Comparison of commercial DNA extraction kits and quantitative PCR systems for better sensitivity in detecting the causative agent of paratuberculosis in dairy cow fecal samples.

Mycobacterium avium ssp. paratuberculosis (MAP) causes ruminant paratuberculosis (Johne's disease) worldwide. Oral-fecal contamination is the most important mode of transmission of paratuberculosis, so eradicating MAP-shedding animals could prevent disease propagation. Fecal culture, a well-known method for MAP diagnosis, requires costly specialized media and a long incubation time that sometimes ends in disappointing bacterial contamination. To facilitate the efforts of control programs, we evaluated the performance of direct fecal quantitative PCR (qPCR) assays for their sensitivity and robustness for MAP detection. Commercial kits use different strategies for extracting DNA, combined with qPCR systems, to detect the presence of MAP in fecal samples. In this study, we compared the sensitivity of 3 commercially available DNA extraction kits (A, B, and C) combined with 2 qPCR systems (T and V) for the detection of MAP in infectious cows. A total of 49 dairy cows from 5 herds were sampled twice a year for 3 yr and diagnosed using fecal culture and ELISA. Eight replicates of their fecal samples from the first sampling were tested using each DNA extraction method and qPCR detection system. Although all 3 of the commercial DNA extraction kits have been previously described as very efficient for the diagnosis of paratuberculosis, kit B provided the highest sensitivity. Indeed, 89% of the cows declared positive for paratuberculosis by both fecal culture and ELISA were identified with kit B, whereas only 23 and 43% of the cows were identified with kits A and C, respectively. Interestingly, kit B was able to detect some low-MAP shedders. The qPCR detection system also played a critical role: system T yielded qPCR with the highest sensitivity. The results of this study suggest that DNA extraction kit B combined with detection system T provides the best amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. Although 1 DNA extraction and qPCR analysis should be adequate to confirm that an animal with diarrhea or other signs of paratuberculosis is positive, detecting low shedders at the highest sensitivity should include repetitive testing. This study demonstrates the importance of repetitions using the most appropriate method for extracting DNA from fecal samples, combined with a compatible qPCR system for identifying MAP-shedding animals.

Fock-Chow-Tho D ，Topp E ，Ibeagha-Awemu EA ，Bissonnette N ... -  《-》

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 ... -  《-》

Evaluation of three commercial PCR kits for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces.

Mycobacterium avium subsp. paratuberculosis which is more commonly referred to as MAP is the causative agent of Johnes's disease in ruminants. While cultivation of MAP from faecal samples remains the reference standard diagnostic test for the disease, faecal culture is expensive, slow and not widely available in Ireland. The current study evaluated three commercial kits that combine both DNA extraction and real-time PCR amplification of specific targets for direct MAP detection in cattle faeces. In total, 100 positive samples were tested which consisted of 25 high shedders, 25 medium shedders, 25 low shedders and 25 very low shedders. Also included were 100 negative faecal samples obtained from two Irish herds known to be free of Johne's disease. The kits evaluated in this study showed significant differences in identifying MAP DNA from animals shedding various concentrations of the bacterium. Kit C had the highest specificity, followed by kits B and A. Sensitivity in kits A, B and C was 73.5%, 81% and 93%, respectively, and specificity was 99%, 97% and 100%, respectively. Sensitivity decreased with lower concentrations of MAP in faeces but this was more significant for kits A and B than for kit C. The results of this study demonstrate that PCR can provide an accurate and rapid detection of MAP faecal shedding and kit C was selected for further evaluation of the role of PCR in the confirmation of animal infection in cattle.

Prendergast DM ，Pearce RA ，Yearsley D ，Ramovic E ，Egan J ... -  《-》

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 ... -  《-》

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 ... -  《-》