Q Fever in the Netherlands since 2005

Figure 1

Figure 1. Density of 1,133 reported cases of acute Q fever in humans per municipality, the Netherlands, January 1–June 10, 2009. Area outlined in red is where vaccination of dairy goats and sheep...

Human Q fever cases in the Netherlands increased from 168 in 2007 to 1,000 in 2008 and 2,355 in 2009, mainly in Noord-Brabant Province (11). A campaign of voluntary vaccination of dairy goats began at the end of 2008 in the area of the 2007 human case cluster and was followed by mandatory vaccination of all dairy goat and dairy sheep on farms with >50 animals in a larger area in 2009. This vaccination zone included Noord-Brabant Province and parts of adjacent provinces because the supply of vaccine was not sufficient for all small ruminant farms in the Netherlands and because most human cases had occurred in that area (Figure 1) (13).

Additional control measures implemented in the fall of 2009 were a bulk milk test every 2 weeks to detect C. burnetii–infected herds and to monitor C. burnetii–negative herds, movement and breeding bans for dairy goats or sheep, and culling of all pregnant dairy goats or sheep on infected farms. Health authorities considered a farm to be infected when 2 consecutive bulk milk samples were positive by PCR, as tested by 2 laboratories, including the national reference laboratory (17). Thus, culling included pregnant goats in vaccinated herds and pregnant goats in unvaccinated herds located outside the vaccination zone. Culling was conducted from the end of December 2009 through May 2010.

Vaccine

The vaccine used was Coxevac (Ceva Santé Animale, Libourne, France). This vaccine was not registered in the Netherlands at the time of the study, but authorities had issued a temporary exemption. The vaccine is a phase I vaccine containing inactivated C. burnetii strain Nine Mile (18). It was recommended that uninfected animals be vaccinated twice over a 1-month interval before pregnancy. Although efficacy in dairy goats was not shown, the expected effects in vaccinated animals were reduced infection, abortion, and bacterial shedding if animals were infected after vaccination (19–21).

Study Design

For various reasons related to regulations of the national culling operation, unvaccinated dairy goats from 5 farms, vaccinated dairy goats from 7 farms, and unvaccinated dairy sheep from 1 farm were included in this study. Farms were not randomly selected but were selected on the basis of convenience of culling date, vaccination status, and agreement of farmers to participate in the study. We sampled 100 animals per farm, 50 pregnant and lactating animals (old animals), and 50 nulliparous animals (young animals). With this sample size, we expected to be able to detect a 20% difference in C. burnetii prevalence between vaccinated and unvaccinated animals and between old and young animals. We tested 3 types of samples: 1) uterine fluid, to detect animals with a high risk for shedding around parturition; 2) vaginal mucus, to be consistent with test results of other studies (19–21); and 3) milk, because herds were monitored on the basis of results of bulk milk tests.

On the day before culling, animals were selected and marked on the farm by the study team; authorities identified pregnancies by using sonography. We selected pregnant animals that were closest to giving birth because it was expected that these animals had the highest number of C. burnetii in birth fluids, which would facilitate detection of infection (4). After animals were humanely killed on farms, marked animals were transported in a separate container to a rendering plant (Rendac BV, Son, the Netherlands), where they were unloaded onto a concrete floor and prepared for sampling.

Sampling

Uterine fluid was obtained by using a 9-mL monovette EDTA blood collection system (Sarstedt, Nümbrecht, Germany) and a Bovivet 2.10 mm × 60 mm needle (Terumo Europe NV, Leuven, Belgium). Before obtaining uterine fluid, we made an incision in the linea alba cranial from the udder, moved part of the uterus to an extraabdominal position, and cleaned the uterus with alcohol-soaked cotton balls. We also cleaned the vulva with alcohol-soaked cotton balls and then obtained a swab sample from the vagina wall by using a dry and sterile cotton-tipped Cultiplast swab (LP Italiana SPA, Milan, Italy). These 2 samples were obtained from all selected animals. Additionally, from old animals we obtained a milk sample, which was collected into a 30-mL sterile tube. The teat was cleaned with alcohol-soaked cotton balls before sampling, and the first few streams of milk were discarded. All samples were frozen at −40°C within a few hours after sampling and were sent to the laboratory to be analyzed after the end of the culling period.

Diagnostic Test

Quantitative real-time PCR was performed for all samples. Milk samples were analyzed at the Animal Health Service by using the Taqvet Coxiella burnetii TaqMan Quantitative PCR (Laboratoire Service International, Lissieu, France). Swabs and uterine samples were analyzed by the national reference laboratory by using an in-house real-time PCR specific for the C. burnetii insertion sequence 1111a gene (22). Results for the 3 sample types were given as positive, negative, or doubtful on the basis of cycle threshold (C_t) values, in which a value <36.01 was considered positive and a value >40 was considered negative. A negative result indicated that no specific signal was detected in a maximum of 40 cycles. Values between 36.01 and 40 were reported as doubtful on the basis of <100% reproducibility. For additional analysis, we considered all samples with C_t <40 as positive.

Statistical Analyses

Vaccine efficacy was calculated for young and old animals separately for all 3 sample types according to the following equation: [% (positive test result, unvaccinated) – % (positive test result, vaccinated)] / [% (positive test result, unvaccinated)] (23). This efficacy can be interpreted as the percentage of positive samples (C_t <40) prevented by vaccination in a vaccinated population.

Influence of vaccination and parity on test results of individual animals was examined by using logistic regression (24) for the 3 sample types. We included vaccination status and parity group in the model as explanatory variables. Herd was included as a random factor to incorporate the fact that observations within a herd are dependent in the model. For uterine samples and vaginal swabs, we used the equation logit (fraction of positive test results) = parity (old) + vaccination status stratified by parity (young or old vaccinated) + random herd effect stratified by vaccination status (vaccinated or unvaccinated herds). For milk samples, we used the equation logit (fraction of positive test results) = vaccination status (vaccinated) + random herd effect stratified by vaccination. Vaccine effect was quantified by calculating the odds ratio (OR).

For positive samples only, we tested whether vaccination had an effect on the relative amount of bacteria present in each sample type, as indicated by the C_t value. A C_t value closer to 0 indicates a higher bacterial concentration in the sample relative to a C_t value closer to 40. We performed survival analysis on samples with C_t values for which the C_t value at which a sample result becomes positive is considered the event. Hazard ratio (HR) indicates the rate at which samples from unvaccinated animals become positive compared with samples from vaccinated animals (25). No correction for herd level was necessary and no correction for parity was possible because of the low number of bacterial shedders per group. For each of the 3 samples types, we used the equation C_t value (of positive samples only) = vaccination status (vaccinated).

Kaplan-Meier curves were plotted to show bacterial load in samples from old vaccinated, young vaccinated, old unvaccinated, and young unvaccinated animals. Statistical analyses were performed by using R software (26). For logistic regression, the function glmer() in lme4 in R software (27) was used. For survival analysis, the functions Surv() and coxph() in Survival in R software (28) were used. The model fit of all models was assessed by using the likelihood ratio test.

Background Information for Individual Farms

Information for each farm is shown in Table 1. Three farms (B, F, and K) did not have a history of animals with Q fever before the end of 2009 when their bulk milk PCR results changed from negative to positive during the monitoring period, which suggested a recent infection. Abortion caused by Q fever had been diagnosed in 2008 on sheep farm X. On all other farms, >1 bulk milk ELISA or PCR results were positive for C. burnetii in 2008 or 2009. Animals in vaccinated herds were supposedly vaccinated twice in 2009, with the exception of farm M, where the first vaccination was given after abortions had occurred.

Effect of Vaccination on Bacterial Shedding

Figure 2

Figure 2. Kaplan-Meier curves for cycle threshold (C_t) values of all samples (A–C) and for samples with positive and doubtful results for Coxiella burnetii (C_t <40) (D–F), the Netherlands, January 1–June 10,...

Crude test results are summarized in Table 2. The percentage of C. burnetii–positive animals on each farm is shown in Figure 2. For vaccinated animals, 0.43% of uterine samples, 30% of vaginal swabs, and 4% of milk samples were positive (C_t <36.01). For unvaccinated animals, 26% of uterine samples, 76% of vaginal swabs, and 33% of milk samples were positive. Prevalences within vaccinated herds and unvaccinated herds varied substantially (Table 2).

Vaccine efficacy for uterine sample results was 98% for young animals and 90% for old animals. Vaginal sample vaccine efficacy was much lower (57% and 28%) for young and old animals, respectively. Vaccine efficacy for milk sample test results was 72% (Table 3). All logistic regression model fits and survival model fits were better than those of null models according to likelihood ratio tests.

For vaccinated animals, uterine samples from young animals were 0.5% as likely to be positive for C. burnetii (OR 0.005, 95% CI 0.0002–0.1200), and uterine samples from old animals were 3.2% as likely to be positive (OR 0.032, 95% CI 0.002–0.580) than samples from unvaccinated young animals. For unvaccinated animals, old animals were 44% as likely to be positive than young animals (OR 0.44, 95% CI 0.25–0.78) (Table 4). Results from the vaginal swabs were comparable; vaccinated young animals were 1.5% as likely to be positive for C. burnetii than unvaccinated young animals (OR 0.015, 95% CI 0.0006–0.3500). Milk from vaccinated old animals was 4% as likely to be positive for C. burnetii than milk from unvaccinated old animals (OR 0.04, 95% CI 0.003–0.380) (Table 5).

Effect of Vaccination on C_t Value

In uterine fluid, vaccinated animals had an HR that was half that of unvaccinated animals (HR 0.49, 95% CI 0.34–0.70), which indicated that unvaccinated C. burnetii–positive animals had higher relative amounts of bacteria on the basis of C_t value. This effect was similar for vaginal mucus (HR 0.34, 95% CI 0.28–0.42) and milk (HR 0.54, 95% CI 0.39–0.75) (Table 6).

C_t values for uterine fluid and vaginal mucus were lowest for C. burnetii–positive, unvaccinated young animals, which suggested that they had the highest relative amount of bacteria (Figure 2). C_t values were similar in bacteria-positive vaccinated animals, regardless of parity group, which indicated lower but similar shedding levels in all vaccinated animals. For milk samples, C_t values were lower for unvaccinated animals than for vaccinated animals.