Development and evaluation of LAMP-coupled lateral flow device for the detection of MAP in livestock at point of care resource-limited areas.

The Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (Johne's disease), a systemic and chronic inflammation of intestine that affects bovine, small ruminants like goat and sheep. The disease has a greater economic importance in cattle and in small ruminants. But its effective control is impeded due to lack of rapid and accurate diagnostics. The present study is aimed at developing a LAMP-coupled lateral flow device (LFD) for rapid detection of paratuberculosis in livestock animal species such as cattle and in small ruminants at resource-limited areas. LAMP primers with biotin and FITC end tags were designed for IS900 gene specific for MAP. To determine sensitivity of LAMP assay, 10-fold serial dilutions were made from 10 ng/µl MAP stock DNA and were compared with PCR. The detection limits of LAMP-coupled LFD were defined and reactions were repeated for reproducibility. The specificity was evaluated using other infectious bacteria such as M. bovis, M. tuberculosis, Brucella abortus, Leptospira interrogan, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogens, and Staphylococcus aureus. A total of 95 samples turned positive for LAMP-coupled LFD out of 389 fecal samples. All the cultural-positive and PCR-positive samples showed positive in LAMP-coupled LFD. Nine samples with negative cultures turned positive in LAMP assay. The overall sensitivity and specificity of the LAMP-coupled LFD assays were 100% and 97.02% respectively in comparison with the culture as the gold standard method. The sensitivity detection limit of developed assay was 10 fg/µl and specificity was 100%. This assay successfully detected MAP not only by using bacterial DNA but also in clinical fecal samples. The clear band formation at control and test positions was observed on LAMP-coupled LFD. The developed assay is a simple, rapid, easy to perform, and is very useful in early diagnosis of Mycobacterium avium subsp. paratuberculosis at point of care resource-limited areas.

[Two-stage control of paratuberculosis: Herd-status surveillance as the basis for operational measures to reduce the prevalence. - Experiences from Lower Saxony, Hesse, Thuringia and Tyrol]. / Zweistufige Paratuberkulosebekämpfung in der Praxis: Überwachung auf Herdenebene als Basis für betriebliche Maßnahmen zur Prävalenzsenkung.

Economic losses caused by paratuberculosis (Johne's disease) can be high in infected herds. A universally accepted concept for the surveillance or control of paratuberculosis in cattle herds has not yet been established.In the course of the program for the reduction of MAP (Mycobacterium avium subsp. paratuberculosis) infections in Lower Saxony, Germany, dairy farms are obliged to test bulk tank milk samples for the presence of MAP-antibodies every 6 months. In case of a non-negative result, testing is required at the single animal level. Farmers can than decide whether they join a program to control MAP-infections in their herd. Within the voluntary certification program for paratuberculosis in Hesse, Germany, the MAP-herd status is evaluated using boot swab sampling. On positive farms, animals are tested at 6-month intervals by milk or blood serology with timely culling of positive individuals. The program for the abatement of paratuberculosis in cattle herds in Thuringia, Germany, is based on a yearly fecal examination for MAP-shedding of all adult cattle within a herd. Fecal MAP-positive animals should be culled as soon as possible. The basis of the surveillance and control program for MAP in Tyrol, Austria, is the biennial survey of the MAP-herd status by boot swab sampling. Farms with a MAP-positive boot swab result have the option to have their adult animals tested for MAP by single animal fecal sampling. On the basis of the results of these samples, farmers can decide whether they wish to join the MAP-control program.The programs presented above show that a two-stage approach consisting of the evaluation of the MAP-herd level, followed by the testing of single animals, represents a feasible approach for the surveillance and control of paratuberculosis in cattle herds.

Potential biomarkers as an indicator of vertical transmission of Johne's disease in a Korean native cattle farm.

Paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases in cattle. After birth, calves are raised with natural breast feeding without separation from their mothers in most Korean native cattle (Hanwoo breed) farms. Vertical transmission of PTB has been reported, but the exact PTB infection route has not been revealed in Hanwoo farms. Calves of MAP seropositive dams were tested for MAP presence and MAP antibodies in feces and tissues. MAP was detected in calf tissues by using polymerase chain reaction. Expressions of genes reported to be prognostic biomarkers of MAP infection changed in both calves and cows (p ＜ 0.05). Expression of two genes (HGF and SERPINE1) were significantly decreased in MAP-infected cattle and their offspring (p ＜ 0.01). The results suggest that biomarker gene expression profiles can be useful in detecting early stage MAP infection. Based on the results, complete eradication of MAP may be possible if accurate diagnostic methods to detect infected calves are added to the current PTB eradication strategy, which, because infected individuals are likely to develop into fecal MAP shedders at any time, includes isolation of new born calves and feeding sterilized colostrum.

Opportunities and challenges when pooling milk samples using ELISA.

Testing large quantities of samples in order to detect one or more test-positive sample(s) is expensive and time-consuming. It is possible to optimize this process by pooling samples. Two frameworks to produce different hierarchical and non-hierarchical pooling schemes were tested and compared to standard pooling. Their efficiency and the potential savings were determined as a function of prevalence and the number of pooled samples. The potential benefit of pooling samples is dependent upon the changes in the analytical sensitivity and specificity of the test used when diluting test-positive samples by pooling. To illustrate this, the sensitivity of antibody ELISA on pooled samples of bovine milk for Salmonella Dublin, Mycobacterium avium spp. paratuberculosis, and bovine virus diarrhea was tested. For these milk assays, the analytical sensitivity decreased rapidly with increasing pool sizes. The efficiency of pooling is usually only measured by the number of tests performed, yet real savings depend on all the costs involved in the pooling process. These may differ between laboratories depending on the available equipment and the salaries of the technicians, among other factors. Therefore, several cost parameters were introduced to describe the total cost and thereby calculate the total savings. In terms of overall savings, both tested schemes were potentially optimal depending on the prevalence, possible pool size, and the cost of retesting. For the pool sizes of interest in this study, the three-stage hierarchical pooling scheme was often marginally more efficient in terms of the total number of tests. However, if the price of re-pooling was high, the two-stage scheme performed better in terms of total savings. In addition, for low prevalences and the possibility of pooling a large number of samples, the two-stage non-hierarchical test may be more efficient, both in terms of number of tests and overall cost. In order to apply these results in different laboratory settings, a free Shiny WebApp was developed, to compare several pooling schemes with different cost parameters.

Non-tuberculous mycobacterial infections of veterinary relevance.

Mycobacteria play an important role in human and animal health fields. We here examine the place of non tuberculous mycobacteria (NTM) infections in the veterinary context. Relevant aspects of a reference laboratory experience and a literature review are presented in this article. Importance is given both to productivity and to economic losses due to misdiagnosis with bovine tuberculosis and paratuberculosis. The impact NTM may have is relative to geographical location, ecology, husbandry, extent of surveillance programs and bovine tuberculosis and paratuberculosis prevalence. The role of the most relevant NTM in animal disease is summarized with a special focus on Mycobacterium avium subsp. paratuberculosis, given its role as causative agent of paratuberculosis, a disease with huge economic consequences for ruminant livestock.

A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis.

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.

Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 µl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

Serum samples can be substituted by plasma samples for the diagnosis of paratuberculosis.

Employing plasma samples rather than serum samples for serological paratuberculosis diagnosis is practical, especially when bovine TB is assessed in the same cattle herd with the gamma interferon bovine avian (IFN-γ BA) test. We demonstrate that antibody titers in serum and plasma samples, utilizing the PARACHECK(®) ELISA kit, are highly comparable (Cohen's kappa test, k=0.955). We conclude that serum can be replaced with plasma in this commercially available antibody detection assay resulting in working hour savings for sampling and blood sample work-up and cost reductions for materials and sample storage.

The effect of alternative testing strategies and bio-exclusion practices on Johne's disease risk in test-negative herds.

Herd classification is a key component of national Johne's disease (JD) control programs. Herds are categorized on the basis of test results, and separate sub-programs are followed for test-positive and test-negative herds. However, a test-negative herd result does not necessarily equate to JD freedom for reasons relating to disease pathogenesis and available diagnostic tests. Thus, in several countries, JD control programs define test-negative herds as having a "low risk" of infection below a specified prevalence. However, the approach is qualitative, and little quantitative work is available on herd-level estimates of probability of freedom in test-negative herds. This paper examines the effect over time of alternative testing strategies and bio-exclusion practices on JD risk in test-negative herds. A simulation model was developed in the programming language R. Key model inputs included sensitivity and specificity estimates for 3 individual animal diagnostic tests (serum ELISA, milk ELISA, and fecal culture), design prevalence, testing options, and testing costs. Key model outputs included the probability that infection will be detected if present at the design prevalence or greater (herd sensitivity; SeH), the probability that infection in the herd is either absent or at very low prevalence (i.e., less than the design prevalence; ProbF), the probability of an uninfected herd producing a false-positive result [P(False+)], and mean testing cost (HerdCost) for different testing strategies. The output ProbF can be updated periodically, incorporating data from additional herd testing and information on cattle purchases, and could form the basis for an output-based approach to herd classification. A high ProbF is very difficult to achieve, reflecting the low sensitivity of the evaluated tests. Moreover, ProbF is greatly affected by any risk of introduction of infection, decreasing in herds with poor bio-exclusion practices despite ongoing negative test results. The value of P(False+) was substantial when tests with imperfect specificity were used. Testing strategies can substantially influence testing costs but with little effect on test performance. This study illustrates an output-based approach to herd classification, with potential for national and field applications.

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world's ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne's disease in animals and might be implicated in cases of human Crohn's disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.

Cost-effectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis super-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture.

Diagnostic strategies to detect Mycobacterium avium subsp. paratuberculosis (MAP) super-shedder cows in dairy herds have been minimally studied. The objective of the current study was to compare the cost-effectiveness of strategies for identification of MAP super-shedders on a California dairy herd of 3,577 cows housed in free-stall pens. Eleven strategies that included serum or milk enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) or culture of environmental samples, pooled or individual cow fecal samples, or combinations thereof were compared. Nineteen super-shedders (0.5%) were identified by qPCR and confirmed by culture as cows shedding ≥ 10,000 colony forming units (CFU)/g feces (median of 30,000 CFU/g feces). A stratified random sample of the study herd based on qPCR results of fecal pools was the most sensitive (74%) strategy and had the highest cost ($5,398/super-shedder). The reference strategy with the lowest cost ($1,230/super-shedder) and sensitivity (47%) included qPCR testing of fecal samples from ELISA-positive lactating (milk) and nonlactating (serum) cows housed in pens with the highest MAP bioburden. The most cost-effective alternative to the reference was to perform qPCR testing of fecal samples from ELISA-positive cows (milk and serum for milking and dry cows, respectively) for a sensitivity of 68% and cost of $2,226/super-shedder. In conclusion, diagnostic strategies varied in their cost-effectiveness depending on the tests, specimen type, and labor costs. Initial qPCR testing of environmental samples from free-stall pens to target cows in pens with the highest MAP bioburden for further testing can improve the cost-effectiveness of strategies for super-shedder identification.

Accuracy assessment and screening of a dairy herd with paratuberculosis by three different ELISAs.

Although the culture of Mycobacterium avium subsp. paratuberculosisis is the gold standard for the diagnosis of paratuberculosis, this bacterium is difficult to grow. In contrast, serological tests like ELISAs are inexpensive, rapid, and easy to perform. The aims of this study were to evaluate the accuracy of three different ELISAs: one with the commercial antigen PPA-3, another one with L5P (a recently described lipopentapeptide), and a third one with an in-house antigen whole cell lysates (WCL) of M. avium (MAA) strain D4ER (Study 1), and to compare them with other tests for paratuberculosis (PTB) diagnosis (Study 2). In Study 1, the sensitivities of the three ELISAs tested were 74.1%, 37% and 74.1%, respectively, whereas their specificities were 98.9%, 100% and 100%, respectively. In Study 2, we compared the three above-mentioned ELISAs with the intradermal reaction test using Avian PPD (PPDa) and fecal culture associated with Ziehl-Neelsen stain and PCR tests, in a dairy herd with 4.6% of cows with clinical signs of PTB. The results showed that fecal samples from 14 cows (16%) were culture-positive and that fecal samples from nine cows (10%) were PPDa-positive. Most of these animals (culture-positive and PPDa-positive) were detected as positive with any of the three ELISAs tested. Serological results showed that 31% of the animals were positive to ELISA-PPA-3, 17% to ELISA-L5P and 42.5% to ELISA-WCL. The combination of these three ELISAs identified 50.6% of the animals as positive in the infected herd. In particular, the results show that the locally developed ELISA seems to be useful for identifying many infected animals in a herd.

Changes in management practices and apparent prevalence on Canadian dairy farms participating in a voluntary risk assessment-based Johne's disease control program.

The objectives of this study were (1) to describe the change in Mycobacterium avium ssp. paratuberculosis (MAP) antibody milk ELISA-positive prevalence in Canadian dairy herds that participated in a risk assessment (RA)-based Johne's disease (JD) control program; (2) to describe the distribution of so-called high-risk management practices on Canadian dairy farms; and (3) to assess if compliance with selected recommendations translated into changes in the scores of associated RA questions. In Ontario and western Canada, 226 herds voluntarily participated in a RA-based JD control program for several years. In 2005-2007, a previsit survey, RA, and MAP-antibody milk ELISA of the entire milking herd were conducted. Therefore, the interpretation of the results of this study is strictly for the MAP-antibody milk ELISA status of cows or herds, because no culture of MAP (of fecal or environmental samples) was conducted due to economic restrictions. In early 2008, a telephone interview was used to determine compliance with recommended management changes after the first RA. In 2008-2009, a second RA and another whole-herd MAP antibody milk ELISA were performed. At both herd tests, about 35% of the farms had at least one MAP-antibody milk ELISA-positive cow, classifying them as a MAP-antibody milk ELISA-positive herd. However, 28.8% of herds had changed their MAP-antibody milk ELISA status between the 2 tests, demonstrating that a single herd test was insufficient to determine the long-term MAP-antibody ELISA status of a herd. The average within-herd MAP-antibody milk ELISA-positive prevalence changed from 5.4 to 4.2% over the study period, but management practices did not change much throughout the 2- to 3-yr period and were similar to those reported in other parts of North America. The overall RA scores decreased at the second RA, in particular for management practices in the calving and preweaned calf area, and when herds were test-positive at the first test. This was not surprising, because many of the recommendations at the first RA focused on these management areas and compliance with some recommended farm-specific management practices in this area might be linked to reduced scores for associated RA questions. In conclusion, the participating farms did, on average, decrease their within-herd MAP-antibody milk ELISA positive-prevalence and RA total scores. Changes in RA scores could be linked to improved management practices, indicating that the RA questions appropriately reflected management practices. Some herds changed their MAP-antibody milk ELISA status between tests, which underlines that a current test of the entire milking herd is necessary to determine the present MAP-antibody milk ELISA status of a dairy herd.

Strategies for time of culling in control of paratuberculosis in dairy herds.

Effect of time for culling cows infected with Mycobacterium avium ssp. paratuberculosis on prevalence and profitability was identified through simulations. Seven test-and-cull strategies with different culling criteria and no attempts to close infection routes were compared with strategies with (1) no control and (2) closure of infection routes and no culling. The effects on true prevalence and gross margin were evaluated in a herd with typical reproduction management (heat detection rate of 38%). This was repeated in a herd with poor reproduction management (heat detection rate of 28%), because poor reproduction leads to lack of replacement animals, which was hypothesized to affect the economic effects of culling. Effects of varying prices of milk, replacement heifers, and hourly wages were also evaluated. The simulated results predicted that immediate culling after the first positive antibody ELISA test would be the most effective culling strategy to reduce prevalence. However, closing transmission routes was even more effective in reducing the prevalence. In the first 3 to 6 yr, all test-and-cull strategies reduced gross margin by US$5 to 55/stall per year. These losses were fully compensated by increased gross margin in yr 6 to 19. In the short run (7 yr with typical reproduction and 10 yr with poor reproduction), it was most profitable to cull test-positive cows when their milk yield decreased below 85% of that expected according to their parity and lactation stage, especially in herds with poor reproduction management. However, this strategy only stabilized the prevalence and did not reduce it. In the long term (>7 yr from implementation of a strategy), it was most profitable to cull cows immediately or as soon as possible after testing positive the first time. Varying milk prices did not affect the ranking between the different culling strategies. Increased market price (20%) of replacement heifers made all culling strategies less profitable and made culling based on a milk yield criterion the most profitable culling strategy for a longer period (11 to 13 yr). A 20% reduction in heifer price made immediate culling after a positive test the most profitable strategy overall in herds with typical reproduction, and after 9 yr in herd with poor reproduction. To conclude, the ideal culling strategy depends on the aim of intervention, the time horizon, and the reproductive capabilities combined with prices of replacement animals.

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10(4)-10(-1)) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10(2) and 10(3)MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6weeks by real-time PCR.

Effect of classifying disease states in genetic association studies for paratuberculosis.

Genetic association studies are a means to elucidate underlying genetic regulation of host-pathogen interaction, immune response, and the fate of infection. Diseases such as paratuberculosis in cattle lack definitive diagnostic criteria, thereby complicating the definition of infection status as an outcome for genetic association studies. A study was performed to evaluate the potential bias in estimates of effect and differences in statistical power associated with parallel test interpretation, latent probability of infection adjusted for imperfect test sensitivity and specificity, and multinomial outcomes in cohorts of cattle simulated using Monte Carlo sampling methods. Test results were simulated for microbial culture of feces for Mycobacterium avium subsp. paratuberculosis (MAP) and serum ELISA for anti-MAP antibody using estimates of test sensitivity and specificity. A range of disease allele frequencies and levels of association were considered. Case-control study populations were drawn from the simulated cohorts and the association between the disease allele and infection status was evaluated using logistic regression for binary outcomes and polytomous regression for multinomial outcomes. For the majority of the classification and analytical methods evaluated, estimates of effect were biased toward the null. Frequentist approaches to analysis of the latent probability of infection and multinomial classifications based upon results of culture of feces for MAP demonstrated the smallest degree of bias. Power to detect associations was generally low for all models, but improved with larger effects and higher allele frequencies. Imperfect specificity of serum ELISA was a major factor in the degree of bias observed and statistical power. The results of this study indicate that the method of classifying infection status must be considered carefully in genetic association studies for paratuberculosis and other diseases with similar challenges in defining infection status, and study designs should be modified to accommodate relative advantages and disadvantages of available methods.

Effect of changes in testing parameters on the cost-effectiveness of two pooled test methods to classify infection status of animals in a herd.

Monte Carlo simulation was used to determine optimal fecal pool sizes for identification of all Mycobacterium avium subsp. paratuberculosis (MAP)-infected cows in a dairy herd. Two pooling protocols were compared: a halving protocol involving a single retest of negative pools followed by halving of positive pools and a simple protocol involving single retest of negative pools but no halving of positive pools. For both protocols, all component samples in positive pools were then tested individually. In the simulations, the distributions of number of tests required to classify all individuals in an infected herd were generated for various combinations of prevalence (0.01, 0.05 and 0.1), herd size (300, 1000 and 3000), pool size (5, 10, 20 and 50) and test sensitivity (0.5-0.9). Test specificity was fixed at 1.0 because fecal culture for MAP yields no or rare false-positive results. Optimal performance was determined primarily on the basis of a comparison of the distributions of numbers of tests needed to detect MAP-infected cows using the Mann-Whitney U test statistic. Optimal pool size was independent of both herd size and test characteristics, regardless of protocol. When sensitivity was the same for each pool size, pool sizes of 20 and 10 performed best for both protocols for prevalences of 0.01 and 0.1, respectively, while for prevalences of 0.05, pool sizes of 10 and 20 were optimal for the simple and halving protocols, respectively. When sensitivity decreased with increasing pool size, the results changed for prevalences of 0.05 and 0.1 with pool sizes of 50 being optimal especially at a prevalence of 0.1. Overall, the halving protocol was more cost effective than the simple protocol especially at higher prevalences. For detection of MAP using fecal culture, we recommend use of the halving protocol and pool sizes of 10 or 20 when the prevalence is suspected to range from 0.01 to 0.1 and there is no expected loss of sensitivity with increasing pool size. If loss in sensitivity is expected and the prevalence is thought to be between 0.05 and 0.1, the halving protocol and a pool size of 50 is recommended. Our findings are broadly applicable to other infectious diseases under comparable testing conditions.

Economy, efficacy, and feasibility of a risk-based control program against paratuberculosis.

Long-term effects of paratuberculosis on within-herd prevalence and on-farm economy of implementing risk-based control strategies were compared with alternative strategies by using a herd-simulation model. Closing transmission routes is essential for effective control of paratuberculosis. However, many farmers lack the resources to carry out these procedures for all cows in the herd. When using risk-based control strategies 1) all cows are tested quarterly with a milk ELISA, 2) specific cows with a high risk of being infectious are identified, and 3) the farmer can focus only on these infectious animals to close infection routes. In this way the workload can be reduced, making these control strategies more feasible. This study evaluates potential long-term effects of the risk-based approach compared with non-risk-based strategies by simulations conducted with the herd-simulation model PTB-Simherd. Seven control strategies were simulated in herds with initial true herd prevalences of 5, 25, and 50%, respectively. The results predicted the risk-based control strategies to be very efficient and comparable to the best whole-herd strategies in reducing the within-herd prevalence of paratuberculosis with considerably less labor. If infection routes are closed efficiently, prevalence can be reduced to 10% of initial prevalence within 5 to 7 yr. Test-and-cull strategies without closing infection routes were found, by simulation, to be ineffective in reducing prevalence and were not cost-effective methods. The profitability of the various control strategies depends on hourly wages and time spent per cow/calving. Furthermore, simulations show that immediate culling of highly infectious cows is only necessary and cost-effective if infection routes from these cows are not efficiently closed. The risk-based control strategies are recommended in the Danish voluntary control program "Operation Paratuberculosis," which was initiated in February 2006 and now includes 1,220 dairy farmers in Denmark.

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 (PrLow) 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 cows4 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 PrLow in infected herds, however, PrLow 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 PrLow values>0.9 for most herds, depending on herd size and age-structure. None of the strategies provided enough power to establish a high PrLow 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.

Receiver operating characteristic-based assessment of a serological test used to detect Johne's disease in Israeli dairy herds.

The overall accuracy of an enzyme-linked immunosorbent assay (ELISA) used to detect Johne's disease at herd level was explored in relation to an imperfect test (fecal culture) in 57 Israeli dairy herds. Receiver-operating characteristic (ROC) analysis indicated an area under the curve (AUC) that corresponded to a test accuracy of 82.0% (69.5% to 90.9%; 95% confidence), with optimized herd sensitivity and herd specificity of 70.4% and 83.3%, respectively; and predictive values of 79.2 (+) and 75.8% (-). The optimal ELISA cutoff was 3.16% (> 3.16% seropositive cows in a herd), which was associated with likelihood ratios (LR) of 4.22 (+LR) and 0.36 (-LR), and post-test probabilities of 0.79 (+) and 0.17 (-). For herds with < or = 200 cows (n = 19 herds), the 95% confidence interval (CI) for the AUC was 0.62-0.97 and the optimal cutoff was 3.33% (HSe = 87.5, HSp = 81.8); for herds with > 200 but < or = 270 cows (n = 19 herds), the 95% AUC CI was 0.62-0.97 and the optimal cutoff was 1.13% (HSe = 90.0, HSp = 77.78); and for herds with > 270 cows (n = 19 herds), the 95% AUC CI was 0.69-0.99 and the optimal cutoff was 0.7% (HSe = 100.0, HSp = 70.0). The AUC was not influenced by across-herd prevalence [R2 (adjusted) = 0.0, P > 0.05]. Findings may be applied to facilitate targeted sampling of herds similar to those evaluated. For instance, a test cutoff of 0.76% could be considered for "ruling disease in," while a cutoff of 3.7% could be used for "ruling disease out." Caveats that may influence this analysis are discussed.