Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds.

Laurin EL ，Sanchez J ，Chaffer M ，McKenna SLB ，Keefe GP ... -  《-》

Longitudinal relationship between fecal culture, fecal quantitative PCR, and milk ELISA in Mycobacterium avium ssp. paratuberculosis-infected cows from low-prevalence dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of ruminant Johne's disease, presents a particular challenge with regard to infection mitigation on dairy farms. Diagnostic testing strategies to identify and quantify MAP and associated antibodies are imperfect, and certain facets of the relationship between diagnostic tests remain to be explored. Additional repeated-measures data from known infected animals are needed to complement the body of cross-sectional research on Johne's disease-testing methods. Statistical models that accurately account for multiple diagnostic results while adjusting for the effects of individual animals and herds over time can provide a more detailed understanding of the interplay between diagnostic outcomes. Further, test results may be considered as continuous wherever possible so as to avoid the information loss associated with dichotomization. To achieve a broader understanding of the relationship between diagnostic tests, we collected a large number of repeated fecal and milk samples from 14 infected cows, in addition to bulk milk samples, from 2 low-prevalence dairy herds in the northeast United States. Predominately through the use of mixed linear modeling, we identified strong associations between milk ELISA optical density, fecal quantitative PCR, and fecal culture in individual animals while concurrently adjusting for variables that could alter these relationships. Notably, we uncovered subtleties in the predictive abilities of fecal shedding level on milk ELISA results, with animals categorized as disease progressors reaching higher ELISA optical density levels. Moreover, we observed that spikes in fecal shedding could predict subsequent high ELISA values up to 2 mo later. We also investigated the presence of MAP in individual milk samples via PCR and noted an association between poor udder hygiene and MAP positivity in milk, suggesting some level of environmental contamination. The paucity of positive milk samples and the complete absence of detectable MAP in the bulk tank throughout the study period indicate that contamination of milk with MAP may not be of chief concern in low-prevalence herds. An enhanced understanding of the interrelationships between diagnostic tests can only benefit the development of testing strategies and objectives, which in turn may lessen MAP infection prevalence in dairy herds.

Beaver A ，Sweeney RW ，Hovingh E ，Wolfgang DR ，Gröhn YT ，Schukken YH ... -  《-》

The association of detection method, season, and lactation stage on identification of fecal shedding in Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative organism of Johne's disease. Although fecal culture is considered the standard diagnostic test, the long incubation times, costs, and intermittent shedding of MAP hinder efficient screening programs based on culture results. The primary objectives of this study were to determine the detection ability of solid culture, broth culture, and real-time PCR (qPCR) for MAP in fecal samples and to assess how shedding patterns of MAP in feces vary with lactation stage and season. This knowledge could improve the use of these diagnostic assays in Johne's management programs. For this study, 51 MAP-infectious cows from 7 Atlantic Canadian dairy farms had fecal samples collected monthly over a 12-mo period. Samples were analyzed for MAP bacterial load via solid culture, broth culture, and qPCR. For all fecal samples, 46% [95% confidence interval (CI): 40 to 51%] were positive by solid culture, 55% (95% CI: 50 to 60%) by broth culture, and 78% (95% CI: 73 to 82%) by qPCR. Sensitivity of qPCR was numerically higher in the dry and postpartum lactation periods, and qPCR detection in summer and fall was 85% of that in winter and spring. Furthermore, culture-determined moderate or light shedding categories generally corresponded to qPCR cycle threshold values <35, but heavy shedding categories corresponded to qPCR values <29. Direct fecal qPCR is a MAP detection method that is quick and less costly than culture techniques, and it avoids the use of decontamination steps that could decrease numbers of bacteria in a sample below the detection limit. This study indicates that, for known MAP-positive cows, fecal qPCR had high sensitivity of MAP detection, thereby supporting the use of direct fecal qPCR as part of a Johne's herd control program.

Laurin EL ，Chaffer M ，McClure JT ，McKenna SL ，Keefe GP ... -  《-》

Long-term detection of Mycobacterium avium subspecies paratuberculosis in individual and bulk tank milk from a dairy herd with a low prevalence of Johne's disease.

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease (JD) in ruminants and is shed into the milk of infected cows, which contributes to the controversial discussion about a possible link between MAP and Crohn's disease in humans. The aim of the study was to investigate the risk for the entry of MAP in the food chain via milk from dairy farms with subclinical JD. Therefore, the occurrence of MAP in the milk of a dairy herd with a low prevalence of JD was studied in single and bulk tank milk samples over a period of 23 mo and compared with MAP shedding into feces. Milk, fecal, and blood samples were taken from all cows older than 1.5 yr of age at the beginning and the end of the trial and analyzed for MAP or specific antibodies. In addition, 63 cows (33 MAP infected and 30 MAP noninfected) were selected for monthly sampling. Raw and pasteurized bulk tank milk samples were collected on a monthly basis. The milk samples were tested for MAP by real-time quantitative PCR (qPCR), and the fecal samples were tested for bacterial shedding by qPCR or solid culture. Based on the results of the herd investigations, the prevalence of cows shedding MAP was around 5%; no cases of clinical JD were observed during the study period. The results of the ELISA showed high variation, with 2.1 to 5.1% positive milk samples and 14.9 to 18.8% ELISA-positive blood samples. Monthly milk sampling revealed low levels of MAP shedding into the individual milk samples of both MAP-infected and noninfected cows, with only 13 cows shedding the bacterium into milk during the study period. Mycobacterium avium ssp. paratuberculosis was not detected by qPCR in any raw or pasteurized bulk tank milk sample throughout the study. A significant positive association could be found between MAP shedding into milk and feces. From the results of the present study, it can be concluded that MAP is only shed via milk in a small proportion of cows with subclinical JD for a limited period of time and is diluted below the detection level of qPCR within the bulk tank milk of these herds. These findings indicate that dairy herds subclinically infected with JD pose only a minor source for human MAP consumption with milk and milk products.

Khol JL ，Wassertheurer M ，Sodoma E ，Revilla-Fernández S ，Damoser J ，Osterreicher E ，Dünser M ，Kleb U ，Baumgartner W ... -  《-》

Age at occurrence of Mycobacterium avium subspecies paratuberculosis in naturally infected dairy cows.

Nielsen SS ，Ersbøll AK 《-》