H.I.J. Roest

The Q fever epidemic in The Netherlands: history, onset, response and reflection

The 2007-2009 human Q fever epidemic in The Netherlands attracted attention due to its magnitude and duration. The current epidemic and the historical background of Q fever in The Netherlands are reviewed according to national and international publications. Seroprevalence studies suggest that Q fever was endemic in The Netherlands several decades before the disease was diagnosed in dairy goats and dairy sheep. This was in 2005 and the increase in humans started in 2007. Q fever abortions were registered on 30 dairy goat and dairy sheep farms between 2005 and 2009. A total of 3523 human cases were notified between 2007 and 2009. Proximity to aborting small ruminants and high numbers of susceptible humans are probably the main causes of the human Q fever outbreak in The Netherlands. In general good monitoring and surveillance systems are necessary to assess the real magnitude of Q fever.

Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii. One of the largest reported outbreaks of Q fever in humans occurred in the Netherlands starting in 2007; epidemiologic investigations identified small ruminants as the source. To determine the genetic background of C. burnetii in domestic ruminants responsible for the human Q fever outbreak, we genotyped 126 C. burnetii–positive samples from ruminants by using a 10-loci multilocus variable-number tandem-repeat analyses panel and compared them with internationally known genotypes. One unique genotype predominated in dairy goat herds and 1 sheep herd in the human Q fever outbreak area in the south of the Netherlands. On the basis of 4 loci, this genotype is similar to a human genotype from the Netherlands. This finding strengthens the probability that this genotype of C. burnetii is responsible for the human Q fever epidemic in the Netherlands.

Reduction of Coxiella burnetii Prevalence by Vaccination of Goats and Sheep, the Netherlands

Recently, the number of human Q fever cases in the Netherlands increased dramatically. In response to this increase, dairy goats and dairy sheep were vaccinated against Coxiella burnetii. All pregnant dairy goats and dairy sheep in herds positive for Q fever were culled. We identified the effect of vaccination on bacterial shedding by small ruminants. On the day of culling, samples of uterine fluid, vaginal mucus, and milk were obtained from 957 pregnant animals in 13 herds. Prevalence and bacterial load were reduced in vaccinated animals compared with unvaccinated animals. These effects were most pronounced in animals during their first pregnancy. Results indicate that vaccination may reduce bacterial load in the environment and human exposure to C. burnetii.

Genotypic Diversity of Coxiella burnetii in the 2007-2010 Q Fever Outbreak Episodes in The Netherlands

ABSTRACT The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.

Coxiella burnetii infections in sheep or goats: an opinionated review

Q fever is an almost ubiquitous zoonosis caused by Coxiella burnetii, which is able to infect several animal species, as well as humans. Cattle, sheep and goats are the primary animal reservoirs. In small ruminants, infections are mostly without clinical symptoms, however, abortions and stillbirths can occur, mainly during late pregnancy. Shedding of C. burnetii occurs in feces, milk and, mostly, in placental membranes and birth fluids. During parturition of infected small ruminants, bacteria from birth products become aerosolized. Transmission to humans mainly happens through inhalation of contaminated aerosols. In the last decade, there have been several, sometimes large, human Q fever outbreaks related to sheep and goats. In this review, we describe C. burnetii infections in sheep and goats, including both advantages and disadvantages of available laboratory techniques, as pathology, different serological tests, PCR and culture to detect C. burnetii. Moreover, worldwide prevalences of C. burnetii in small ruminants are described, as well as possibilities for treatment and prevention. Prevention of shedding and subsequent environmental contamination by vaccination of sheep and goats with a phase I vaccine are possible. In addition, compulsory surveillance of C. burnetii in small ruminant farms raises awareness and hygiene measures in farms help to decrease exposure of people to the organism. Finally, this review challenges how to contain an infection of C. burnetii in small ruminants, bearing in mind possible consequences for the human population and probable interference of veterinary strategies, human risk perception and political considerations.

Genotyping Reveals the Presence of a Predominant Genotype of Coxiella burnetii in Consumer Milk Products

ABSTRACT Real-time PCR shows the widespread presence of Coxiella burnetii DNA in a broad range of commercially available milk and milk products. MLVA genotyping shows that this is the result of the presence of a predominant C. burnetii genotype in the dairy cattle population.

Search for Possible Additional Reservoirs for Human Q Fever, the Netherlands

To the Editor: Q fever is a zoonosis caused by the bacterium Coxiella burnetii. The Q fever outbreak in the Netherlands affected ≈4,000 humans during 2007–2010 (1). In this outbreak, 1 genotype of C. burnetii appeared to be responsible for abortions in small ruminants and for clinical disease in humans (2,3). However, little is known about the outbreak genotype and the prevalence of C. burnetii in possible additional reservoirs for human Q fever (i.e., cats, dogs, horses, sheep, and cattle) in the Netherlands. We aimed to search for possible additional reservoirs for human Q fever in the Netherlands. Placentas from 15 cats, 54 dogs, and 31 horses were collected in 2011 at 5 veterinary practices. Placentas were collected by targeted sampling at breeding facilities and during parturition with veterinary assistance. In addition, 27 ovine, 11 caprine, 16 porcine, 8 equine, and 139 bovine placentas (originating from aborting animals from throughout the Netherlands that were submitted in 2011 to investigate the abortion cause) were included in the study. Samples were stored at −20°C before testing. DNA was extracted from the allantochorion of the placenta and analyzed as described (2). Samples with sufficient DNA load (cycle threshold [Ct] value <32) were typed by using 2 multilocus variable-number tandem-repeat analyses (MLVA) genotyping methods (MLVA-12 and MLVA-6), and the multispacer sequence typing method (3–5). Two C. burnetii strains from the Netherlands representing the outbreak genotype (X09003262, 3345937) and the Nine Mile RSA 493 were included as reference. For prevalence calculations, the Netherlands was divided in a southern part, comprising the Q fever hot spot area of notified cases in humans and small ruminants during the 2007–2010 epidemic (1,6), and a northern part, comprising the rest of the country. C. burnetii DNA was not detected in placentas from cats, goats, or pigs. C. burnetii DNA was detected in 4 (7% [95% CI 0.4–14.4]) of 54 canine placentas; 3 from the north and 1 from the south of the Netherlands. C. burnetii DNA was detected in 3 (8% [95% CI 0.0–16.1]) of 39 equine placentas, all from the north of the country, without known abortion history. C. burnetii DNA was detected in 7 (26% [95% CI 9.4–42.5]) of 27 ovine and in 33 (24% [95% CI 16.7–30.8]) of 139 bovine placentas. The prevalence of C. burnetii DNA–positive ovine and bovine placentas from the north and the south did not differ significantly. The C. burnetii DNA load in the placentas from dogs (Ct value 37.4–38.0) and horses (Ct value 35.4–37.4) was too low to be suitable for genotyping. Typing of 1 positive sheep sample resulted in an incomplete genotype, which is related to the outbreak genotype (sheep 192, Figure). Seven of the 33 C. burnetii DNA–positive bovine placentas were suitable for typing. One sample had a genotype similar to the outbreak genotype (2,3). Six other samples revealed a (partial) genotype related to bovine genotypes from the Netherlands (2,5,7), including a novel one. MLVA-6 and multispacer sequence typing results were consistent with the MLVA-12 results (Figure). Figure Phylogenetic tree of the genotypes of Coxiella burnetii from the samples of this study based on multilocus variable-number tandem-repeat analyses (MLVA) including 12 loci (MLVA-12). Repeats per locus are shown, and open spots indicate missing values. ... Results give no indication for major reservoirs of C. burnetii in cats, goats, and pigs in the Netherlands in 2011. However, the low numbers of placentas may have biased the results. Dogs and horses should be considered as reservoirs for C. burnetii. The detection of C. burnetii DNA–positive placentas in dogs and horses in the northern part of the country indicates the presence of a true reservoir rather than a spillover effect from the contaminated environment in the south. This observation is consistent with a reported seroprevalence of 13% in dogs in the Netherlands in 1992 (1). Until now, horses had been discussed as a risk factor in the Q fever outbreak in the Netherlands (8). Prevalence data from sheep and cattle suggest that C. burnetii is present in placentas in 25% of the abortion cases in these species. Presence of the outbreak genotype of C. burnetii in sheep has been observed (2,5), indicating sheep are a reservoir for Q fever in humans. Genotyping data show a distinct genotype in 6 of the 7 cattle samples in accordance with previous work (2,5,7). However, the outbreak genotype was detected in 1 sample from a cow. Whether this is an incidental finding or the first observation of the outbreak genotype being transferred to the cattle population is not clear. If the latter, exposure to cattle also possibly might become a risk factor for human Q fever, in addition to goats and sheep.

Specific Interferon-γ detection for the diagnosis of previous Q fever

BACKGROUND Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. METHODS One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. RESULTS In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). CONCLUSIONS Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.

Q fever in pregnant goats: humoral and cellular immune responses

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. Both humoral and cellular immunity are important in the host defence against intracellular bacteria. Little is known about the immune response to C. burnetii infections in domestic ruminants even though these species are the major source of Q fever in humans. To investigate the goat’s immune response we inoculated groups of pregnant goats via inhalation with a Dutch outbreak isolate of C. burnetii. All animals were successfully infected. Phase 1 and Phase 2 IgM- and IgG-specific antibodies were measured. Cellular immune responses were investigated by interferon-gamma, enzyme-linked immunosorbent spot test (IFN-γ Elispot), lymphocyte proliferation test (LPT) and systemic cytokines. After two weeks post inoculation (wpi), a strong anti-C. burnetii Phase 2 IgM and IgG antibody response was observed while the increase in IgM anti-Phase 1 antibodies was less pronounced. IgG anti-Phase 1 antibodies started to rise at 6 wpi. Cellular immune responses were observed after parturition. Our results demonstrated humoral and cellular immune responses to C. burnetii infection in pregnant goats. Cell-mediated immune responses did not differ enough to distinguish between Coxiella-infected and non-infected pregnant animals, whereas a strong-phase specific antibody response is detected after 2 wpi. This humoral immune response may be useful in the early detection of C. burnetii-infected pregnant goats.

Cell-free propagation of Coxiella burnetii does not affect its relative virulence.

Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.