Presence of Coxiella burnetii DNA in inflamed bovine cardiac valves.

BACKGROUND: Bacterial endocarditis is a recognised disease in humans and animals. In humans, infection with Coxiella burnetii can cause endocarditis, but this has not been investigated thoroughly in animals. Endocarditis in cattle is a common post-mortem finding in abattoirs and studies have identified Trueperella pyogenes as a major cause. Despite exposure of cattle to C. burnetii, the significance of this particular bacterium for development and progression of endocarditis has not been studied in detail. Cardiac valves of cattle affected with endocarditis (n = 100) were examined by histology, fluorescence in situ hybridization (FISH) and real time quantitative polymerase chain reaction (PCR). Serum was examined for anti-C. burnetii antibodies by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serology revealed that 70% of the cattle were positive for antibodies to C. burnetii, while PCR analysis identified 25% of endocarditis valve samples as being positive. C. burnetii was not detected by FISH, probably due to the low infection levels. Most cattle had chronic valvular vegetative endocarditis with lesions being characterised by a core of fibrous tissue covered by significant amounts of fibrin, sometimes with areas of liquefaction, and with a coagulum covering the surface. In a few cases, including the case with the highest infection level, lesions were characterized by extensive fibrosis and calcification. Histologically, bacteria other than C. burnetii were observed in most cases. CONCLUSIONS: The presence of C. burnetii DNA is relatively common in cattle affected with valvular endocarditis. The role of C. burnetii remains however unknown as lesions did not differ between C. burnetii infected and non-infected cattle and because T. pyogenes-like bacteria were present in the inflamed valves; a bacterium able to induce the observed lesions. Heart valves of normal cattle should be investigated to assess if C. burnetii may be present without preexisting lesions.

Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis.

The accuracy of three diagnostic tests for paratuberculosis was evaluated using maximum-likelihood estimation of sensitivity and specificity. We also explored the variety of estimates that can be obtained if the tests are to be used in populations of different composition with regard to infection and disease states. Two enzyme-linked immunosorbent assays (ELISAs) were evaluated separately with the faecal culture (FC). The study was carried out as a cross-sectional field study to cover all likely states of infection with Mycobacterium avium subsp. paratuberculosis.The three basic assumptions for the maximum-likelihood technique were evaluated to validate the results. Our accuracy estimates for the ELISAs were not very different from those previously published, but those for faecal culture differed if a different cut-off value was chosen for the ELISA. If faecal culture was used for screening in a Danish dairy region where the median ELISA reading was a measure of the general disease situation, the sensitivity of the faecal culture was 20-25%. If faecal culture was used as a confirmatory test on cows with a high ELISA reading (and thus high level of antibodies), the sensitivity of the faecal culture would be in the range 60-70%. These results emphasise the importance of the composition of a target population before selecting a specific diagnostic test for a given purpose. We concluded that faecal culture is useful for confirmation but not for screening purposes.

Prevalence and risk factors of Coxiella burnetii seropositivity in Danish beef and dairy cattle at slaughter adjusted for test uncertainty.

Antibodies to Coxiella burnetii have been found in the Danish dairy cattle population with high levels of herd and within herd seroprevalences. However, the prevalence of antibodies to C. burnetii in Danish beef cattle remains unknown. The objectives of this study were to (1) estimate the prevalence and (2) identify risk factors associated with C. burnetii seropositivity in Danish beef and dairy cattle based on sampling at slaughter. Eight hundred blood samples from slaughtered cattle were collected from six Danish slaughter houses from August to October 2012 following a random sampling procedure. Blood samples were tested by a commercially available C. burnetii antibody ELISA kit. A sample was defined positive if the sample-to-positive ratio was greater than or equal to 40. Animal and herd information were extracted from the Danish Cattle Database. Apparent (AP) and true prevalences (TPs) specific for breed, breed groups, gender and herd type; and breed-specific true prevalences with a random effect of breed was estimated in a Bayesian framework. A Bayesian logistic regression model was used to identify risk factors of C. burnetii seropositivity. Test sensitivity and specificity estimates from a previous study involving Danish dairy cattle were used to generate prior information. The prevalence was significantly higher in dairy breeds (AP=9.11%; TP=9.45%) than in beef breeds (AP=4.32%; TP=3.54%), in females (AP=9.10%; TP=9.40%) than in males (AP=3.62%; TP=2.61%) and in dairy herds (AP=15.10%; TP=16.67%) compared to beef herds (AP=4.54%; TP=3.66%). The Bayesian logistic regression model identified breed group along with age, and number of movements as contributors for C. burnetii seropositivity. The risk of seropositivity increased with age and increasing number of movements between herds. Results indicate that seroprevalence of C. burnetii is lower in cattle sent for slaughter than in Danish dairy cows in production units. A greater proportion of this prevalence is attributed to slaughtered cattle of dairy breeds or cattle raised in dairy herds rather than beef breeds.

Bayesian estimation of sensitivity and specificity of Coxiella burnetii antibody ELISA tests in bovine blood and milk.

Serological tests for Coxiella burnetii (the causative agent of Q fever) antibodies are usually based on enzyme linked immunosorbent assay (ELISA) although this method is not thoroughly evaluated. The objective of this study was to determine the sensitivity and specificity of an ELISA for detection of C. burnetii antibodies in milk and blood samples, using latent class models in a Bayesian analysis. Blood and milk samples of 568 lactating cows from 17 Danish dairy cattle herds collected in 2008 were used. The best combination of sensitivity and specificity estimates was revealed at a sample to positive (S/P) cut-off of 40 for both blood and milk ELISAs. At this cut-off, sensitivity of milk ELISA was 0.86 (95% posterior credibility interval [PCI] [0.76; 0.96]). This was slightly but insignificantly higher than sensitivity of blood ELISA (0.84; 95% PCI [0.75; 0.93]). The specificity estimates of the ELISA methods on milk and blood were equal at 0.99. No conditional dependence was observed between the specificity estimates of the two test methods. However, the sensitivity estimates of both tests were significantly reduced when conditional covariances ≥ 40 were used. Collection of milk samples from lactating cows is relatively easy, non-invasive and inexpensive and hence milk ELISA may be a better option for screening lactating cows. But, blood ELISA is an option for screening non-lactating cattle.

Factors associated with Coxiella burnetii antibody positivity in Danish dairy cows.

The aim of the study was to identify associations between the level of Coxiella burnetii (C. burnetii) antibodies in individual milk samples and cow and herd level factors in Danish dairy cows. The study, designed as a prospective cross sectional study with follow up, included 24 herds identified by a stratified random sampling procedure according to the level of C. burnetii antibodies in one bulk tank milk (BTM) sample at the beginning of the study. Ten herds were BTM positive, ten herds were BTM negative and four herds had an intermediate level. The samples were tested with an ELISA and results determined as S/P (sample to positive control) values. Three cross sectional studies of all lactating cows within each herd were then conducted during an 11 months follow up period with collection of a total of 5829 milk samples from 3116 cows. Each sample was tested with the same ELISA as used for BTM testing, and cows were considered test positive for S/P values ≥ 40, and otherwise negative. Individual cow information was extracted from the Danish Cattle Database and herd information was obtained from a telephone interview with each farmer. From multivariable logistic regression analysis accounting for hierarchical structures in the data it was concluded that odds for seropositivity increased with Danish Holstein breed, increasing number of parity and high milk protein contents, but decreased with increasing milk yield and high milk fat contents. Cows were at a higher risk during summer than other seasons. Among the herd level factors, herd size, tie stall housing system, quarantine of newly purchased animals and good hygienic precautions taken by the veterinarian before entering into the stable were also significantly associated with reduced odds of C. burnetii antibody positivity. The prevalence of test positive cows was almost constant during the study period in herds which were initially BTM positive and BTM intermediate, whilst the prevalence of positive cows in a few of the initial BTM negative herds changed from almost zero to higher than 60%. This indicates that herd infections last quite long and that test negative herds may convert to positive due to a few latently infected cows or due to transmissions from other herds.

Association between antibodies to Coxiella burnetii in bulk tank milk and perinatal mortality of Danish dairy calves.

BACKGROUND: Coxiella burnetii is a well-known cause of placentitis and subsequent abortion in ruminants, but there are no reports on the relationship with perinatal mortality. The study was performed to determine the influence of level and change of bulk tank milk (BTM) antibodies to C. burnetii on two outcomes associated with parturition in cattle: a) stillbirth; and b) stillbirth and neonatal mortality combined (perinatal death). METHODS: Twenty-four Danish dairy herds were tested repeatedly for antibodies to C. burnetii in BTM using a commercial ELISA. Samples were collected monthly from July 2008 to July 2009. Information on the 2,362 calvings occurring in the study period was obtained from the Danish Cattle Database. Two multilevel logistic regression models were created for the two outcomes stillbirth and perinatal mortality. One model included the level of BTM antibodies in a specified period before or after the outcome had occurred. The other model included the change in antibodies over time. These predictors were included both at herd and animal level. Furthermore, all models included parity and breed. RESULTS: The individual monthly BTM antibody levels were highly correlated within herds. Consequently, changes in BTM antibody levels were not found to be associated with neither risk of stillbirth nor the risk of perinatal mortality. However, the risk of stillborn calves and perinatal death was higher with high level of BTM antibodies 8 to 9 months after the incident, but not outside this period. CONCLUSION: We conclude that the level of antibodies to C. burnetii in BTM may be associated with perinatal mortality, but the association was not persistent and should be investigated further.

Coxiella burnetii associated placental lesions and infection level in parturient cows.

Cotyledons (n=170) from dairy cattle were analysed for Coxiella burnetii by real-time (rt) PCR targeting the IS1111a and icd genes. Positive cases (n=90) and a random selection of negative cases (n=20) were examined by histology, immunohistochemistry and, if infection level was high, by fluorescence in situ hybridisation. PCR results were compared to bulk tank milk (BTM) antibody levels. Placental infection was detected in cows from herds at all BTM antibody levels. However the likelihood of placental infection was generally higher in herds with intermediate or high BMT antibody levels than in herds with low antibody levels. Histological examination revealed a range of mostly mild cotyledonary changes; C. burnetii infection was only rarely associated with inflammation. This may explain why bovine Q fever is usually not clinically apparent. Nevertheless, infected cattle will shed C. burnetii at calving and this can occur even in herds without BTM antibodies.

Prevalence of Coxiella burnetii antibodies in Danish dairy herds.

During recent years in Denmark higher rates of antibodies to Coxiella burnetii have been detected in animals and humans than previously reported. A study based on bulk tank milk samples from 100 randomly selected dairy herds was performed to estimate the prevalence and geographical distribution of antibody positive dairy herds. Using the CHEKIT Q-Fever Antibody ELISA Test Kit (IDEXX), the study demonstrated a prevalence of 59% antibody positive herds, 11% antibody intermediate herds and 30% antibody negative herds based on the instructions provided by the manufacturer. The geographical distribution does not indicate a relationship between the regional density of dairy farms and the prevalence of antibody positive dairy farms. The result supports the hypothesis of an increase in the prevalence of positive dairy herds compared to previous years.

Managerial and environmental determinants of clinical mastitis in Danish dairy herds.

BACKGROUND: Several management and environmental factors are known as contributory causes of clinical mastitis in dairy herd. The study objectives were to describe the structure of herd-specific mastitis management and environmental factors and to assess the relevance of these herd-specific indicators to mastitis incidence rate. METHODS: Disease reports from the Danish Cattle Data Base and a management questionnaire from 2,146 herds in three Danish regions were analyzed to identify and characterize risk factors of clinical mastitis. A total of 94 (18 continuous and 76 discrete) management and production variables were screened in separate bivariate regression models. Variables associated with mastitis incidence rate at a p-value < 0.10 were examined with a factor analysis to assess the construct of data. Separately, a multivariable regression model was used to estimate the association of management variables with herd mastitis rate. RESULTS: Three latent factors (quality of labor, region of Denmark and claw trimming, and quality of outdoor holding area) were identified from 14 variables. Daily milk production per cow, claw disease, quality of labor and region of Denmark were found to be significantly associated with mastitis incidence rate. A common multiple regression analysis with backward and forward selection procedures indicated there were 9 herd-specific risk factors. CONCLUSION: Though risk factors ascertained by farmer-completed surveys explained a small percentage of the among-herd variability in crude herd-specific mastitis rates, the study suggested that farmer attitudes toward mastitis and lameness treatment were important determinants for mastitis incidence rate. Our factor analysis identified one significant latent factor, which was related to labor quality on the farm.