Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR

Effect of sodium humate and zinc oxide used in prophylaxis of post-weaning diarrhoea on faecal microbiota composition in weaned piglets

“…Faecal DNA was isolated from 200 mg of faecal samples, using a commercially available kit (Stool DNA Isolation kit, Qiagen, Germany) with previously described modifications (Kralik et al 2011). The DNA was subsequently mixed from all the animals within a group and used for amplification.…”

Detection of Mycobacterium avium subspecies paratuberculosis excretion in bovine feces, using quantitative real time polymerase chain reaction (Q-PCR)

“…The results agree with those of Khol et al (2010) in that Map shedding was detected from 12 months of age and that as time advances shedding increased logarithmically from 10 2 to 10 6 copies of IS900 of Map (bovines 1, 10 and 25; Table 4). Kralik et al (2011) developed a predictive model to determine the shedding of Map using Q-PCR with DNA extracted from feces, establishing that the minimum detectable shedding is 10 2 copies of IS900 of Map per gram of feces. The results obtained agree with those of kralik et al (2011), because in the first sampling three animals were detected as positive and the quantification was obtained as 1.9 to 5.30 × 10 2 copies of IS900 of Map per gram of feces.…”

“…Nevertheless, mistakes in DNA extraction or sample contamination could also explain the results and it cannot be excluded (Khol et al, 2010). Correlation between bacteriological culture and Q-PCR depends on the stage of the infection present in the individual at the time of sample collection which has an effect on the amount of bacilli shed (Kralik et al, 2011;Aly et al, 2010;Pinedo et al, 2008). The ELISA used in the study detected less number of animals as positive, this is because, in early infection stages the predominant immune response is cell type, and the elimination of bacillus in feces begins in an intermediate way.…”

“…Another important disadvantage of bacteriological culture is the high risk of contamination with other microorganisms, especially fungi, and the decrease in viable bacilli after the sample decontamination process. By using Q-PCR, results are obtained within at least two days, depending on the amount of samples to be processed, representing a clear advantage in time-to-diagnosis when compared with bacteriological isolation allowing the quantification of bacilli shedding in feces (Kralik et al, 2011;Khol et al, 2010). With the simultaneous use of IS900 Q-PCR, fecal culture, and the ELISA test, it is possible to detect a greater number of infected animals.…”

“…Most of the PCR protocols, for DNA/RNA Map detection, use primers that amplify the 900-insertion sequence (IS900), and therefore it is been accepted as a standard Map marker. IS900 is a 1451 base-pair sequence that codifies for a hypothetical transposase of 399 amino acids, with 14 to 20 copies in the MAP genome inserted in a consensus region within highly conserved loci in the Map genome (Kralik et al, 2011;Bull et al, 2000). It is thought that, for every animal diagnosed with the clinical profile of paratuberculosis, there are approximately 25 other individuals that have a subclinical profile and that these will intermittently shed the bacillus through feces.…”