Estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis in a sample of Minnesota dairy herds using bacterial culture of pooled fecal samples

The objective of this study was to describe the estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis (Map) in a sample of infected dairy herds in Minnesota (N = 66) using test results from bacterial culture of pooled fecal samples. Fecal samples were collected from up to 100 cows in each herd and were tested using bacterial culture in pools of 5 cows based on age order. The mean herd size was 222 (44 to 1500) milking cows; the cows were predominantly Holstein. Using a frequentist approach, the within-herd mean individual fecal prevalence was 10% [95% confidence interval (CI) = 4% to 16%] assuming 70% test sensitivity and 99.5% test specificity. Using Bayesian methods, the estimated true within-herd individual cow prevalence was 14% (95% CI = 7% to 27%). Within-herd prevalence was higher in larger dairy herds than in herds with fewer cows. As Map is the causative agent of Johne's disease (JD), the results of this study could contribute to the success of a nationwide control program for this disease.     

Simulation model for evaluation of testing strategies for detection of paratuberculosis in midwestern US dairy herds

We developed a stochastic simulation model to compare the herd sensitivity (HSe) of five testing strategies for detection of Mycobacterium avium subsp. paratuberculosis (Map) in Midwestern US dairies. Testing strategies were ELISA serologic testing by two commercial assays (EA and EB), ELISA testing with follow-up of positive samples with individual fecal culture (EAIFC and EBIFC), individual fecal culture (IFC), pooled fecal culture (PFC), and culture of fecal slurry samples from the environment (ENV). We assumed that these dairies had no prior paratuberculosis-related testing and culling. We used cost-effectiveness (CE) analysis to compare the cost to HSe of testing strategies for different within-herd prevalences. HSe was strongly associated with within-herd prevalence, number of Map organisms shed in feces by infected cows, and number of samples tested. Among evaluated testing methods with 100% herd specificity (HSp), ENV was the most cost-effective method for herds with a low (5%), moderate (16%) or high (35%) Map prevalence. The PFC, IFC, EAIFC and EBIFC were increasingly more costly detection methods. Culture of six environmental samples per herd yielded >or=99% HSe in herds with >or=16% within-herd prevalence, but was not sufficient to achieve 95% HSe in low-prevalence herds (5%). Testing all cows using EAIFC or EBIFC, as is commonly done in paratuberculosis-screening programs, was less likely to achieve a HSe of 95% in low than in high prevalence herds. ELISA alone was a sensitive and low-cost testing method; however, without confirmatory fecal culture, testing 30 cows in non-infected herds yielded HSp of 21% and 91% for EA and EB, respectively.     

Effects of prevalence and testing by enzyme-linked immunosorbent assay and fecal culture on the risk of introduction of Mycobacterium avium subsp. paratuberculosis-infected cows into dairy herds.

A stochastic simulation model was developed to assess the risk of introduction of Mycobacterium avium subsp. paratuberculosis infection into a dairy herd through purchase of female replacement cattle. The effects of infection prevalence in the source herd(s), number of females purchased, and testing by enzyme-linked immunosorbent assay (ELISA) alone or ELISA and fecal culture as risk mitigation strategies were evaluated. Decisions about negative test results were made on a lot and individual basis. A hypothetical dairy herd, free from M. a. paratuberculosis, which replaced 1 lot (10, 30, or 100) of cows per year, was considered. Probability distributions were specified for the sensitivities and specificities of ELISA and fecal culture, the proportion of infected herds and within-herd prevalence for randomly selected replacement source herds (high prevalence) and herds in level 2 (medium prevalence) and level 3 (low prevalence) of the Voluntary Johne's Disease Herd Status Program (VJDHSP). Simulation results predicted that 1-56% of the lots had at least 1 M. a. paratuberculosis-infected cow. Assuming that ELISA sensitivity was 25%, simulation results showed on a lot basis that between 0.4% and 18% and between 0.1% and 9% were predicted to have at least 1 infected cow not detected by ELISA and by a combination of ELISA and fecal culture, respectively. On an individual cow basis, between 0.1% and 8.3% of ELISA-negative cattle in ELISA-positive lots were estimated to be infected. In both the lot and individual analyses, the probability of nondetection increased with larger lot sizes and greater prevalence. Sensitivity analysis indicated that the effect of a lower ELISA sensitivity (10%) was a variable decrease in mean detection probabilities for all combinations of prevalence and lot size. The benefit of testing introduced cattle with ELISA alone or in combination with fecal culture was found to be minimal if cows were purchased from known, low-prevalence (level 3) herds. The value of testing by ELISA alone or in combination with fecal culture was greatest in high-prevalence herds for all lot sizes. Testing of random-source cattle, bought as herd replacements, can partially mitigate the risk of introduction of M. a. paratuberculosis but not as well as by using low-prevalence source herds (level-3 VJDHSP), with or without testing.     

Evaluation of test-strategies for estimating probability of low prevalence of paratuberculosis in Danish dairy herds

Paratuberculosis is a chronic infection affecting cattle and other ruminants. In the dairy industry, losses due to paratuberculosis can be substantial in infected herds and several countries have implemented national programmes based on herd-classification to manage the disease. The aim of this study was to develop a method to estimate the probability of low within-herd prevalence of paratuberculosis for Danish dairy herds. A stochastic simulation model was developed using the R^® programming environment. Features of this model included: use of age-specific estimates of test-sensitivity and specificity; use of a distribution of observed values (rather than a fixed, low value) for design prevalence; and estimates of the probability of low prevalence (Pr_Low) based on a specific number of test-positive animals, rather than for a result less than or equal to a specified cut-point number of reactors.

Using this model, five herd-testing strategies were evaluated: (1) milk-ELISA on all lactating cows; (2) milk-ELISA on lactating cows ≤4 years old; (3) milk-ELISA on lactating cows >4 years old; (4) faecal culture on all lactating cows; and (5) milk-ELISA plus faecal culture in series on all lactating cows.

The five testing strategies were evaluated using observed milk-ELISA results from 19 Danish dairy herds as well as for simulated results from the same herds assuming that they were uninfected.

Whole-herd milk-ELISA was the preferred strategy, and considered the most cost-effective strategy of the five alternatives. The five strategies were all efficient in detecting infection, i.e. estimating a low Pr_Low in infected herds, however, Pr_Low estimates for milk-ELISA on age-cohorts were too low in simulated uninfected herds and the strategies involving faecal culture were too expensive to be of practical interest. For simulated uninfected herds, whole-herd milk-ELISA resulted in median Pr_Low values >0.9 for most herds, depending on herd size and age-structure. None of the strategies provided enough power to establish a high Pr_Low in smaller herds, or herds with a younger age-structure. Despite this, it appears as if the method is a useful approach for herd-classification for most herds in the Danish dairy industry.     

Simulation of alternatives for the Dutch Johne's disease certification-and-monitoring program

To identify optimal method(s) for certification and subsequent monitoring of Mycobacterium avium subsp. paratuberculosis (Map)-unsuspected herds, certification-and-monitoring schemes were studied using a stochastic simulation model ("JohneSSim"). JohneSSim simulated the within-herd transmission and economic aspects of Map in closed Dutch dairy herds. The model was validated with field observations on Map-unsuspected herds. The current Dutch certification-and-monitoring schemes were compared with 11 alternative schemes in which individual and pooled fecal culture, ELISA, Johnin-intradermal test and gamma-IFN ELISA were used, varying the test frequency, tested age group and number of tested animals. On reaching the 'Map-free' status with the standard certification scheme, 11% of the simulated herds were not truly Map-free. Therefore, the designation 'Map-free' should be changed into, for instance, 'low-risk Map'. In the most-attractive alternative certification scheme, the 'Map-free' status was reached after four herd examinations (at 2-year intervals) consisting of serial testing of all cattle > or = 2 years of age with a pooled fecal culture and individual fecal culture of positive pools. This scheme resulted in lower total and annual discounted costs and a lower animal-level prevalence at reaching the 'Map-free' status compared to the standard scheme, assuming that there was no new introduction of the infection. Schemes to monitor the 'Map-free' status were compared, assuming that this status was reached with the standard certification scheme. In comparison to the standard monitoring scheme, none of the alternative monitoring schemes resulted in both a lower animal-level prevalence of undetected pre-existing Map infections in closed herds, and lower median annual discounted costs. Results of the model were very sensitive to the assumed sensitivity of the fecal culture test and to management measures that prevent within-herd transmission of Map. If these preventive measures were taken, the probability of undetected Map infections in closed 'Map-free' herds was decreased substantially.     

Spatial differences in occurrence of paratuberculosis in Danish dairy herds and in control programme participation

Paratuberculosis is a chronic infection of economic importance to the cattle industry and a voluntary control programme is offered to Danish dairy farmers. Our objective was to evaluate spatial differences in both control programme participation and paratuberculosis prevalence in Denmark. The study included 4414 dairy herds: 1249 were participating in the control programme, and 1503 were tested for antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). Spatial differences were evaluated by kernel smoothing, kriging, and cluster analysis. Participation was lowest among herds on the island Zealand (≤23%). The risk of a herd being infected with MAP was found to be high on most of Zealand, but the uncertainty of this result was large due to a limited number of tested herds. In the rest of the country, the south western part of the peninsula Jutland had the highest risk of MAP (≥91%). The risk of MAP was also high (86-91%) in the northern part of both Jutland and Funen. The predicted apparent within-herd prevalence was highest (5-8.5%) in some local areas across Jutland, in the north western part of the island Funen, and in the south and western part of Zealand. Scan statistics located the primary cluster of herds with high apparent within-herd prevalence in the western part of Funen. Furthermore, a number of significant clusters were found in Jutland and a single significant cluster in Zealand. Consistency was found between kriging and scan statistics results with respect to location of areas with high apparent within-herd prevalence of MAP. Potential explanations for differences in participation include herd size and local herd health advisers, whereas for example soil characteristics might influence prevalence. Further studies are needed to evaluate these and other risk factors.     

Sensitivity of test strategies used in the Voluntary Johne's Disease Herd Status Program for detection of Mycobacterium paratuberculosis infection in dairy cattle herds

OBJECTIVE: To evaluate sensitivities at the herd level of test strategies used in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and alternative test strategies for detecting dairy cattle herds infected with Mycobacterium paratuberculosis. DESIGN: Nonrandom cross-sectional study. SAMPLE POPULATION: 64 dairy herds from Pennsylvania, Minnesota, Colorado, Ohio, and Wisconsin. Fifty-six herds had at least 1 cow shedding M. paratuberculosis in feces; the other 8 herds were free from paratuberculosis. PROCEDURE: For all adult cows in each herd, serum samples were tested for antibodies to M. paratuberculosis with an ELISA, and fecal samples were submitted for bacterial culture for M. paratuberculosis. Sensitivities at the herd level (probability of detecting infected herd) of various testing strategies were then evaluated. RESULTS: Sensitivity at the herd level of the testing strategy used in level 1 of the VJDHSP (use of the ELISA to test samples from 30 cows followed by confirmatory bacterial culture of feces from cows with positive ELISA result) ranged from 33 to 84% for infected herds, depending on percentage of cows in the herd with positive bacterial culture results. If follow-up bacterial culture was not used to confirm positive ELISA results, sensitivity ranged from 70 to 93%, but probability of identifying uninfected herds as infected was 89%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the testing strategy used in the VJDHSP will fail to identify as infected most dairy herds with a low prevalence of paratuberculosis. A higher percentage of infected herds was detected if follow-up bacterial culture was not used, but this test strategy was associated with a high probability of misclassifying uninfected herds.     

Milk quality assurance for paratuberculosis: simulation of within-herd infection dynamics and economics

A bulk milk quality assurance programme for Mycobacterium avium subsp. paratuberculosis (Map) in dairy herds was simulated with a stochastic simulation model (JohneSSim). The aim of this study was to evaluate the epidemiological and economic effects of preventive management measures and various test schemes in a simulated population of closed Dutch dairy herds over a 20-year period. Herds were certified as ;low-Map bulk milk' if, with a certain probability, the concentration of Map in bulk milk did not exceed a maximum acceptable concentration of 10(3) Map organisms per litre (based on pasteurisation studies). The programme started with an initial assessment; test-negative herds entered a surveillance procedure and test-positive herds a control procedure. The simulations showed that herd examinations by ELISA for the initial assessment, surveillance and control procedures effectively ensure the quality of ;low-Map bulk milk': > 75% of simulated herds were certified and > 96% of certified herds produced bulk milk with < 10(3) Map/L if the initial herd-level prevalence was 30%. Preventive management measures only had a minor effect on bulk milk quality of certified herds. Culling based on biennial faecal culture was more effective than culling based on annual ELISA. Average total discounted costs for 20-year participation in a programme consisting of initial assessment by ELISA, surveillance by biennial ELISA and control by biennial faecal culture were 16 Euro x 10(3) per herd. In conclusion, this study shows that a bulk milk quality assurance programme for closed Dutch dairy herds is feasible and provides information on the cost-effectiveness of different programmes. The concepts of this study equally apply to other countries because mechanisms of paratuberculosis infection, disease, and testing are comparable in other dairy cattle populations.     

Effects of infectious young stock on results of certification, surveillance and control programmes for paratuberculosis in dairy herds

In many epidemiological models for paratuberculosis, it is assumed that infected young stock (<2 years of age) do not shed Mycobacterium avium subsp. paratuberculosis (MAP) before adulthood. If this assumption were true, the effective separation of young stock from adult cattle (≥ 2 years) would largely prevent postnatal infections, provided that uninfected adult cattle are highly resistant to infection. However, this assumption is in contrast with observed faecal shedding of MAP in young stock. Consequently, this assumption may have resulted in an underestimation of the effects of MAP transmission in herds participating in certification-, surveillance-, and control programmes for paratuberculosis. Therefore, the aim of the present study was to evaluate the long-term effects of transmission of MAP amongst young stock on key output parameters of certification-, surveillance-, and control programmes for paratuberculosis in simulated closed dairy herds. Closed Dutch dairy herds participating in a paratuberculosis programme were simulated with a stochastic model, JohneSSim. Various test schemes, preventive management measures, distributions of age at onset of faecal shedding and rates of effective contacts between young stock were simulated. The results indicate that transmission of MAP amongst young stock has no relevant effects on the animal-level prevalence and milk quality of herds that are certified in a paratuberculosis programme. However, transmission of MAP amongst young stock increased the economic losses due to paratuberculosis and costs of participation in a programme. Moreover, it substantially decreased the beneficial effect of the separation of young stock from adult cattle on the probability of being certified. However, even in the presence of transmission of MAP amongst young stock, preventive management measures to separate young stock from adult cattle remain important.     

A simulated surveillance program for bovine paratuberculosis in dairy herds in Norway

Monte Carlo simulation models were used to evaluate the feasibility and potential results of a proposed national survey of the prevalence of bovine paratuberculosis (PTB) in dairy herds in Norway. The expected herd prevalence was assumed to be 0.2% in the simulations. The low sensitivity of the ELISA test, the assumed low herd prevalence, the typical low within-herd prevalence of PTB and the small herd sizes all present problems in detection of the disease. Simulations with 500, 1000, 2500 and 6000 herds tested were done. Our results suggest that a national survey would not be feasible at present, due to the low probability of detecting infected herds and because of the high number of false-positive reactions that would be expected to occur.     

Bayesian mixture models for within-herd prevalence estimates of bovine paratuberculosis based on a continuous ELISA response

Diagnostic inference by use of assays such as ELISA is usually done by dichotomizing the optical density (OD)-values based on a predetermined cut-off. For paratuberculosis, a slowly developing infection in cattle and other ruminants, it is known that laboratory factors as well as animal specific covariates influence the OD-value, but while laboratory factors are adjusted for, the animal specific covariates are seldom utilized when establishing cut-offs. Furthermore, when dichotomizing an OD-value, information is lost. Considering the poor diagnostic performance of ELISAs for diagnosis of paratuberculosis, a framework for utilizing the continuous OD-values as well as known coavariates could be useful in addition to the traditional approaches, e.g. for estimating within-herd prevalences.

The objective of this study was to develop a Bayesian mixture model with two components describing the continuous OD response of infected and non-infected cows, while adjusting for known covariates. Based on this model, four different within-herd prevalence indicators were considered: the mean prevalence in the herd; the age adjusted prevalence of the herd for better between-herd comparisons; the rank of the age adjusted prevalence to better compare across time; and a threshold-based prevalence to describe differences between herds. For comparison, the within-herd prevalence and associated rank using a traditional dichotomization approach based on a single cut-off for an OD corrected for laboratory variation was estimated in a Bayesian model with priors for sensitivity and specificity.

The models were applied to the OD-values of a milk ELISA using samples from all lactating cows in 100 Danish dairy herds in three sampling rounds 13 months apart. The results of the comparison showed that including covariates in the mixture model reduced the uncertainty of the prevalence estimates compared to the cut-off based estimates. This allowed a more informative ranking of the herds where low ranking and high ranking herds were easier to identify.     

Pooled fecal culture sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and prevalence.

A stochastic spreadsheet model was developed to obtain estimates of the costs of whole herd testing on dairy farms for Mycobacterium avium subsp. paratuberculosis (Map) with pooled fecal samples. The optimal pool size was investigated for 2 scenarios, prevalence (a low-prevalence herd [< or = 5%] and a high-prevalence herd [> 5%]) and for different herd sizes (100-, 250-, 500- and 1,000-cow herds). All adult animals in the herd were sampled, and the samples of the individuals were divided into equal sized pools. When a pool tested positive, the manure samples of the animals in the pool were tested individually. The individual samples from a negative pool were assumed negative and not tested individually. Distributions were used to model the uncertainty about the sensitivity of the fecal culture at farm level and Map prevalence. The model randomly allocated a disease status to the cows (not shedding, low Map shedder, moderate Map shedder, and heavy Map shedder) on the basis of the expected prevalence in the herd. Pooling was not efficient in 100-cow and 250-cow herds with low prevalence because the probability to detect a map infection in these herds became poor (53% and 88%) when samples were pooled. When samples were pooled in larger herds, the probability to detect at least 1 (moderate to heavy) shedder was > 90%. The cost reduction as a result of pooling varied from 43% in a 100-cow herd with a high prevalence to 71% in a 1,000-cow herd with a low prevalence. The optimal pool size increased with increasing herd size and varied from 3 for a 500-cow herd with a low prevalence to 5 for a 1,000-cow herd with a high prevalence.     

Farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds on the New York State Paratuberculosis Control Program

An extensive questionnaire was developed and used to collect data from 33 herds that were on the New York State Paratuberculosis Control Program, to study farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds. The results of the last whole herd paratuberculosis fecal culture were used to indicate presence of infection in a herd, with herds having one or more animals positive classified as ‘infected’. The average prevalence within herds was 5.2%. Fourteen herds were uninfected and 19 herds had prevalences ranging from 0.7%–28.2%. Data on 31 continuous and 67 categorical risk factors were collected by questionnaire. Ten factors were significantly associated with prevalence risk of infection in the univariable logistic regression. These factors were: the type of farm operation (commercial/registered or both); earlier diagnosis of the disease before entering the control program; number of clinical cases in the previous year; whether clinical cases were raised or purchased animals; typical signs in clinical cases; exposure of calves 0–6 weeks of age to feces of adult cows; contact of young stock with adult animal feces from using the same equipment to clean the housing for both groups of animals; spreading feces on fields from which forage is later harvested and fed to animals of any age group; what is done with animals that are suspected of having paratuberculosis or test positive on culture; and frequency of cleaning the cow barn. Stepwise logistic regression was used to determine the significance of each risk factor while controlling simultaneously for the effect of other factors. The significant factors were the type of farm operation, clinical signs, and exposure of calves to feces of adult cows. Commercial herds, presence of clinical signs typical of paratuberculosis in animals, and exposure of calves 0–6 weeks old to feces of adult cows all indicate a higher likelihood that a herd is infected with M. paratuberculosis.     

Comparative sensitivity of various faecal culture methods and ELISA in dairy cattle herds with endemic Johne's disease

In three New South Wales dairy cattle herds with endemic Johne's disease, prevalence rates by faecal culture were determined to be 12, 18 and 22%, respectively. Whole herd faecal culture was shown to detect markedly more infected cattle than whole herd testing by the EMAI absorbed ELISA, particularly in the two herds with greatest prevalence. In the three study herds, five methods for whole herd faecal culture were compared in each. These included two methods based on primary culture on Herrold's egg yolk medium with mycobactin J (HEYM): (1) conventional decontamination with sedimentation and primary culture on HEYM; (2) Whitlock decontamination and culture on HEYM. The remaining three methods were based on radiometric (BACTEC) culture: (3) decontamination and filtration to BACTEC medium; (4) modified Whitlock decontamination to BACTEC medium and (5) Whitlock decontamination to BACTEC medium. For BACTEC cultures, two methods were compared as confirmatory tests for Mycobacterium paratuberculosis: mycobactin dependence on conventional subculture to HEYM and IS900 PCR analysis of radiometric media.Among 179 cattle tested simultaneously by all five culture methods, 38 cattle were confirmed to be shedding M. paratuberculosis. In identifying shedder cattle, method 5 was the most sensitive, followed by methods 2, 4, 1, and 3 was the least sensitive. The number of BACTEC cultures confirmed by mycobactin dependence or PCR was similar.     

Prevalence of paratuberculosis (Johne's disease) in the Belgian cattle population

The national bovine paratuberculosis (PTB) seroprevalence (apparent prevalence) in the Belgian cattle population was determined by a serological survey that was conducted from December 1997 to March 1998. In a random sample of herds (N=556, 9.5%), all adult cattle of 24 months of age or older (N=13,317, 0.4%) were tested for the presence of antibodies using a commercially available absorbed ELISA test kit. The PTB median within-herd seroprevalence (proportion of detected animals within the seropositive herds) and the PTB individual-animal seroprevalence (proportion of detected animals) were, respectively, 2.9% (quartiles=1.6-5.6) and 0.87% (95% confidence interval (CI)=0.71-1.03). The PTB herd seroprevalence (proportion of detected herds) was 18% (95% CI=14-21).Assuming a test sensitivity and specificity of 45 and 99% [Sweeney et al., 1995. J. Vet. Diagn. Invest. 7 (4), 488; Sockett et al., 1992. J. Clin. Microbiol. 30 (5), 1134], respectively, the median true within-herd prevalence and the true individual-animal were estimated to be 7 and 2%, respectively. The true herd prevalence of Mycobacterium paratuberculosis infection was first estimated according to currently accepted methodology. This calculation revealed that the specificity of the used test has a dramatic effect on the estimation; assuming a test sensitivity of 45% and a true within-herd prevalence of 7%, the true herd prevalence estimation decreased from 36 to 0.8% if the test specificity decreased from 99. 9 to 99%, respectively. This sensitivity analysis showed that the practical limits of the accuracy of the used screening test jeopardize the estimation of the true herd prevalence within reasonable confidence limits, because the within-herd PTB true prevalence was low.For this reason we augmented the herd specificity for herds with larger adult herd size (>5). This was done by increasing the cut-off number of positive cattle required (>/=2) to classify a herd truly positive and including herds with one positive test result if there was historical evidence of PTB (previous diagnosis and/or clinical signs). This approach resulted in an estimated true herd prevalence of M. paratuberculosis infection of 6%. The true herd prevalence for dairy, mixed and beef herds was, respectively, 10, 11 and 3%.     

Culture of strategically pooled bovine fecal samples as a method to screen herds for paratuberculosis.

Fecal samples from 733 cows in 11 dairy herds with a low prevalence of paratuberculosis were cultured for the presence of Mycobacterium avium subsp. paratuberculosis both individually and after combining (pooling) in groups of 5. The culture procedure was the modified Jorgensen method, which uses NaOH and oxalic acid for decontamination and modified Lowenstein-Jensen agar slants for cultivation. Pooling was performed by mixing fecal samples from 5 animals ordered by age, herein referred to as strategic pooling. Culture of individual fecal samples detected M. a. paratuberculosis infections in 43 of the 733 cows and 7 of 11 infected herds (herd sensitivity = 64%). Culture of pooled fecal samples detected M. a. paratuberculosis in 28 of 151 pooled samples representing 8 of the infected 11 herds (herd sensitivity = 73%). Feces of the 43 culture-positive cows was included in 32 pools: of these 32 pools, 26 were culture positive and 6 were culture negative. In addition to the 26 positive pools containing feces from cows that were found culture positive on individual fecal samples, another 2 pools were culture positive, although comprised of feces from cows with negative results after culture of individual fecal samples. From the total of 45 infected cows that were found (43 by individual fecal culture and an additional 2 by pooled fecal culture), individual fecal culture detected 43 of these 45 (96%), while pooled fecal culture detected 39 (87%). Culture of strategically pooled fecal samples using the modified Jorgensen method was equivalent in herd sensitivity to the culture of individual fecal samples and is significantly less expensive.     

Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds

OBJECTIVE: To evaluate sensitivity of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis (MAP) in large dairy herds and assess the use of the method for estimation of MAP prevalence. ANIMALS: 1,740 lactating cows from 29 dairy herds in California. PROCEDURE: Serum from each cow was tested by use of a commercial ELISA kit. Individual fecal samples were cultured and used to create pooled fecal samples (10 randomly selected fecal samples/pool; 6 pooled samples/herd). Sensitivity of MAP detection was compared between Herrold's egg yolk (HEY) agar and a new liquid culture method. Bayesian methods were used to estimate true prevalence of MAP-infected cows and herd sensitivity. RESULTS: Estimated sensitivity for pooled fecal samples among all herds was 0.69 (25 culture-positive pools/36 pools that were MAP positive). Sensitivity increased as the number of culture-positive samples in a pool increased. The HEY agar method detected more infected cows than the liquid culture method but had lower sensitivity for pooled fecal samples. Prevalence of MAP-infected cows was estimated to be 4% (95% probability interval, 2% to 6%) on the basis of culture of pooled fecal samples. Herd-level sensitivity estimate ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity for culture of pooled fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Use of pooled fecal samples from 10 cows was a cost-effective tool for herd screening and may provide a good estimate of the percentage of MAP-infected cows in dairy herds with a low prevalence of MAP.     

Dairy goat demography and Q fever infection dynamics

Between 2007 and 2009, the largest human Q fever epidemic ever described occurred in the Netherlands. The source was traced back to dairy goat farms, where abortion storms had been observed since 2005. Since one putative cause of these abortion storms is the intensive husbandry systems in which the goats are kept, the objective of this study was to assess whether these could be explained by herd size, reproductive pattern and other demographic aspects of Dutch dairy goat herds alone. We adapted an existing, fully parameterized simulation model for Q fever transmission in French dairy cattle herds to represent the demographics typical for Dutch dairy goat herds. The original model represents the infection dynamics in a herd of 50 dairy cows after introduction of a single infected animal; the adapted model has 770 dairy goats. For a full comparison, herds of 770 cows and 50 goats were also modeled. The effects of herd size and goat versus cattle demographics on the probability of and time to extinction of the infection, environmental bacterial load and abortion rate were studied by simulation. The abortion storms could not be fully explained by demographics alone. Adequate data were lacking at the moment to attribute the difference to characteristics of the pathogen, host, within-herd environment, or a combination thereof. The probability of extinction was higher in goat herds than in cattle herds of the same size. The environmental contamination was highest within cattle herds, which may be taken into account when enlarging cattle farming systems.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

Prevalence and transmission of Neospora caninum within infected Swedish dairy herds

In this long-term study, the changes in within-herd Neospora caninum seroprevalences and the relative importance of horizontal and vertical parasite transmission in Swedish dairy herds were investigated. The within-herd prevalences varied between 5.8% and 65.0% when the herds were first sampled. Comparing the prevalences of year 2000 with those found 2-3 years later, nine out of 14 investigated herds had a similar or lower within-herd prevalence while five herds had an increased portion of seropositive individuals. Three herds were free or almost free from infection at their last sampling. Changes in prevalence were not related to the level of prevalence that each herd had when first sampled. Antibody avidity measurements revealed that the majority of seropositive individuals in all herds were chronically infected. Dam-calf pair investigations and inspection of genealogic trees supplemented with antibody test results showed that vertical transmission was by far the dominant transmission route in all herds. Taken together, these results indicate that the prevalence of N. caninum infection in Swedish dairy cattle can be successfully reduced if actions to eliminate infection in a herd are combined with preventive measures to avoid re-infection.