Development of a method for detecting Coxiella burnetii in cheese samples

Coxiella burnetii is the causative agent of Q fever, and the main route of infection in humans is inhalation of contaminated aerosols. Although oral transmission by contaminated raw milk or dairy products is also a possible route of human infection, there have been few studies investigating the presence of C. burnetii in dairy products. We developed a new method of extracting DNA from cheese and detecting C. burnetii DNA in cheese samples with a nested PCR assay. The limit of detection was 6.0 × 10(2) C. burnetii particles per gram. We subsequently used this method to examine the presence of C. burnetii in cheese at commercial markets in Tokyo from June 2005 to December 2008. Twenty-eight of 147 cheese samples were found to be positive for C. burnetii DNA. However, when we assessed the viability of C. burnetii by inoculating mice with DNA-positive samples, all of the samples were found to be negative. Thus, the viability of C. burnetii appears to have been lost in these cheese samples.     

Prevalence of antibodies to Coxiella burnetii (Q fever) in bulk tank milk in England and Wales.

In the United Kingdom, the infection of people with Coxiella burnetii, the causative agent of Q fever, is of significant public health importance and is associated with contact with dairy cattle. An ELISA was developed for the detection of IgG antibodies against C burnetii in bulk tank milk, and in a survey of randomly selected samples from dairy herds in England and Wales, 21 per cent showed serological evidence of C burnetii infection.     

Occurrence, distribution, and role in abortion of Coxiella burnetii in sheep and goats in Sardinia, Italy

Between 1999 and 2002, 9349 sera and 517 aborted samples (422 foetuses and 95 placenta) were analysed from 675 sheep and 82 goat farms distributed all over the island of Sardinia. After abortion notification, sera collected at random from adult animals were examined to detect antibodies specific to Coxiella burnetii by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 255 (38%) sheep farms and in 39 (47%) goat herds whereas 40 ovine (10%) and 3 (6%) caprine foetuses were C. burnetii PCR-positive. Although C. burnetii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. Seroprevalence analysis indicates that C. burnetii distribution in sheep and goats is very high, but PCR results demonstrate that C. burnetii has a relatively low role in abortion, especially in goats.     

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.     

Relevance of the positron emission tomography in the diagnosis of vascular graft infection with Coxiella burnetii

Coxiella burnetii, the causative agent of Q fever, may cause culture-negative vascular graft infections that can be diagnosed by serology and molecular biology. We present a case of vascular graft infection detected by positron emission tomography (PET) scanner. The presence of C. burnetii was confirmed by high antibody titers and positive polymerase chain reaction specific for C. burnetii. This report emphasizes the relevance of the PET scanner in the diagnosis of infection when used in association with Q fever serology and molecular biology for the etiological identification of C. burnetii.     

Association between Coxiella burnetii shedding in milk and subclinical mastitis in dairy cattle

The objective of this research was to explore the potential association between Coxiella burnetii shedding in milk and chronic subclinical mastitis in dairy cattle. In two separate studies, we identified an association between PCR-based detection of C. burnetii in milk and chronic subclinical mastitis in lactating dairy cows. These studies were conducted in a commercial dairy herd where there was ongoing intensive monitoring of subclinical mastitis by aerobic bacteriology, but no prior knowledge or management of C. burnetii infections. In a case-control study, quarter level C. burnetii status determined by real-time quantitative PCR (RT-qPCR) was strongly associated with chronic subclinical mastitis as measured by milk somatic cell counts. In a subsequent cross sectional study, 147 (45%) of 325 lactating cows were positive for C. burnetii by RT-qPCR of composite milk samples. In a generalized linear model, accounting for the effect of covariates including aerobic intramammary infection status, C. burnetii PCR status was a significant predictor of linear somatic cell count score. In agreement with a small number of previous reports, this research provides evidence that there may be mammary gland specific manifestations of C. burnetii infections in dairy cattle.     

Determination of Coxiella burnetii seroprevalence in macropods in Australia

Many animal species, including macropods, have the potential to act as atypical reservoirs of the causative agent of Q fever, Coxiella burnetii. The objective of this study was to determine the seroprevalence of C. burnetii in various macropod species in Australia. Competitive and indirect ELISAs were developed for the testing of macropod sera for antibodies to phase II and I C. burnetii antigens separately. A total of 500 macropod serum samples from selected species sampled in eastern and western coastal states of Australia were screened for the presence of anti-C. burnetii antibodies. An overall seroprevalence of 20.8% (95% CI 20.8-20.9%) was observed with 30.4% (30.2-30.9%) in northern Queensland, 13.0% (12.9-13.1%) in southern Queensland, 7.1% (7.1-8.0%) in western Queensland and 22.8% (22.7-22.9%) in south-western Western Australia. These data indicated that macropods represented a potential reservoir for zoonotic transmission of C. burnetii to domestic animals and the human population.     

Serological evidence of Q fever in cattle in Malawi

The serological prevalence of Coxiella burnetii in cattle in Malawi is unknown. Serum samples from 200 Malawian zebu cattle were tested for C. burnetii antibodies using the complement fixation test. The percentage of positive and suspicious titres was 1.5% and 5% respectively.     

Spread of Coxiella burnetii infection in a flock of sheep after an episode of Q fever

In October 1998, two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes in the sheep flock belonging to INRA Tours-Nouzilly. The flock was kept in groups of approximately 40 ewes, which were housed together in the same accommodation. The prevalence of C burnetii infection in the groups was investigated by using ELISA and PCR tests, which revealed a high prevalence of C burnetii. The ewes were treated with oxytetracycline to reduce the shedding of C burnetii and to prevent further abortions. Nevertheless, five abortions attributed to C burnetii occurred in January and March 1999 in three groups of ewes, and 24 of the ewes still shed the bacteria into their vaginal tracts. In addition, a serological study was carried out during the first year of life of the female lambs born in 1999 and 2000; 12 per cent of 113 lambs born in 1999 were seropositive for C burnetii by ELISA, and half of the ELISA-positive lambs were born either to serologically positive ewes or to dams that excreted the pathogen into their vaginal tracts. However, all the 150 lambs born in 2000 were ELISA-negative, suggesting that the preventive measures undertaken had suppressed both the abortions and the shedding of C burnetii, and reduced the transmission of the agent.     

Serological survey of Q fever in Crete, southern Greece

Coxiella burnetii, the causative agent of Q fever, is an obligatory intracellular bacterium with worldwide distribution. The aim of this study was to determine the prevalence of C. burnetii phase II antibodies in two different groups (high and low risk) of healthy human population and investigate the epidemiological characteristics of the infection in the island of Crete (southern Greece). Collection and testing by IFA of 493 sample sera for IgG and IgM antibodies against C. bumetii phase II antigen indicated a prevalence of IgG antibodies of 48.7%. Of the seropositive individuals, 34% also revealed IgM seropositive antibody titers. Analysis of 225 sample sera by IFA from high risk population presented a prevalence for C. burnetii of 62.2%. Our findings revealed that C. burnetii is highly endemic in Crete, indicating a high exposure of the population to the pathogen regardless of occupation or place of residence.     

Coxiella burnetii infections and infections with bacteria of the genus Chlamydia in dairy cattle

Comparative studies on the prevalence of infections caused by Coxiella burnetii (C. burnetii) and Chlamydia were carried out with 592 cattle older than 2 years and 234 cattle younger than 2 years. Of these 477 originated from 24 dairy herds with considerable fertility problems (positive herds) and 349 from 14 dairy herds without major fertility problems (control herds). For the direct detection of these pathogens in the genitals capture ELISAs were employed, for the demonstration of antibodies the complement fixation test (CFT). Direct detection of C. burnetii and Chlamydia single as well as mixed infection revealed significant higher values for cattle from positive herds compared with those from the control herds. Animals revealing insemination ratios of > or = 2 showed significantly more frequent excretion of Chlamydia via the genitals and antibodies against C. burnetii than cattle with an insemination ratio of < 2. Investigations of cows which had had an abortion showed no indications of significantly more frequent C. burnetii or chlamydial infections. Inseminated but non-pregnant cows excreted significantly more C. burnetii and Chlamydia than pregnant cows. Clinical signs of endometritis were associated with an enhanced excretion of Chlamydia. Animals younger than 2 years excreted significantly more frequently C. burnetii but not Chlamydia via the genitals than animals older than 2 years. Indirect test showed results vice versa.     

The incidence of Coxiella burnetii antibodies in cattle in the Transvaal

With the use of the complement fixation test, 8,900 cattle were tested for antibodies to Coxiella burnetii. These were randomly selected from 178 different farms in 37 districts in the Transvaal. The percentage of cattle in the sample with positive antibody titres was equal to 7.78%, with a standard error of 0.28%. Because of the large size of the sample, asymptotic normality can be relied upon and the population confidence interval calculated. This was found to be greater than or = 0.07 and less than or = 0.085 at a 99% confidence level. Hence we are 99% confident that between 7% and 8.5% of the cattle in the Transvaal had antibodies to Coxiella burnetii during the period March 1985 to July 1986. The proportion of cattle with C. burnetii antibodies was also estimated for each of the 37 districts tested. Every district tested had some evidence of C. burnetii. The percentage of positive titres ranged from less than 1%-30% per district. This suggests that C. burnetii is probably an endemic disease of the cattle population of the Transvaal. A higher proportion of cattle had antibody titres in the central and south-eastern parts of the Transvaal. This distribution may be linked to the distribution of Boophilus species ticks which occur in the same areas of the Transvaal.     

Seroepidemiologic survey of Coxiella burnetii and attempt to detect Coxiella DNA in aged non-laying chickens in a prefecture of Japan where poultry farming prospers

The indirect fluorescent antibody test (IFAT) revealed seropositivity to Coxiella burnetii in aged non-laying chickens in poultry farms in a prefecture in the central part of Japan. Seropositivity was 7%, and antibody titers ranged from 16 to 64. No DNA fragment specific for C. burnetii was detected in the chickens by nested-PCR. The prevalence of C. burnetii infection in a prefecture of Japan in which poultry farming prospers was 7%.     

Detection of Coxiella burnetii in the air of a sheep barn during shearing

Local epidemics of Q fever occur sporadically in Germany, mainly in rural residential communities. There is increasing evidence that these outbreaks, which are caused by Coxiella burnetii, are related particularly to the lambing season and shearing periods of nearby sheep holdings. It is assumed that this zoonotic agent is massively emitted from the placenta of infected ewes at birth and during shearing of wool contaminated with infected faeces of ticks. However, little is known about the airborne transmission and travel distance of this infectious agent, and only few attempts have been made to isolate it directly from the air. This paper describes for the first time the isolation and detection of C. burnetii in the air of an enclosed sheep barn during shearing of a herd which had tested positive for C. burnetii serologically and by PCR. Samples of inhalable dust samples were taken using I.O.M. samplers with glass fibre and polycarbonate filters at a flow rate of 2.5 l/min. The sampling time was nearly 4.5 h. Two sampling positions were set up on both sides of the shearing place at a distance of 3 m and 1.5 m above the ground. A third position with the same sampling equipment was not activated and served as a sampling and transport control. In the laboratory, the glass fibre filters were used to determine the dust concentration. The polycarbonate filters were treated in a specific breakdown procedure which inactivates PCR inhibitors, followed by amplification and sequencing of a specific DNA section of C. burnetii, which was found in the dust from both active sampling positions. The investigation clearly shows that the sampling and detection methods used in this small field study are suitable for the detection of C. burnetii in the air of sheep barns. The results confirm experimentally the high risk of airborne transmission of C. burnetii from sero-positive sheep herds during shearing. However, little is known about the effective travel distance of infective airborne C. burnetii particles. There is an urgent need for more detailed investigations on the emission and airborne dispersion of infectious C. burnetii particles in order to improve our understanding of the health risks caused by this zoonotic agent originating from sheep herds.     

Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004

The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.     

Seroprevalence of Coxiella burnetii in selected populations of domestic ruminants in Newfoundland

The seroprevalence of Coxiella burnetii among cattle, sheep, and goats in Newfoundland was determined by microimmunofluorescence. Seropositivity to phase II antigen increased in sheep from 3.1% in 1997 to 23.5% in 1999-2000 (P < 0.001). Cows (24%) had antibodies to phase I antigen; goats (15.6%) had antibodies to phase II antigen. Seroprevalence of C. burnetii is increasing among sheep.     

Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever

Q fever has emerged as an important human and veterinary public health problem in the Netherlands with major outbreaks in three consecutive years. Goat farms are probably the prime source from which Coxiella burnetii have spread throughout the environment, infecting people living in the vicinity. Coxiella burnetii infection not only spilled over from animal husbandry to humans but could also have spread to neighbouring wildlife and pets forming novel reservoirs and consequently posing another and lingering threat to humans, companion animals and livestock. In these cases, transmission routes other than airborne spread of contaminated aerosols may become significant. Therefore, the role of ticks in the transmission of Coxiella burnetii in the current situation was investigated. A total of 1891 questing Ixodes ricinus ticks and 1086 ticks feeding on pets, wildlife and livestock were tested by a recently developed multiplex Q-PCR. All ticks were negative, except for a few ticks feeding on a herd of recently vaccinated sheep. Coxiella-positive ticks were not detected after resampling this particular herd three months later. Based on these data we conclude that the current risk of acquiring Q fever from questing ticks in the Netherlands is negligible. However, for future risk assessments, it might be relevant to sample more ticks in the vicinity of previously C. burnetii infected goat farms and to assess whether C. burnetii can be transmitted transovarially and transstadially in I. ricinus ticks.     

Serological evidence of Coxiella burnetii infection in dogs in a regional centre

OBJECTIVE Investigate the seroprevalence of the causative agent of Q fever, Coxiella burnetii in domestic dogs in the Townsville region, North Queensland, Australia. METHOD Blood samples were collected from dogs attending veterinary clinics for routine procedures. RESULTS An overall seropositivity of 21.8% (95% confidence interval (CI) 21.6-22.1%) was observed. A retrospective study of samples collected in the same region during 1984-85 was also performed, with an overall seropositivity of 16.0% (95% CI 15.9-16.2). CONCLUSION Evidence of C. burnetii infection in domestic dogs may have public health implications for dog owners, as well as veterinarians because of occupational exposure. This study is the first known investigation of C. burnetii seroprevalence in dogs in Queensland.     

The behavior of the Q-fever agent, Coxiella burnetii, in birds. 3. Experimental infection of quail]

After simultaneous aerogenic and alimentary infection of quail by intra-nasal inoculation of a suspension of C. burnetii, the agent was reisolated 6 h after infection from lung and gut, from the 3rd day on from the spleen, at the 8th and 10th day from blood, and from the 8th day on from liver and kidney. C.burnetii was found in various organs up to 21 days after infection. In the majority of birds agglutinating antibodies could be demonstrated from the 18th day up to termination of the experiment 66 days after infection. On the basis of these results the course of a C. burnetii infection in birds is discussed.     

The occurrence of Coxiella burnetii in sheep and ticks of the genus Dermacentor in Baden-Wuerttemberg

This study examined the occurrence of Coxiella burnetii (C. burnetii), the infectious agent of Q-fever, in sheep and sheep-ticks in Baden-Wuerttemberg, Germany, as a possible source of infection in Q-fever outbreaks. Using PCR, we examined a total of 1066 Dermacentor ticks from 23 herds and 49 samples of tick excrement from 18 herds for C. burnetii. We found the infectious agent in one non-engorged tick and in one sample of tick excrement from the same herd, in Efringen-Kirchen (district Loerrach). Sequencing the PCR-products confirmed the amplifications as specific for C. burnetii. Further serological tests of random samples of the four districts of Baden-Wuerttemberg showed a seroprevalence from 0 to 1.4% using complement fixation test (CFT), as well as a 0.9 to 10.2% seroprevalence, using ELISA test. Serum samples from a Q-fever-suspicious herd resulted, however, in 6% (CFT) and 53% (ELISA) positive reactions. A comparison between CFT and ELISA showed both a correlation of the two test methods that increased with higher CFT titration levels and positive reactions using ELISA for 9.4% of the serums that had tested negative using CFT. The results of the present study reveal that ticks and their excrements are important vectors of transmission of Q-fever in Baden-Wuerttemberg. Investigations on C. burnetii using PCR as well as serological surveys of sheep are important instruments for diagnosis and disease control of Q-fever.