Prevalence estimates for paratuberculosis adjusted for test variability using Bayesian analysis

The ELISA tests that are available to detect an infection with Mycobacterium avium subsp. paratuberculosis (MAP) have a limited validity expressed as the sensitivity (Se) and specificity (Sp). In many studies, the Se and Sp of the tests are treated as constants and this will result in an underestimation of the variability of the true prevalence (TP). Bayesian inference provided a natural framework for using information on the test variability (i.e., the uncertainty) in the estimates of test Se and Sp when estimating the TP.

Data from two prevalence studies for MAP using an ELISA in several regions in two locations were available for the analyses. In location 1, all cattle of at least 3 years of age were sampled in approximately 90 randomly sampled herds in each of the four regions of the country. In location 2, in 30 randomly sampled herds in each of three regions, approximately 30 randomly selected cows were sampled. Information about the unknown test Se and Sp and MAP prevalence was incorporated into a Bayesian model by joint prior probability distributions. Posterior estimates were obtained by combining the actual likelihood with the prior distributions using Bayes’ formula.

The corrected cow-level TP (proportion of infected cows in a herd) was low, 5.8 and 3.6% in locations 1 and 2, respectively. Certain regions within a location differed significantly in herd-level TP (proportion of infected herds). The herd-level TP was 54.3% in location 1 (95% credible interval (CI) 46.1, 63.3%) and 32.9% in location 2 (95% CI: 14.4, 73.3%). The variation in the herd-level TP estimate for location 2 was more than three times as large as the variation in location 1 mainly because of the relatively small number of investigated herds in location 2. In future prevalence studies for MAP, sample size calculations should be based on a very low cow-level prevalence. Approximately 50 and 90% of the herds in the current study had an estimated cow-level TP below 4 and 10%, respectively.     

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.     

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

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

Dairy farmers' reasons for participation in the Danish control programme on bovine paratuberculosis

The Danish dairy industry initiated a voluntary, producer-paid control programme on Mycobacterium avium subsp. paratuberculosis (MAP) in 2006. Approximately 28% of the dairy herds, including 40% of the cows, are part of the programme. Farmers' reasons for participation are of great importance for the success of voluntary disease control programmes. This study was carried out to determine these reasons. Questionnaires with seven close-ended questions were mailed to the 1177 voluntary participants in 2008. A total of 1013 (86%) responded, and multiple reasons could be given by each farmer. The distribution of reasons for participation in the programme was: (1) control to increase animal health (91%); (2) certification "free of MAP infection" within 4-10 years (87%); (3) control to avoid production losses associated with MAP infections (86%); (4) control of MAP infections to increase consumer safety (64%); (5) certification for sale of livestock (58%); (6) control following production losses (48%); and (7) certification "free of MAP infection" within 1-3 years (31%). It was not possible to rank the reasons for participation since weighting of responses was not included. Therefore, the relative importance of the reasons given could not be determined. Based on the farmers' responses, control of MAP infections to increase animal health and avoid production losses were frequent reasons for participating in the Danish paratuberculosis control programme. The results also indicate that implementation of a certification programme would be desired by farmers. Animal health advisors should consider the diversity of participation reasons and address the specific reasons of individual farmers when establishing a strategy for control of MAP infections in a herd.     

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

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

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

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

Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle

Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4 cfu per tube less than placebo treated cows (P = 0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P = 0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR = 0.27; P = 0.03). Monensin sodium administered to infected animals at 335 mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown.     

Association between cow reproduction and calf growth traits and ELISA scores for paratuberculosis in a multibreed herd of beef cattle

The objective of this research was to assess the association between 4 cow reproductive and weight traits, and 2 preweaning calf traits and ELISA scores for paratuberculosis (0 = negative, 1 = suspect, 2 = weak-positive, and 3 = positive) in a multibreed herd of cows ranging from 100% Angus (A) to 100% Brahman (B). Cow data were 624 gestation lengths (GL), 358 records of time open (TO), 605 calving intervals (CI), and 1240 weight changes from November to weaning in September (WC) from 502 purebred and crossbred cows. Calf data consisted of 956 birth weights (BWT), and 923 weaning weights adjusted to 205 d of age (WW205) from 956 purebred and crossbred calves. Traits were analyzed individually using multibreed mixed models that assumed homogeneity of variances across breed groups. Covariances among random effects were assumed to be zero. Fixed effects were year, age of cow, sex of calf, year × age of cow interaction (except WC), age of cow × sex of calf interaction (only for WC), and covariates for B fraction of sire and cow, heterosis of cow and calf, and ELISA score. Random effects were sire (except for TO and CI), dam, and residual. Regression estimates of cow and calf traits on ELISA scores indicated that lower cow fertility (longer TO), lower ability of cows to maintain weight (negative WC), lower calf BWT, and lower calf WW205 were associated with higher cow ELISA scores. Further research on the effects of subclinical paratuberculosis in beef cattle at regional and national levels seems advisable considering the large potential economic cost of this disease.     

A practical approach to calculate sample size for herd prevalence surveys

When designing a herd-level prevalence study that will use an imperfect diagnostic test, it is necessary to consider the test sensitivity and specificity. A new approach was developed to take into account the imperfections of the test. We present an adapted formula that, when combined with an existing piece of software, allows improved planning. Bovine paratuberculosis is included as an example infection because it originally stimulated the work. Examples are provided of the trade-off between the benefit (low number of herds) and the disadvantage (large number of animals per herd and exclusion of small herds) that are associated with achieving high herd-level sensitivity and specificity. We demonstrate the bias in the estimate of prevalence and the underestimate of the confidence range that would arise if we did not account for test sensitivity and specificity.     

Evaluation of Ziehl–Neelsen stained faecal smear and ELISA as tools for surveillance of clinical paratuberculosis in cattle in the Netherlands

Testing cattle suspected of clinical paratuberculosis is an important element of surveillance of paratuberculosis. The aim of this study was to evaluate the diagnostic-test characteristics of microscopic examination of Ziehl–Neelsen stained faecal smears for acid-fast Mycobacteria (ZN-test) and serum-ELISA in cattle suspected of clinical paratuberculosis in the Netherlands.Results of all samples submitted for ZN-test and serum-ELISA between April 2003 and April 2006 to our laboratory were retrieved. Results from cattle for which both tests were performed were analysed using two Bayesian latent-class models for evaluation of diagnostic tests in two populations without a gold standard, assuming (a) conditional independence of tests, or (b) conditional dependence of tests in both infected and non-infected cattle. Sampled cattle were divided into two populations in different ways using four known risk factors for clinical paratuberculosis: region, soil type, clinical signs, and age.For 892 cattle suspected of clinical paratuberculosis, both ZN-test and serum-ELISA results were retrieved: 250 ZN-positive and ELISA-positive, 12 ZN-positive and ELISA-negative, 260 ZN-negative and ELISA-positive, and 370 ZN-negative and ELISA-negative cattle.With priors based on the available literature, the posterior estimates of sensitivity, specificity, and positive and negative predictive values of the ELISA were always higher than those of the ZN-test. Furthermore, lower limits of the 95% credibility intervals of the posterior positive predictive values of the ELISA were ≥99.7%, and of the negative predictive values of the ELISA ≥56.4%.We conclude that the ELISA is preferred to the ZN-test to confirm the presumptive diagnosis of clinical paratuberculosis in the Netherlands. Little diagnostic information can be gained by performing the ZN-test in addition to the ELISA.     

Factor analysis of a Johne's disease risk assessment questionnaire with evaluation of factor scores and a subset of original questions as predictors of observed clinical paratuberculosis

Factor analysis was used to examine the interrelationships among 38 variables collected as part of a Johne's disease risk assessment questionnaire completed in 2002 on 815 U.S. dairy operations. Eleven factors were extracted, accounting for two-thirds of the variance encountered in the original variables. Responses to many of the risk assessment questions were closely related. Standardized scores on the 11 factors were calculated for operations providing complete information, and were evaluated as predictors in a model-based logistic regression analysis with the outcome being whether operations had observed one or more cows with clinical signs suggestive of paratuberculosis during the previous year. A logistic regression model was also used to evaluate the predictive ability of a reduced subset of approximately one-third of the original variables that was selected to represent the derived factors. The performance of both sets of predictors was comparable with respect to goodness-of-fit and predictive ability. In conclusion, the length of the current risk assessment instrument could be reduced considerably without a substantial loss of information by removing or combining questions that are strongly correlated.     

Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia

The sensitivities and specificities of an absorbed enzyme-linked immunosorbent assay (ELISA) and an agar-gel immuno-diffusion (AGID) test for the detection of Johne’s disease in sheep were estimated using data from six known infected and 12 assumed uninfected sheep flocks. Sensitivities were estimated for all histologically positive sheep, as well as by histological lesion score, lesion type (paucibacillary or multibacillary) and sheep body-condition score, with ELISA sensitivities estimated at 95 and 99% specificity. Logistic-regression analysis was used to test for significant effects of lesion score and condition score, with flock included in the model as a random effect.

Estimated specificities were 95% (95% CI: 93.4, 95.6%) and 99% (98.4, 99.4%) for ELISA cut-point ratios of 2.4 and 3.6, respectively, and 100% (99.7, 100.0%) for the AGID. Estimated sensitivities for all infected sheep were 41.5% (35.0, 48.3%), 21.9% (16.6, 27.9%) and 24.6% (19.1, 30.7%) for ELISA cut-point ratios of 2.4 and 3.6 and for AGID, respectively. Sensitivities of all tests and cut-points varied significantly between flocks and between categories of lesion score and condition score. Sensitivity ranged from 25 to 73, 10 to 47 and 9.2 to 63% between flocks, for the ELISA with cut-points of 2.4 and 3.6, and for the AGID, respectively. Sensitivity was highest in thin sheep and in sheep with multibacillary lesions. The effects of lesion type and condition score on test sensitivity were significant in the logistic regressions for the AGID and ELISA at both cut-points and the flock effect was significant for the AGID but not for the ELISA at either cut-point.     

Tissue predilection sites and effect of dose on Mycobacterium avium subs. paratuberculosis organism recovery in a short-term bovine experimental oral infection model

The objective of this study was to develop a short-term experimental infection model for Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, using small oral doses of organisms. Specifically, the effect of dose size was evaluated, as well as specific tissue predilection sites for recovery of MAP. Oral doses as low as 1.5 x 10(6) CFU reliably produced infection that could be detected 3 weeks following infection. Detection of infection required culture of multiple intestinal samples (jejunum and ileum) for MAP. Histological examination did not permit detection at this early stage. Results from this study suggest intestinal mucosa, rather than tonsil, as the primary portal of entry for MAP. The experimental infection model described here is useful for studying the early effects of preventive and therapeutic interventions for paratuberculosis in cattle.     

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.     

A model (BSurvE) for estimating the prevalence of bovine spongiform encephalopathy in a national herd

We developed the BSurvE spreadsheet model to estimate the true prevalence of bovine spongiform encephalopathy (BSE) in a national cattle population, and evaluate national BSE surveillance programs. BSurvE uses BSE surveillance data and demographic information about the national cattle population. The proportion of each cohort infected with BSE is found by equating the observed number of infected animals with the number expected, following a series of probability calculations and assuming a binomial distribution for the number of infected animals detected in each surveillance stream. BSurvE has been used in a series of international workshops, where analysis of national datasets demonstrated patterns of cohort infection that were consistent with infection-control activities within the country. The results also reflected the timing of known events that were high-risk for introduction of the infectious agent.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).     

Evaluation of a bulk-milk ELISA test for the classification of herd-level bovine leukemia virus status

The results of a commercial bulk-milk enzyme-linked immunosorbent assay (ELISA) test for herd-level bovine leukemia virus (BLV) status were compared to results obtained from individual agar-gel immunodiffussion (AGID) testing on sampled cattle. A positive herd was defined as a herd having one or more AGID-positive animals. The estimated true herd status was based on the sensitivity and specificity of the AGID test and the number of cattle sampled per herd. Ninety-seven herds were used, with a mean of 13 cows sampled per herd. The AGID test indicated an apparent herd prevalence of 70.1%. After accounting for the number of cows sampled and the sensitivity and specificity of the AGID test, the estimated true herd prevalence of BLV was 52.3%. The ELISA test identified 79.4% of herds as positive for BLV, and had an apparent sensitivity and specificity of 0.97 and 0.62, respectively. However, after accounting for the sensitivity and specificity of the AGID test in individual animals, the specificity of the ELISA test was 0.44. The ELISA test was useful for identifying BLV-negative herds (i.e., ruling out the presence of BLV infection in test negative herds). With the moderately low specificity, herds identified as positive by the ELISA test would require further testing at the individual or herd level to definitively establish their BLV status.     

基于单克隆抗体的O型口蹄疫病毒抗体固相竞争ELISA检测方法的建立

为建立评价O型口蹄疫病毒(FMDV)疫苗免疫水平的方法,本研究以单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 8E8作为检测MAb,经过条件优化建立了基于MAb的检测O型FMDV抗体的固相竞争ELISA(SPCE)方法。对该方法进行了特异性、敏感性、重复性试验。结果显示,MAb 3D9的最佳稀释度为1:25 000,灭活O型FMDV抗原的最佳稀释浓度为1:3,HRP标记的MAb 8E8的最佳稀释度为15 000,当血清1:32稀释时,检测的临界值确定为45%。该方法分别检测A型FMDV抗体阳性参考血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清,检测结果均为阴性,未出现交叉反应。经检测,当阳性标准血清的抗体稀释度在1:512时,该方法仍具有较好的敏感性;批内和批间重复性试验的变异系数均小于10%,表明其重复性较好。并将该方法与液相阻断ELISA(LPBE)方法和病毒中和试验(VNT)的相关性进行了比较,结果显示该方法与LPBE和VNT的相关性分别为0.896和0.923。本研究为国内评价O型FMDV疫苗免疫水平建立了一种新的方法。     

Development,validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin

Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro–differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level.