[Show abstract][Hide abstract] ABSTRACT: Culturing of Mycobacterium avium subspecies paratuberculosis (Map) remains difficult and is time consuming. An alternative for the rapid detection of Map in samples is PCR. We have developed a sensitive DNA-extraction method based on sequence capture for the rapid detection of M. avium subspecies paratuberculosis by PCR in fecal and tissue samples. The method detected 10(2)Map/g feces using spiked samples, and reached a diagnostic sensitivity of 33,7% compared to 22% for culture. Analysis of tissue samples gave 65 polymerase chain reaction (PCR)-positive (42.2%) and 49 culture-positive samples (31.8%). Therefore, the detection limit of the DNA-extraction is the same as previously reported for culture, the PCR assay could detect more positive samples than the culture method.
Full-text · Article · Jun 2007 · Veterinary Microbiology
[Show abstract][Hide abstract] ABSTRACT: The amplified fragment length polymorphism (AFLP) technique is a reliable and powerful DNA fingerprint tool for genetic characterisation and analysis. In this paper, we described a modified AFLP with high resolution for Trypanosoma congolense using one enzyme and agarose or Elchrom gel electrophoresis. Eleven allopatric and fourteen sympatric isolates of T. congolense savannah were used to assess the resolution of the method and its ability to characterise T. congolense isolates. Two enzymes (Eco RI or Bgl II) and corresponding non-selective and selective primers were used to identify the most appropriate combination. Patterns generated by Bgl II enzyme and a single selective primer A, C, G or T produced clear profiles. Each of the four selective primers produced different profiles for all the 25 T. congolense isolates. Due to the reduction in the number of bands, profiles could be analysed using agarose or Elchrom gels. Although comparison of a great number of samples could benefit from software help, this technique did not require flurochrome detection methods. The results of the present study demonstrated that this modified AFLP makes the characterisation of T. congolense easier while maintaining high resolution.
Full-text · Article · Sep 2006 · Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.
Full-text · Article · Oct 2005 · The Veterinary Journal
[Show abstract][Hide abstract] ABSTRACT: Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map). The primers used for IS900 detection of Map have amplified these sequences causing false positive results. In this study, we have developed two new PCR assays for the detection of Map. The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence. The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates). All Map strains were positive and all non-Map strains were negative. Serial dilutions of Map bacteria were used to assess the sensitivity of the assays. We achieved a sensitivity of 1CFU per PCR for both assays. In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers. The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done.