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.

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)(20)(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)(20)(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 blood, 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 older 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 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 of 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

Effect of Vaccination on Bacterial Shedding
Crude test results are summarized in Table 2 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.

Effect of Vaccination on C t Value
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.

Discussion
This study showed that vaccination of dairy goats against Q fever with Coxevac reduced the percentage of animals in which bacteria were detected and bacterial load in uterine fluid, vaginal swabs, and milk. Reduced bacterial load was most prominent in uterine fluid and in young animals. Because shedding of bacteria may be quantitatively highest during parturition, abortion, and subsequent periods, these results suggest that vaccination may reduce environmental contamination, thereby contributing to reduction of risk for human exposure and associated human cases of Q fever.
Our findings are consistent with those of other studies. In a clinical trial of cattle, Guatteo et al. (20) demonstrated that vaccine was effective in reducing the probability of becoming a bacterial shedder when given to uninfected animals before pregnancy. Arricau-Bouvery et al. (21) showed that vaccination of 17 goats in a clinical trial decreased excretion of C. burnetii.
Rousset et al. (19) conducted a field study of a goat herd infected with C. burnetii and found that vaccination did not prevent shedding but did reduce bacterial load in vaginal swabs of primiparous animals.
Although these studies provided useful data on the effect of vaccination, these data were based on a limited number of observations. The advantages of our study were that it was based on a larger number of field samples (1,034 animals from 13 herds) obtained from animals vaccinated under field conditions and that it tested uterine fluid, which is likely to be a good proxy for shedding at the time of kidding. A disadvantage of our study was its observational nature, in which vaccination was not conducted randomly at the herd, animal, parity, or infection levels, as would have been conducted in a clinical trial.
In unvaccinated herds C. burnetii was detected more often in uterine fluid of young animals than in old animals. However, no parity difference was observed for vaccinated herds. Rousset et al. (19) observed a reduced bacterial load in vaginal swabs in primiparous goats only.
We also observed that the bacterial load was most reduced in young vaccinated animals.
However, vaccinated young and old animals had similar bacterial loads in uterine fluid and vaginal mucus (Figure 2). Our results suggest that vaccination is more protective in nulliparous animals than in parous animals. Further investigations are required to determine whether the association between vaccination and bacterial shedding depends on vaccination before a first or subsequent pregnancy or on vaccination before or after natural exposure, and to elucidate underlying mechanisms.
As reported by Guatteo et al. (20), the time of vaccination before or during breeding may affect its effectiveness. In our study, whether all animals had been vaccinated before breeding Another study limitation is that the stage of pregnancy can affect the amount of C.
burnetii; bacterial load in secreta may increase sharply during the last stage of pregnancy (4).
Although we attempted to select animals that were closest to giving birth, not all animals sampled were in the same stage of pregnancy, and the average duration of pregnancy may have differed from farm to farm. Because data about gestation stage were lacking, we did not include this factor in our analyses.
Goats and sheep in the Netherlands were vaccinated to reduce the number of human cases of Q fever. However, other countries use a different strategy. In Australia, persons at risk are vaccinated against Q fever (29). In France, cattle are vaccinated to prevent economic losses caused by abortions (30). No substantial numbers of human cases of Q fever have been reported in these countries (31). Our results showed that in uterine fluid, vaginal mucus, and milk, C. burnetii prevalence and load were reduced in vaccinated animals in the Netherlands. These effects were most pronounced in young primiparous animals. We can reasonably assume that vaccination under field conditions contributed to reduction of shedding of C. burnetii by dairy goats and dairy sheep, which in turn may contribute to reduction of the risk for human exposure.