in BMDM did not depend on Nod2 as DNA copy number was similar in control and Nod2 ⁄) BMDM (data not shown). Next, we tested whether the production of cytokines ⁄ chemokines by C. burnetii-stimulated BMDM required Nod2. We assessed the expression of genes encoding pro-inflammatory (TNF) and immunoregulatory cytokines (IL-10). BMDM were stimulated for 8 h with C. burnetii and gene expression was monitored by quantitative RTPCR [2]. In Nod2 ⁄) BMDM, although TNF transcripts were induced, they were significantly reduced compared with control macrophages (Fig. 1a). This result agrees with other studies showing that TNF and IL-12p40 levels are reduced in the absence of Nod2 after stimulation with Mycobacterium tuberculosis [4]. Note that IL10 was not affected by the absence of Nod2 (Fig. 1a). IL-10 is known to interfere with macrophage activation by stimulating the replication of C. burnetii through decreasing TNF production and blocking phagosomal maturation [1]. IL-10 is also associated with the chronic evolution of Q fever [1]. The absence of IL-10 modulation observed in Nod2 ⁄) BMDM is consistent with these data, as we did not observe any increase in bacterial replication. We also measured the expression of RANTES and MCP-1, two chemokines modulated in response to C. burnetii [5]. RANTES expression was fourfold increased in Nod2 ⁄) BMDM whereas MCP-1 expression was decreased by 40% (Fig. 1a). It is likely that RANTES and MCP-1 play a role in Q fever pathophysiology, as their production is Corresponding author and reprint requests: B. Desnues, CNRS UMR 6236, Faculte de Medecine de la Timone, 27 Bd J. Moulin, 13385 Marseille Cedex 05, France. E-mail: benoit.desnues@univmed.fr

Laboratory Maintenance of Coxiella burnetii

AbstractCoxiella burnetii, an obligate intracellular Gram‐negative bacterium, is the agent of Q fever, a self‐limited flu‐like illness that may also present as chronic endocarditis. The ability to persist in the environment at a low infectious dose in aerosols resulted in the classification of C. burnetii as a BSL‐3 select agent. Routine propagation of this agent is in embryonated eggs or tissue culture. Purification from host tissues includes multiple differential centrifugations to separate bacteria from host material. Infection of host cells is routinely verified microscopically by using Gimenez stain, fluorescent dyes, or immunofluorescence antibody (IFA) staining. Identification of C. burnetii DNA in host material is measured by PCR. Quantification of purified C. burnetii is accomplished through conversion of optical density to dry weight or, more precisely, by RT‐PCR to determine genome equivalents. Serum antibody titer to C. burnetii is determined by microagglutination assay or ELISA. Curr. Protoc. Microbiol. 15:6C.1.1‐6C.1.16. © 2009 by John Wiley & Sons, Inc.

A Case of Acute Q Fever with Severe Acute Cholestatic Hepatitis

Minimal hepatic dysfunction can be common in acute Q fever, but severe acute cholestatic hepatitis is rarely reported. We report on a 55-year-old male with acute Q fever and severe acute cholestatic hepatitis. He complained of fever, jaundice, ascites, and restlessness on admission. A liver biopsy revealed the presence of compact fibrin-ring granulomas. Serologic titers for C. burnetii IgM and IgG were 2048:1 and 1024:1, respectively. C. burnetii DNA was detected by a nested polymerase chain reaction on the liver tissue.

Ovine-associated Q fever

In Atlantic Canada, the traditional risk factor for acquisition of Q fever infection has been exposure to infected parturient cats or newborn kittens. In this study we describe the first case of Q fever in Nova Scotia acquired as a result of direct exposure to sheep. A serosurvey of the associated flock was undertaken using an indirect immunofluorescence assay (IFA) testing for antibodies to phase I and phase II Coxiella burnetii antigens. This serosurvey revealed that 23 of 46 sheep (50%) were seropositive for the phase II antibody. Four of these sheep had titres of 1:64 including three nursing ewes, one of which had delivered two lambs that died shortly after delivery. Only one ewe had phase I antibodies but had the study's highest phase II antibody titre (1:128). Molecular studies using polymerase chain reaction (PCR) failed to detect C. burnetii DNA in any of the milk specimens.

Coxiella burnetiiin Wild-caught Filth Flies

To the Editor: Coxiella burnetii, the agent of Q fever, is a bacterium and a potential agent of bioterrorism. The most frequent signs of infection in domestic animals are abortion and reduced fertility (1). Clinical signs of Q fever in humans vary from mild fevers to pneumonia, hepatitis, or death; atypical cases occur as other disorders, such as cholecystitis (1,2). Aerosols are the most common route of exposure, but oral transmission occurs (1). Some flies feed on the feces, milk, carcasses, or blood of domestic animals that can be infected with C. burnetii. These flies regurgitate and defecate when feeding and are mechanical vectors of bacteria (3,4). Flies have been shown to harbor, mechanically transport, and even support the growth of C. burnetii (4–6). It is known that house flies (Musca domestica) are possible mechanical vectors of C. burnetii because this organism survived 32 days in house flies and viable bacteria were shed by flies for 15 days (4). There are no studies of C. burnetii in field-collected flies. To examine the prevalence of C. burnetii in field-collected flies, we tested flies from farms, forests, ranches, and zoos. Flies that develop on animal dung, carcasses, feces, blood, or garbage are often called filth flies. Adult Calliphoridae, Hippoboscidae, Muscidae, and Sarcophagidae were collected from forests, zoos, ranches, and farms (Table). Flies were killed in 95% ethanol or by freezing. DNA was extracted from individual flies as described (7,8). A distilled water negative control was used for each extraction. Table Flies from the United States and Dominica assayed for Coxiella burnetii, 2004–2007 Individual DNA samples were tested, in duplicate, with a previously described TaqMan assay with a lower limit of detection of 1 C. burnetii organism (8). Positive and negative controls were used for all assays. Positive flies were verified by PCR and sequencing of 16S rRNA gene as described (9). Vouchers for each insect species were deposited in the Clemson University Arthropod Collection (Clemson, SC, USA), the University of Georgia Museum of Natural History (Athens, GA, USA), or the University of Wyoming Insect Collection (Laramie, WY, USA). Five of 363 flies were positive for C. burnetii DNA (Table). These flies included Stomoxys calcitrans, in which the adults feed on animal and human blood, and the blowflies Lucilia coeruleiviridis and L. sericata. C. burnetii–positive flies were obtained from carrion (1/12, 8.3%), a garbage bin of elephant feces (3/18, 16.7%), and a barn at a ranch (1/55, 1.8%). We sequenced 1,100 bp of the 16S rRNA gene from select DNA extracts, which were 99% identical with that of C. burnetii strain NC 002971. We detected DNA from C. burnetii in flies from a zoo, a ranch, and carrion in a forest. Laboratory data on house flies, which shed live C. burnetii for 15 days after exposure, suggest that related flies (e.g., S. calcitrans and Lucilia spp.) might also harbor viable C. burnetii. On the basis of our field data, S. calcitrans and Lucilia spp. should be studied as mechanical vectors of C. burnetii. Unlike many enteric bacteria, which require large inocula to cause disease, C. burnetii can be infectious at the level of 1 bacterium (10). If flies transmit C. burnetii, they pose an additional threat to human and animal health. The role of the sheep ked (Melophagus ovinus) in maintenance or transmission of C. burnetii is unknown. This fly is an obligate ectoparasite of sheep. It feeds on sheep blood, and feces from sheep keds can accumulate in the wool of sheep. Testing of sheep keds from infected sheep would help understand whether keds play a role in the epidemiology of C. burnetii.

Prevalence of Coxiella burnetti infection in wild and farmed ungulates

The aim of this study was to evaluate by serology and PCR analyses the prevalence of Coxiella burnetti infection in ungulates in Spain. Sera were collected from red deer ( Cervus elaphus; n = 116), roe deer ( Capreolus capreolus; n = 39), fallow deer ( Dama dama; n = 13) and cattle ( n = 79). Sera were tested for anti- C. burnetii antibody detection by means of an immunofluorescence antibody assay (IFA) and C. burnetii DNA was amplified by PCR in samples from ungulates that had antibodies to phase II antigens. Twenty-nine, 15 and 39 percent of the red deer, roe deer and cattle had antibodies against C. burnetii, respectively. None of the fallow deer sera tested positive. Seroprevalence was statistically higher in farmed than in wild red deer and higher in northern than in southern populations, whereas an inverse pattern was observed for the roe deer. Most of the seropositive animals had only anti- C. burnetii phase II antibodies, thus showing the acute nature of infections in the sampled ungulates. These results show that C. burnetii circulates in wild ungulates in Spain and suggest that they can act as pathogen reservoirs for both domestic animals and humans.

Prevalence of Coxiella burnetii DNA in vaginal and uterine samples from healthy cats of north-central Colorado.

Q fever is a worldwide zoonotic disease caused by Coxiella burnetii. Although traditionally associated with livestock exposure, human infection has also been documented from contact with parturient cats. The goal of this study was to determine the prevalence of C burnetii DNA in uterine and vaginal tissues from healthy client-owned and shelter cats of north-central Colorado using polymerase chain reaction assay. Coxiella burnetii was not amplified from vaginal samples of any cat or uterine biopsies of shelter cats. However, a nucleotide sequence with 99% homology to C burnetii DNA was amplified from four of 47 (8.5%) uterine biopsies of client-owned cats. This study demonstrates that clinically normal cats in north-central Colorado can harbor C burnetii. Care should be taken when attending to parturient cats and contact with parturient secretions should be avoided. Additional studies are indicated to further characterize the role of cats in zoonotic Q fever.

Psoas abscess: An unusual manifestation of Q fever

Q fever may lead to serious complications in chronically infected patients. We report two cases of psoas abscess due to Coxiella burnetii associated with lumbar osteomyelitis secondary to an aortic aneurysmal infection. Diagnosis was based on serology, and PCR detected C. burnetii DNA in an abscess sample.

Goats may experience reproductive failures and shed Coxiella burnetii at two successive parturitions after a Q fever infection

Q fever is a zoonosis caused by the obligate intracellular bacterium, Coxiella burnetii. Aborting domestic ruminants are the main source of human infection. In January 2003, an abortion episode occurred in a dairy caprine herd where 18/60 (30%) goats experienced reproductive problems: 4/60 (7%) aborted and 14/60 (23%) had stillbirths. Serological screening for abortion-related infectious diseases suggested Q fever. The diagnosis of C. burnetii infection was confirmed with PCR based on the occurrence of C. burnetii shedding into vaginal mucus, faeces and colostrums taken after kidding from the affected animals. The pregnancy following this episode resulted in one abortion and four stillbirths; three of those goats had already experienced reproductive failure during the previous kidding season. The seroprevalence of C. burnetii infection and the bacteria shedding were investigated using both ELISA and PCR assays, respectively, during the course of the initial and subsequent kidding seasons. Serological testing, performed on the whole herd 6 weeks after the abortion episode, showed 48/60 (80%) of ELISA positive goats. PCR assay performed on both vaginal swab and milk samples showed that the bacterium was shed for almost four months after the outbreak. C. burnetii DNA was also amplified from vaginal swab and milk samples taken from goats after the second kidding season. Furthermore, the bacteria were found into 14 vaginal swabs and 12 milk samples taken from infected females at both kidding seasons.

Incidence of Ovine Abortion by Coxiella burnetii in Northern Spain

The infectious causes of ovine abortion occurring in 148 farms in northern Spain between 1999 and 2003 were investigated. Laboratory analysis included microbiological, serological, pathological and molecular techniques. Border disease was diagnosed in 16% of the flocks, toxoplasmosis in 15%, chlamydiosis in 12%, salmonellosis in 10%, Q fever in 3%, miscellaneous infections in 7% (Yersinia spp., Listeria spp., Brucella spp.), and inflammatory lesions compatible with an infectious cause were seen in 7% of the flock. In an additional 1% of the flocks non-infectious causes were identified, and a diagnosis was not reached in 38% of the flocks. When a PCR retrospective study was carried out to investigate the possible implication of Coxiella burnetii in the cases without diagnosis, including those with inflammatory lesions, the prevalence of this pathogen increased from 3% up to 9% of the flocks, revealing the importance of this zoonotic pathogen as a small-ruminant abortifacient agent. Placenta was the most commonly positive sample, but other fetal tissues were also of value for C. burnetii DNA detection. The present results update information about the situation of abortion in sheep farms in northern Spain, and highlight the relevance of molecular diagnostic tools in routine laboratory analysis of abortions by C. burnetii.