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 ... -  《-》

Enhanced sensitivity and fast turnaround time in laboratory diagnosis for bovine paratuberculosis in faecal samples.

Bovine paratuberculosis (Johne's disease in cattle) is caused by the pathogen Mycobacterium avium subsp. paratuberculosis (MAP) and is a widespread chronic bacterial infectious disease in cattle. Due to the peculiarities of the pathogen, detection of MAP in faeces remains difficult. DNA extraction and real-time PCR for detection of MAP in bovine faeces (direct PCR) have been refined and feasible procedures for rapid, sensitive and automatable detection of the pathogen agent have been developed. Accordingly, in a first step we tested 20 faecal samples using two MAP complete kits (DNA extraction kits based on magnetic beads combined with real-time PCR assays) and six other DNA extraction kits for faeces. MAP-specific DNA was detected by real-time PCR assays. Cultivation of MAP on the solid medium HEYM and in the liquid medium M7H9C served as reference standards. The two complete kits detected significantly more MAP-DNA positive samples than the other procedures applied (p < 0.04). Ct values of 37 and 38 served as cut-off for the respective real-time PCR assays calculated on the basis of standard curves and droplet digital PCR (ddPCR). In a second step, the two MAP complete kits were employed for a comprehensive study including 107 positive and 50 negative faecal samples which had been previously tested on HEYM cultivation. The MAP complete kits yielded sensitivity values of 86% and 89% and specificity values of 100% compared to cultivation of MAP in the liquid medium M7H9C. In detail, cultivation of MAP in M7H9C detected the pathogen in 97% and 100% of the samples tested after an incubation period of six and twelve weeks, respectively. However, the cultivation of MAP on HEYM succeeded in only 74% after twelve weeks of incubation. In all these solid culture positive samples, MAP was also detected using the two complete kits. Additionally, the impact of repeated freezing and thawing of samples on re-cultivation of MAP was tested using 20 faecal samples and resulted in a reduction to 75% and 25% of bacterial growth when using liquid medium M7H9C and solid medium HEYM, respectively. The results of this study show that complete kits with refined automatable protocols for DNA extraction in combination with real-time PCR assays for detection of MAP can compete with sensitive cultivation of the pathogen in liquid medium.

Schwalm AK ，Obiegala A ，Pfeffer M ，Sting R ... -  《-》

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 ... -  《-》

Evaluation of PMS-PCR technology for detection of Mycobacterium avium subsp. paratuberculosis directly from bovine fecal specimens.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with <100 MAP per 2g feces (30.06) (p<0.05). PMS-PCR technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection.

Salgado M ，Steuer P ，Troncoso E ，Collins MT ... -  《-》

Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction.

Leite FL ，Stokes KD ，Robbe-Austerman S ，Stabel JR ... -  《-》