Pooled Faecal Culture Research Articles

Three-year monitoring of paratuberculosis in dairy cattle by pooled faecal culture and individual prevalence estimation

Abstract The objective of this study was to assess the implementation of a three-year Mycobacterium avium subsp. paratuberculosis monitoring programme using pooled faecal culture in small and mediumsized dairy herds to classify them as infected or non-infected and apply proper hygiene and biosecurity measures. Over a three-year period, 35 dairy herds were analysed annually by faecal culture of ten pooled samples. In addition, proper hygiene and biosecurity protocols were implemented in the farms after the first testing round. Considering a herd as infected with at least one culture positive in any of the three years, the accumulated percentage of infected herds was 25.7%, 40% and 45.7%, for each year respectively. Assuming that all infected herds had been detected at the end of the study, the percentage of infected herds detected each year was 56.25% and 87.5% for the first and second year, respectively. Using frequentist and Bayesian approaches, the estimated individual prevalence revealed a downward trend from 3.30-3.65% in the first year to 1.66-1.86% in the third year. The results of this study indicate that pooled faecal culture allowed for proper classification of the herds and can be a useful tool for monitoring dairy herds against paratuberculosis. In addition, statistical analysis of pooled faecal culture results can be used to evaluate the evolution of individual prevalence in the population and therefore the function of the implemented control programmes.

Changes in prevalence of ovine paratuberculosis following vaccination with Gudair®: Results of a longitudinal study conducted over a decade

Since its registration in 2002, Gudair® vaccine has become the key tool for managing paratuberculosis in sheep in Australia. This study was conducted to evaluate its effectiveness in reducing paratuberculosis prevalence in 12 flocks over more than one decade. Flocks with variable initial prevalence (5, 4 and 3 with low, medium, and high prevalence, respectively) were enrolled in the study in 2003–04. Six biennial faecal samplings were conducted in these flocks over a 10year period. At each sampling, faeces from four age groups (3, 4, 5 and 6year-old sheep) were collected from each flock. The aim was to select 7 or 14 pools of faeces constituted with pellets from 50 or 25 sheep from each age group, respectively, but the numbers and sizes of pools did vary due to logistical issues. Samples were cultured using pooled faecal culture approach and sheep level paratuberculosis prevalence was estimated. Changes in probability of a pool being positive and in paratuberculosis prevalence over time were evaluated by fitting generalised-linear and linear mixed models. The proportion of positive faecal pools significantly declined over time from 50.3% at the first sampling in 2003–04 to only 3.1% at the last sampling in 2013–14, suggestive of a 30-fold reduction in the odds of a pooled faecal sample to be positive (p<0.001). Similarly, the average animal level prevalence in flocks dropped from 7.64% at the first sampling to 0.12% at the last sampling. However, faecal shedding persisted and was still present in 3 of the 8 flocks (37.5%) that remained in the study at the last sampling conducted in 2013–14. This is the longest study ever conducted to evaluate paratuberculosis vaccine effectiveness. The results will enable sheep producers to make informed decisions on managing paratuberculosis, and in evaluating the risks of purchasing and trading vaccinated sheep.

Effectiveness of Gudair™ vaccine for the control of ovine Johne's disease in flocks vaccinating for at least 5 years.

Examine the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (Mptb) at least 5 years after starting vaccination with Gudair™ in flocks of varying initial prevalence of ovine Johne's disease (OJD) and identify risk factors for variation in vaccine efficacy. Pooled faecal culture (PFC) was conducted for 41 flocks from southern NSW and Victoria to determine estimates of current OJD prevalence. The data were compared to estimates of prevalence at or prior to commencement of vaccination at least 5 years earlier, based on available serological or PFC tests when vaccination commenced. A cross-sectional study was conducted to identify risk factors for differing prevalence levels in 36 of the flocks. Historical data enabled classification of 37 flocks as high (13; 35.1%), medium (10; 27.0%) or low (14; 37.8%) estimated initial OJD prevalence. Results of PFC in 2008-09 identified that 81.1% (30/37) of flocks had detectable shedders, with 48.6% (18/37) flocks still classified as medium or high OJD prevalence, including 50% (7/14) of flocks initially classified as low prevalence. Shedding was not detected in 18.9% (7/37) flocks. Flocks with OJD prevalence exceeding 1% at 5 years or more following the commencement of vaccination were associated with reports of sheep straying and introduction of new sheep. Despite significant declines in estimated OJD prevalence following vaccination for ≥5 years, 81.1% of flocks were shedding Mptb and considered at risk of spreading the disease or suffering recrudescence of losses if vaccination were to cease. Flock managers are advised to persist with vaccination.

Managing control programs for ovine caseous lymphadenitis and paratuberculosis in Australia, and the need for persistent vaccination.

Ovine caseous lymphadenitis (CLA) and ovine Johne’s disease (OJD) or paratuberculosis have been serious diseases in the Australian sheep industry, mainly causing losses from abattoir condemnations from CLA or mortalities on the farm from OJD. CLA is now a disease of minimal concern, with clinical cases reported rarely. Although OJD continues to spread through parts of the sheep population, the catastrophic losses in flocks occurring prior to the introduction of vaccination are now uncommon. Change-management factors relevant to the improvements in both prevalence and producer concerns for CLA and OJD were examined, including drivers and motivation for change, resistance to change, knowledge management, farming system dimensions and leadership. Although extension programs addressing disease risk factors are likely to be of relevance to improved knowledge and attitudes towards disease risk management of producers, improvements in disease-control practices were considered largely attributable to the introduction of vaccination programs for CLA in 1983 and OJD in 2002. Inclusion of the CLA antigen within clostridial vaccines (“6 in 1” vaccine) enabled routine annual CLA vaccination to occur in an increasing proportion of the national flock, with estimates of CLA prevalence suggesting a decline from 26% in 1995 to 5.2% in 2009. Encouraging the routine vaccination of lambs for OJD (Gudair vaccine) in infected flocks to reduce or avoid losses significantly reduced the within-flock prevaccination–postvaccination median prevalence from 2.72% to 0.72%, based on estimated shedding rates of Mycobacterium avium subsp. paratuberculosis determined by pooled fecal culture in 37 infected flocks vaccinating for at least 5 years. Although persistent use of CLA vaccine is a convenient intervention for producers, promoting the persistent use of OJD vaccination to continue disease suppression when clinical cases are undetectable, plus improvements in biosecurity, remain a challenge for animal-health authorities. Despite concerns of vaccine efficacy and safety issues with OJD vaccination, persistent vaccination has produced a profound improvement in the health of Australian sheep, and is a positive development of relevance to sheep production in other countries.

Evaluation of milk ELISA fordetection of Mycobacterium avium subspecies paratuberculosis indairy herds and association with within-herd prevalence

Cow-level milk ELISA results can be used to determine herd Mycobacterium avium ssp. paratuberculosis (MAP) status. Milk sample collection is minimally invasive and ELISA results can be obtained quickly and economically. The objectives were to evaluate the herd-level test characteristics of 3 commercial milk ELISA, and to determine the impact of within-herd MAP prevalence on the performance of the milk ELISA herd test. A total of 32 purposively selected herds with a median herd size of 66 milking cows were used in this 2-yr project. Fecal and milk samples were collected from all milking cows at 6-mo intervals. Fecal samples were pooled by cow age, with 5 cow samples per pool; individual fecal culture was completed on cow samples from positive pools. Herd MAP status was defined as MAP positive if, at any point during the longitudinal study, a pooled fecal culture from the herd was positive. Milk samples were analyzed using each of 3 commercial milk ELISA kits; a cow-level result from each ELISA was classified as positive following the respective manufacturer’s recommended threshold for a positive result. Herd-level milk ELISA test characteristics were estimated using generalized estimating equations logistic models, which accounted for repeated measurements. Using a cutoff of 2% milk ELISA-positive cows, milk ELISA herd sensitivity relative to a herd MAP status based on all pooled fecal culture results collected during the study was as follows: ELISA A: 59% [95% confidence interval (CI): 36–78%), ELISA B: 56% (95% CI: 32–77%), and ELISA C: 63% (95% CI: 41–81%). Herd specificity for ELISA A, B, and C was 80% (95% CI: 71–88%), 96% (95% CI: 89–98%), and 92% (95% CI: 86–96%), respectively. The remainder of the analyses focused on results from ELISA B. Herd sensitivity of ELISA B increased as MAP prevalence increased. In herds with a mean MAP prevalence ≤5%, the herd sensitivity of the milk ELISA was low, ranging from 11% when MAP prevalence was 1%, to 62% when MAP prevalence was 5%. Categorical likelihood ratios based on milk ELISA within-herd prevalence predicted that herds with milk ELISA prevalence above 0 but <2% had a similar likelihood to be MAP positive or MAP negative, whereas herds with a milk ELISA prevalence between 2 and 4% were 3.7 times more likely to be MAP positive than MAP negative. All herds with a milk ELISA prevalence >4% were MAP positive. Although milk ELISA B worked well to establish herd MAP status in high-prevalence herds, interpretation was unreliable in MAP-negative and low-prevalence herds.

Comparison of pre- and post-vaccination ovine Johne's disease prevalence using a Bayesian approach

This study was conducted to evaluate the effectiveness of Gudair™ vaccine in decreasing the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in flocks of varying initial prevalence. Thirty-seven self-replacing Merino flocks from New South Wales and Victoria (Australia) that had been vaccinating lambs with Gudair™ for at least five years were enrolled in the study. These flocks had been tested prior to or at commencement of vaccination using pooled faecal culture, agar gel immunodiffusion or both tests. These pre-vaccination test results were used to estimate pre-vaccination prevalence. Post-vaccination prevalence was estimated from culture of usually 7 pools of 50 sheep collected from the enrolled flocks in 2008–2009, approximately five or more years after commencement of vaccination.A Bayesian model was developed to estimate and compare the pre- and post-vaccination prevalences for the enrolled flocks. Apparent pre- and post-vaccination prevalences for flocks were modelled as functions of the true pre- and post-vaccination prevalences, respectively, and the sensitivities and specificities of the respective diagnostic tests. Logit-normal models were specified on pre- and post-vaccination true prevalences and were then used to make inferences about the median and 90th percentile of the prevalence distributions and their differences. Priors were mostly specified based on published literature or analysis of abattoir surveillance data for this population of flocks.The analysis found a significant decline in ovine Johne's disease prevalence from a pre-vaccination median prevalence of 2.72% [95% probability interval (PI): 1.40; 6.86%] to a post-vaccination median prevalence of 0.72% (0.39; 1.27%). However 30 of the 37 flocks still contained sheep that were shedding MAP in their faeces. The results suggest that vaccination with Gudair™ is usually effective in reducing the prevalence of faecal shedding but the response to vaccination is variable among flocks. The Bayesian approach reported here could be implemented in similar situations to compare prevalences where information from multiple diagnostic tests with varied sensitivities and specificities is available.

Effect of vaccination and natural infection with Mycobacterium avium subsp. paratuberculosis on specificity of diagnostic tests for bovine tuberculosis in farmed red deer (Cervus elaphus)

AIM: To assess the effect of vaccination against paratuberculosis (Johne's disease) on reactivity to diagnostic tests for bovine tuberculosis (Tb) in deer exposed to natural challenge with Mycobacterium avium subsp. paratuberculosis (Map), and to investigate Map infection as a factor in Tb test cross-reactivity at the herd level. METHODS: In Study 1, yearling deer (n=180 vaccinates and n=181 controls) were randomly selected from three commercial deer herds participating in a trial of a commercial vaccine against paratuberculosis. The deer were subjected to the comparative cervical skin test (CCT) for Tb at 44 weeks post-vaccination. Interpretation as a mid-cervical tuberculin skin test (MCT) was also recorded. Serum from deer positive to the CCT was collected 3–4 weeks after tuberculin injection and tested using the ELISA Tb test (ETB), with both standard and the modified-ETB interpretations. In Study 2, 102 herds were categorised as infected or uninfected with Map based on results of pooled faecal culture, and positive to MCT if one or more deer gave a positive reaction to the MCT following testing in 2005. Information on other potential risk factors for MCT reactivity was collected using a standardised questionnaire conducted on each farm. The data were analysed using a multivariable logistic regression model. RESULTS: In Study 1, 79/180 (44%) vaccinates and 42/181 (23%) controls were positive to the MCT (p<0.001). Two vaccinates (1.1%) that were CCT-positive were both positive to the standard ETB and negative to the modified ETB. One of three CCT-positive controls was negative to the standard ETB, and the other two were positive; both controls were modified ETB-positive. In Study 2, significantly more MCT-positive (41/58; 71%) than MCT-negative (18/44; 41%) herds were infected with Map (p=0.003). The OR for a positive MCT for herds infected with Map was 3.1 (95% CI=1.3–7.5), compared with uninfected herds. Herd size was positively associated with a positive MCT result (p=0.004). CONCLUSIONS: Infection with Map and vaccination increased the risk of non-specificity of the MCT in deer herds. The CCT and ancillary testing of CCT-positive animals using the modified ETB are effective tools to address the reduced specificity of the MCT. However, where use of these tests is not permitted, non-specificity related to infection and vaccination will be more difficult to resolve.

A Bayesian Approach to Estimate OJD Prevalence From Pooled Fecal Samples of Variable Pool Size

This paper describes a Bayesian approach to prevalence estimation based on pooled samples that accommodates variation in pool size and adjusts for test imperfection. A logistic model was developed for pooled fecal culture (PFC) sensitivity as a function of pool size and a logistic mixed model for ovine Johne’s disease (OJD) prevalence as a function of covariates that were found significant in a recent OJD risk factor study conducted in Australia. Available data on these factors and prior information about prevalence and sensitivity were incorporated into a Bayesian model to estimate OJD prevalence from PFC data. Overall, posterior cohort OJD prevalence was estimated to be 0.16 (range of prevalences across cohorts 0.002 to 0.72). The average prevalence was higher in wethers than ewes. PFC sensitivities for pool sizes 10, 30 and 50 were estimated to be 0.91 (95% probability intervals 0.80, 0.96), 0.85 (0.80, 0.90) and 0.77 (0.65, 0.88), respectively. Posterior specificity of PFC was almost perfect though based primarily on the prior. Results suggest the Bayesian model successfully estimated the animal-level prevalence after accounting for variable pool size and imperfect test parameters. The method can be easily adapted for other conditions and diseases where pooled samples are collected. WinBugs code for the article is available online.

Estimation of sensitivity and flock-sensitivity of pooled faecal culture for Mycobacterium avium subsp. paratuberculosis in sheep

Pooled faecal culture (PFC) is a widely used test in ovine Johne's disease (OJD) control programmes in Australia but information about its characteristics is limited. We conducted this study to estimate sensitivity and flock-sensitivity of PFC in sheep with different OJD histopathological lesions in simulated flocks with a range of infection prevalence levels. Initially, a known quantity of faeces from sheep with pauci- or multibacillary lesions was pooled with uncontaminated faeces from confirmed non-infected sheep and cultured using PFC technique. PFC sensitivity, calculated as a proportion of the pools of a particular size that tested positive, was determined to be 90% in sheep with the multibacillary form of the disease but varied with pool size in sheep with the paucibacillary form of OJD. Subsequently, probabilistic models were developed to estimate overall pool-sensitivity achieved in a flock (Sek) and flock-sensitivity of PFC (FSe) in various simulated scenarios. In flocks with a given ratio of multi- to paucibacillary sheep and with low to moderate infection prevalence level, Sek decreased with increase in pool size, but increased with pool size in flocks with ≥10% prevalence. FSe, in contrast, increased with pool size in all the tested scenarios. Both Sek and FSe increased with infection prevalence, ratio of multi- to paucibacillary sheep and the number of pools sourced from flocks. Sensitivity analyses indicated that the estimates and trends were robust to moderate changes in input parameters. The results suggest that the current testing of seven pools of size 50 is adequate for most scenarios, however, for very low prevalence flocks, a gain in FSe can be made by increasing the number of pools tested and a higher Sek can be achieved by reducing pool sizes.

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low‐shedder cattle

To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.

Sensibilidad del cultivo de pool fecal para detectar infección por Mycobacterium avium subsp. paratuberculosis en rebaños bovinos de leche y su relación con la prueba de ELISA

SUMMARY The aim of the present study was to evaluate the sensitivity and specificity of pooled faecal culture as a better alternative for the diagnosis of paratuberculosis in dairy herds compared to the conventional faecal culture and the ELISA test. Individual faecal and blood samples were collected from 50 cows in each one of 12 dairy herds (n = 598) with a history of paratuberculosis. Faecal samples were cultured on Herrold's medium individually and in pools of 5 and 10 animals strategically grouped according to age. Sensitivity and specificity of both tests were evaluated by means of two by two tables using the conventional faecal culture as the reference test. 10 (83.3%) herds and 42 (7%) animals resulted positive for Map, and 15.8% and 22% of pooled faecal samples (5 and 10 animals, respectively) were also positive. The sensitivity of the pooled faecal samples was 43.2% for the 5 animal pools and 46.4% for the 10 animal pools whilst the sensitivity of the ELISA test was 42.9%. The cost of using pooled faecal culture in 5 animals was similar to that of the ELISA test but four times less than the conventional faecal culture; however, the sensitivity and specificity of this pool was similar to the conventional faecal culture. These results suggest that faecal culture with pooled samples could be successfully used in combination with ELISA test for detection of Map infection in dairy herds.

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 ≥99% HSe in herds with ≥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.

A comparison of methods to estimate the prevalence of ovine Johne's infection from pooled faecal samples

To compare estimates of ovine Johne's infection prevalence produced by several alternate methods based on pooled faecal culture (PFC) results with prevalence estimates based on individual faecal culture (IFC). Seven methods for estimating prevalence of infection based on PFC results were incorporated in a computer program, including methods for imperfect test sensitivity and specificity, for variable pool size and a Bayesian method that incorporates prior knowledge about test performance and prevalence. These methods were then used to analyse PFC data at one observation 30 months post-vaccination in a field trial of a killed vaccine for the control of OJD, undertaken on three farms in New South Wales. Prevalence estimates, for three methods that assume a perfect test, were close to the IFC estimate, whereas for three other methods that assume an imperfect test, the estimated prevalence was generally higher than the IFC estimate. In comparison, the Bayesian approach produced more variable estimates that were substantially higher than the IFC estimate when an inappropriately high prior estimate of prevalence was used. Despite the limitations of each method, two methods provided accurate and reasonable estimates of the prevalence assessed by IFC in all instances, and are appropriate for the analysis of data from this vaccine trial. One of these methods also has the advantage of allowing for variable pool size. However, further research is needed to develop a method that will simultaneously account for variation in pool size and in test sensitivity and specificity.

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Risk factors for ovine Johne's disease in infected sheep flocks in Australia

We conducted a cross-sectional study in 2004–2005 to investigate risk factors for ovine Johne's disease (OJD) involving 92 infected Merino sheep flocks in Australia. In each enrolled flock we collected pooled faecal-samples from 3- to 5-year-old sheep and cultured them for Mycobacterium avium subsp. paratuberculosis (MAP) to determine their OJD status. Based on pooled faecal-culture (PFC) results, three outcome variables representing different facets of disease biology were derived: pool OJD status (binomial: positive or negative), log pool MAP number (continuous) and cohort OJD prevalence level (ordinal: low (<2%), medium (2–10%) and high (>10%) prevalence). We used these outcomes in three separate multivariable analyses to identify risk factors, which were based on a questionnaire administered during a face-to-face interview with the farmer. We found higher OJD infection in sheep whose dams had been in poor condition and kept at a high stocking rate during lambing and in sheep which had experienced a longer period of growth retardation during their lifetime. Flocks that had vaccinated for >2 years (rather than only 1–2 years) with a killed MAP vaccine had significantly lower OJD infection. In addition, practices including culling low body weight sheep or selling sub-flocks experiencing high losses, sharing of roads between neighbouring farms, and greater frequency of application of super phosphate fertilizers were associated with higher OJD. Of the confounders investigated, infection was higher in flocks experiencing high mortalities; in wethers compared to ewes; and in 3-year-old sheep compared to 4-year-old sheep.

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.

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Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS 900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS 900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 × 10 6 viable M. avium subsp. paratuberculosis ( Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 × 10 5 viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 × 10 5 viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.

Comparison of BACTEC 460 and MGIT 960 systems for the culture of Mycobacterium avium subsp. paratuberculosis S strain and observations on the effect of inclusion of ampicillin in culture media to reduce contamination

To compare the utility and diagnostic accuracy of BACTEC and MGIT culture systems, a total of 41 pooled faecal samples, each containing faeces from one sheep infected with the S strain of Mycobacterium paratuberculosis and four uninfected sheep was cultured. The MGIT culture system did not support the growth of the S strain of M. paratuberculosis from faeces within the time frame of the experiments, although a laboratory adapted S strain grew slowly in MGIT provided that sufficient bacteria were inoculated. In contrast, C strain grew rapidly in MGIT. The sensitivity of culture was calculated relative to the infection status of the animals, none of which had clinical signs of ovine Johne's disease. The overall sensitivity of pooled faecal culture in the BACTEC culture system was 21.9% (95% confidence limits, 10.5–37.6), a figure dependant on the proportion of multibacillary cases. The sensitivities of the BACTEC culture system for pools containing animals with multibacillary and paucibacillary lesions were 100.0% (95% confidence limits, 47.2–100.0) and 17.8% (95% confidence limits 6.06–36.8), respectively. The contamination rate of BACTEC cultures was 9.7% compared to 14.3% for MGIT. The effect of 100 μg/ml ampicillin on the S strain of the M. paratuberculosis was examined and in both BACTEC and MGIT media it delayed growth by about 1 week. The composition of MGIT medium, particularly presence of vancomycin hydrochloride, slowed the growth of the S strain. The low content of egg yolk was considered to be another possible factor. The radiometric BACTEC culture system remains the best alternative for the culture of S strain and is recommended in circumstances where the genotype (s) of the strains present in a region/farm is either unknown or S strain.

Cattle transfers between herds under paratuberculosis surveillance in The Netherlands are not random

The rate and structure of cattle transfers between 206 Dutch cattle herds with a ‘ Mycobacterium avium subsp. paratuberculosis (Map)-free’ status by November 2002, were analyzed over a 3-year period (November 1999–November 2002). Of the 206 ‘Map-free’ herds, 184 were closed herds during the period studied. In total, 280 cattle had been introduced into 22 herds at an average rate of 0.33 animals per year per 100 cattle present in the 206 herds. Assuming a random herd-contact structure, the observed rate of cattle transfers between certified ‘Map-free’ herds was sufficiently low to relax the surveillance scheme to biennial herd examinations by pooled fecal culture of all cattle ≥2 years of age. The cattle transfers were not randomly distributed over the herds. Forty-four of the 280 cattle originated from 12 other ‘Map-free’ herds. The other 236 cattle did not originate from a ‘Map-free’ herd and were introduced into a herd before it obtained the ‘Map-free’ status. No cattle were introduced into any of the ‘Map-free’ herds from which cattle were transferred to other ‘Map-free’ herds. Thus, continued propagation of the infection by cattle transfers was impossible in the group of herds studied during the study period. Therefore the surveillance scheme may be further relaxed, and may be differentiated regarding the risk herds pose to other herds.