Apparent prevalence of antibodies to Coxiella burnetii (Q fever) in bulk tank milk from dairy herds in southern Belgium

In 2006, a cross-sectional survey was carried out in 206 herds in southern Belgium to estimate the proportion of herds with Q fever-seropositive bulk tank milk (BTM), i.e. herds with an intra-herd seroprevalence >10%. Of the tested herds, 119 (57.8%; 95% CI: 51.1-64.4%) were seropositive. Most had a low antibody titre. Of these herds, 50 were randomly subjected to a real time Coxiella burnetii PCR analysis. The proportion of herds excreting Coxiella in BTM was 30.0% with only 2.0% of herds having a high level of shedding. An additional questionnaire and logistical regression identified some factors which increased the risk of BTM seroconversion (in heifers, drinking water from watercourse or well water) and some which decreased it (in heifers, tie- and free-stalling, and animals of all age in the same shed; in heifers and cows, shed disinfection).     

Coxiella burnetii shedding by dairy cows

While shedding routes of Coxiella burnetii are identified, the characteristics of Coxiella shedding are still widely unknown, especially in dairy cattle. However, this information is crucial to assess the natural course of Coxiella burnetii infection within a herd and then to elaborate strategies to limit the risks of transmission between animals and to humans. The present study aimed at (i) describing the characteristics of Coxiella burnetii shedding by dairy cows (in milk, vaginal mucus, faeces) in five infected dairy herds, and at (ii) investigating the possible relationships between shedding patterns and serological responses. A total of 145 cows were included in a follow-up consisting of seven concomitant samplings of milk, vaginal mucus, faeces and blood (Day 0, D7, D14, D21, D28, D63, D90). Detection and quantification of Coxiella burnetii titres were performed in milk, vaginal mucus and faeces samples using real-time PCR assay, while antibodies against Coxiella were detected using an ELISA technique. For a given shedding route, and a given periodicity (weekly or monthly), cows were gathered into different shedding kinetic patterns according to the sequence of PCR responses. Distribution of estimated titres in Coxiella burnetii was described according to shedding kinetic patterns. Coxiella burnetii shedding was found scarcely and sporadically in faeces. Vaginal mucus shedding concerned almost 50% of the cows studied and was found intermittently or sporadically, depending on the periodicity considered. Almost 40% of cows were detected as milk shedders, with two predominant shedding patterns: persistent and sporadic, regardless of the sampling periodicity. Significantly higher estimated titres in Coxiella burnetii were observed in cows with persistent shedding patterns suggesting the existence of heavy shedder cows. These latter cows were mostly, persistently highly-seropositive, suggesting that repeated serological testings could be a reliable tool to screen heavy shedders, before using PCR assays.     

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 infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.     

Shedding routes of Coxiella burnetii in dairy cows: implications for detection and control

Reliable detection of Coxiella burnetii shedders is a critical point for the control of the spread of this bacterium among animals and from animals to humans. Coxiella burnetii is shed by ruminants mainly by birth products (placenta, birth fluids), but may also be shed by vaginal mucus, milk, and faeces, urine and semen. However, the informative value of these types of samples to identify shedders under field conditions is unknown. Our aim was then to describe the responses obtained using a real-time PCR technique applied to milk, vaginal mucus and faeces samples taken from 242 dairy cows in commercial dairy herds known to be naturally infected with Coxiella burnetii, and to assess their putative associations. Positive results were found in all types of tested samples even in faeces. No predominant shedding route was identified. Among the shedder cows, 65.4% were detected as shedders by only one route. By contrast, cows with positive results for all three samples were scarce (less than 7%). Testing a cow based on only one type of biological sample may lead to misclassify it with regards to its shedding of Coxiella burnetii and thereby underestimate the risk of bacterial spread within a herd.     

Seroprevalence of Q fever in naturally infected dairy cattle herds

Coxiella burnetii is the causal agent of Q fever, a worldwide spread zoonosis. Prevention of C. burnetii shedding in cattle is critical to control the spread of the pathogen between animals, and from animals to humans. Vaccination with a phase 1 vaccine has been shown to be effective in preventing shedding when implemented in still susceptible animals, even in infected cattle herds. The identification of these animals (dairy cows and nulliparous females) as targets for vaccination consequently is crucial. Hygiene measures conventionally also are implemented, but their relative impact on C. burnetii diffusion remains unknown. The objectives of this study therefore were to (i) describe the distribution of the within-herd apparent seroprevalence among cows and nulliparous females and (ii) to explore the association between management practices and herd characteristics on the one hand, and these seroprevalences on the other. In a sample of 100 naturally and clinically infected dairy herds, blood samples were taken systematically from all nulliparous females (older than 12 months) and cows, and serologically tested. Information on herd characteristics and management practices were collected through a questionnaire filled in by each farmer. The variation in within-herd seroprevalence among cows and the risk for a herd of having at least one seropositive nulliparous female were investigated using multivariate (linear and logistic respectively) regression models. Median within-herd seroprevalence was 0.32 (Q1=0.22; Q3=0.43). We observed a low to null (median=0.01; Q1=0; Q3=0.10) within-herd seroprevalence in nulliparous females contrary to a high value (median=0.42) and variability (Q1=0.28; Q3=0.56) in cows. Only a few herd characteristics and management practices were found to be related to seroprevalence. Within-herd seroprevalence in cows was found to be significantly (P<0.10) higher in herds (i) with a number of cows<46, (ii) with seasonal calving, and (iii) with grazing or contact through the fence with other ruminant herds. The risk of having at least one seropositive nulliparous female was increased in herds (i) with seasonal calving and (ii) where the foetus and/or the placenta of aborted cows were not systematically removed. Our findings support, in addition to the implementation of high level of hygiene measures, the relevance of vaccination (at least in nulliparous females) as a method to control the spread of C. burnetii within an infected herd, as vaccination is effective in susceptible animals and given that nulliparous females are mostly not infected even in infected herds.     

Occurrence of Coxiella burnetii in Polish dairy cattle herds based on serological and PCR tests

The aim of the research was to assess the prevalence of antibodies to Coxiella burnetii in dairy cattle herds in Poland and to compare the results of real-time PCR and ELISA tests performed on bulk tank milk (BTM) samples. In total, 2635 serum samples collected from 969 dairy cattle herds from all provinces were tested using ELISA. Additionally, BTM specimens from 101 herds were analysed by ELISA and real-time PCR targeting IS1111 element. Presence of anti-C. burnetii antibodies was confirmed in 25.39% of serum samples in 237 herds (24.46%) and the herd-level seroprevalence in Voivodeships varied from 2.5% to 61.4%. Moreover, 46 (45.5%) of analysed bulk tank milk samples gave postive result in ELISA and microbial DNA was detected in 40 (39.6%) of tested herds. The comparative analysis of ELISA and real-time PCR results obtained for BTM samples using the chi-square test showed statistically significant relationship between results of both methods.     

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.     

Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp. paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence

Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were to determine: (a) the correlation between repeated BTM reactions; and (b) the association between the BTM antibody ELISA-level and the within-herd prevalence of antibody-positive cows.     

Serological screening for Coxiella burnetii infection and related reproductive performance in high producing dairy cows

The possible relationship between Coxiella-seropositivity and the reproductive performance of cows during the previous year to the serological screening was examined in three high producing dairy herds. The herds had a history of subfertility (<25% of pregnancies for the total number of AI), abortion (>18% abortions) and a positive polymerase chain reaction test for Coxiella burnetii in the bulk tank milk with an excretion higher than 10(4)Coxiella /ml for all three herds. Antibodies against C. burnetii were detected in 50.2% of the 781 parous cows analyzed. Coxiella seropositivity was linked to placenta retention, to changes in the interval from parturition to conception (with the lowest interval parturition-conception for cows with low level of seropositivity), early pregnancy (cows becoming pregnant before Day 90 postpartum) and maintenance of gestation during the early fetal period, while it failed to affect rates of abortion after Day 90 of gestation or stillbirth.     

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.     

Association between management practices and within-herd prevalence of Cryptosporidium parvum shedding on dairy farms in southern Ontario

To identify management practices associated with an increased within-herd prevalence of Cryptosporidium parvum shedding on dairy farms in southern Ontario, fecal samples were taken from 1089 calves aged 7-28 days, from 119 herds. Information on management practices was obtained by administering a questionnaire compiled using a modified Delphi technique. Data were analyzed using univariable and multivariable negative binomial regression. Overall, 30% of the calves in the study were shedding C. parvum oocysts, with at least one positive calf detected in 77% of herds. Within-herd prevalence ranged from 0 to 80%. Predictors significantly associated with an increased prevalence of shedding in multivariable modelling were the use of calf scour prophylaxis in cows (risk ratio [RR] 1.70, P<0.01) and calves (RR 1.38, P=0.02) and the feeding of milk replacer in the first week of life (RR 1.40, P=0.02). In contrast, the presence of concrete flooring in calf housing areas (RR 0.59, P<0.01) and the use of soap or detergent when washing calf feeding utensils (RR 0.61, P<0.01) appeared to be protective.     

Vaccination using phase I vaccine is effective to control Coxiella burnetii shedding in infected dairy cattle herds

The effectiveness of the vaccination of dairy cows combined or not with antibiotics (i.e. oxytetracycline) to control Coxiella burnetii (Cb) shedding at herd level was investigated in 77 Q fever clinically affected herds. In addition to nulliparous heifers’ vaccination, one out of the four following medical strategies was randomly assigned to dairy cows in each herd: vaccination (using a phase I vaccine) alone, vaccination combined with oxytetracycline, oxytetracycline alone or nothing. Their effectiveness to reduce Cb load in quarterly samples of bulk tank milk (BTM) and of pooled milk of primiparous (MP) was assessed through logistic hierarchical models. A significant reduction in Cb load was observed in herds where the vaccination of ≥80% of dairy cows was implemented; whereas the use of antibiotics was uneffective. Our findings support the interest of a whole vaccination strategy and provide evidence for decreasing the use of antibiotics in dairy cattle herds.     

Coxiella burnetii in bulk tank milk samples from dairy goat and dairy sheep farms in The Netherlands in 2008

In 2007, a human Q fever epidemic started, mainly in the south eastern part of The Netherlands with a suspected indirect relation to dairy goats, and, to a lesser degree, to dairy sheep. This article describes the Q fever prevalences in Dutch dairy goat and dairy sheep bulk tank milk (BTM) samples, using a real-time (RT) PCR and ELISA. Results of BTM PCR and ELISA were compared with the serological status of individual animals, and correlations with a history of Q fever abortion were determined. When compared with ELISA results, the optimal cut-off value for the RT-PCR was 100 bacteria/ml. In 2008, there were 392 farms with more than 200 dairy goats, of which 292 submitted a BTM sample. Of these samples, 96 (32.9 per cent) were PCR positive and 87 (29.8 per cent) were ELISA positive. All farms with a history of Q fever abortion (n=17) were ELISA positive, 16 out of 17 were also PCR positive. BTM PCR or ELISA positive farms had significantly higher within-herd seroprevalences than BTM negative farms. In the south eastern provinces, the area where the human Q fever outbreak started in 2007, a significantly larger proportion of the BTM samples was PCR and ELISA positive compared to the rest of The Netherlands. None of the BTM samples from dairy sheep farms (n=16) were PCR positive but three of these farms were ELISA positive. The higher percentage of BTM positive farms in the area where the human Q fever outbreak started, supports the suspected relation between human cases and infected dairy goat farms.     

Cow-level evaluation of a kinetics ELISA with multiple cutoff values to detect fecal shedding of Mycobacterium avium subspecies paratuberculosis in New York State dairy cows

In control programs for Mycobacterium avium subsp. paratuberculosis (Map), the infection status of the cows in a herd is often obtained by testing (a sample of) the herd with an ELISA that may lack some sensitivity and specificity but that is fast and inexpensive. In New York State (NYS), an unabsorbed kinetics ELISA (KELA) has been used extensively for Map control. The objective of this study was to determine the relative sensitivity and specificity of the KELA for detection of fecal shedding of Map for the NYS dairy cow population, taking into account possible confounders such as different antigen batches and Map prevalence in a herd.

The data for the study consisted of all serum samples from NYS dairy cows with concurrent fecal culture results submitted to the NY Animal Health Diagnostic Laboratory (NYAHDL) between 1991 and 1996 (n = 10,562). The data represented cows with different levels of fecal shedding from herds with different within-herd Map prevalence, including herds that were whole herd fecal culture negative on repeated testing.

The cutoff values were based on the predictive value for fecal shedding obtained with a multiple logistic regression model that included variables for the three antigen batches and the Map prevalence in the herd. The KELA could not distinguish between non-shedders and low shedders (≤30 total colony forming units (TCFU)) and thus the predictive value of the KELA to detect moderate to heavy fecal shedders (>30 TCFU) was modeled. The three cutoff values of 65, 135 and 170 were based on low (<0.2), moderate (<0.80) and high (>0.95) probabilities for moderate to heavy fecal shedding. The sensitivity and specificity values relative to culture were 67% and 95.2%, 31% and 99.7%, and 11% and 99.9% for the three cutoff values, respectively. Cutoff values for the KELA decreased for herds with increasing within-herd Map prevalence. For the best positive predictive value of a KELA for moderate to heavy fecal shedding, the cutoff values should be determined based on the apparent within-herd prevalence in a herd.     

Assessing the within-herd prevalence of cows antibody-positive to bovine viral diarrhoea virus with a blocking ELISA on bulk tank milk

Milk samples from 135 herds in Brittany were tested by a blocking ELISA for antibodies to bovine viral diarrhoea virus (BVDV) and used to assess the relationship between the bulk milk result and the within-herd prevalence of antibody-positive lactating cows. This relationship was first quantified by using a general linear model and controlling for the number of cows contributing milk to the bulk tank, for the percentage of primiparous cows in the herds and for the number of milkings contributing to the bulk tank. Receiver operating characteristic (ROC) analysis was then used to define classes of percentage inhibition in the bulk milk associated with minimum intraclass and maximum between-class variances of the within-herd prevalence. Only the percentage inhibition of bulk milk had a significant positive effect on within-herd prevalence (R2 = 0.85). The ROC analysis provided three classes of bulk milk results corresponding to different expected levels of within-herd prevalence. Herds with bulk milk percentage inhibitions of 0 to 35 per cent, 35 to 60 per cent and 60 to 100 per cent had mean (sd) observed prevalences of 4.8 (5.7) per cent, 21.6 (14.6) per cent and 66.0 (29.3) per cent, respectively. Herds with a bulk milk inhibition of 0 to 35 per cent were expected to be BVDV-free. A herd with two consecutive bulk milk results four months apart of 60 per cent or more was likely to have a very high prevalence (median of 93 per cent) and could be suspected of harbouring an active infection.     

Herd prevalence and genotypes of Coxiella burnetii in dairy cattle bulk tank milk in Gyeongsang provinces of South Korea

Coxiella burnetii is the causative agent of the zoonotic Q fever, and its reservoirs include ticks and livestock, which are key sources of transmission to humans. Although there have been several studies on the prevalence of C. burnetii antibodies in dairy cattle bulk tank milk (BTM), there is a lack of information on the molecular detection of C. burnetii in BTM in South Korea. Thus, this study was designed to assess milk shedding of C. burnetii in BTM from dairy cattle herds. Among the 607 BTM samples collected from 41 counties in Gyeongsang provinces in 2015, 108 (17.8%) from 23 (56.1%) counties tested positive for C. burnetii by PCR. Because the 16S rRNA sequences of C. burnetii from all 108 PCR-positive samples were identical, two representative samples (BTM-GB-10 and BTM-GN-63) are described in this paper. These sequences showed high identity (96.9–100%) to other C. burnetii sequences deposited in GenBank. Phylogenetic analysis showed that these two sequences were clustered with existing C. burnetii strains. The relatively high prevalence rates of C. burnetii in BTM detected in this study suggest that C. burnetii is prevalent among dairy cattle herds in South Korea. Thus, implementation of continuous monitoring and control strategies for domestic animals is needed to prevent disease transmission and protect public health.

     

Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA

Background

Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected.

Results

A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %–75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving.

Conclusion

This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.     

Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.     

Studies of Coxiella burnetii infections in dairy herds with special regard to infections in men

Investigations of 1167 dairy cows out of 105 herds with fertility disorders on secretion of Coxiella burnetii (C. burnetii) by the genitals as well as serological studies of these animals using ELISA demonstrated that C. burnetii infections are significantly associated with abortions but not with repeated inseminations without success or vaginal excretions. The demonstration of an increased risk of infections for persons caring for those dairy herds could be shown by a total of 253 serological studies. A comparison of these studies of farmers caring for dairy herds suffering from abortions a seroprevalence of > or = 20% showed that these persons revealed significantly more frequent antibodies against C. burnetii than farmers of the group compared with. Further studies showed that in herds suffering from abortions a seroprevalence of > or = 20% means an additional risk of infections of farmers. Vice versa abortions of the cows in herds with a seroprevalence of > or = 20% imply an additional infectious risk.