Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene
Introduction
Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the etiologic agent of paratuberculosis, also known as Johne’s disease, a chronic inflammatory bowel syndrome in cattle, goats, sheep and other ruminants. The disease spreads slowly and is characterised by granulomatous enteritis, diarrhoea, emaciation and mortality. Paratuberculosis occurs throughout the world and the economic losses is severe because of reduced milk production and poor reproductive performance (Chiodini et al., 1984, Ott et al., 1999). The prevalence of paratuberculosis in Sweden is very low (<1%) (Viske et al., 1996). Early diagnosis of infected cattle is essential for avoiding the spread of infection and eradication is dependent on detection and culling of infected animals as early as possible.
The standard diagnostic method for paratuberculosis is isolation of the organism from faeces or from the intestinal mucosa and the associated mesenteric lymph nodes of infected animals, by culture. This is a slow method requiring up to 4 months to produce visible colonies on solid medium. Detection of IS900, an insertion sequence specific for M. paratuberculosis, by the polymerase chain reaction (PCR) has improved the diagnosis of paratuberculosis (Collins et al., 1989, Green et al., 1989). PCR methods have been developed which can increase the specificity and the sensitivity of the laboratory diagnostics (Vary et al., 1990, Moss et al., 1991, Sanderson et al., 1992, Lisby et al., 1994, Englund et al., 1999). However, difficulties are experienced in recovering DNA from small number of organisms in clinical specimens, especially in complex samples such as faeces.
Inhibitors are commonly present in the processed sample due to insufficient preparation procedures of the specimens (Stevenson and Sharp, 1997). By using nested PCR, a two step amplification procedure, the problems of inhibitory substances can to some extent be overcome. Due to its high sensitivity, false positive results caused by carry-over or cross-contamination, may easily occur. Precautions have to be taken to circumvent this problem (Belak and Ballagi-Pordany, 1993, Kox et al., 1994). Assessment of the sensitivity of the amplification and control of false negative results can be accomplished by using mimics. These are preferably designed to co-amplify with the same primers as the target DNA (Ballagi-Pordány and Belák, 1996). The size difference allows easy discrimination between their PCR products. Analysis of PCR products is commonly performed by agarose gel electrophoresis followed by ethidium bromide staining and visualisation by UV-light transillumination. A more sensitive identification of positive samples can be achieved by combining the use of fluorochrome-labelled oligonucleotide primers for PCR, together with laser-based detection of the fluorescent amplicons (Maher et al., 1995, Rowbotham et al., 1995).
The aim of the present study was to (i) develop an internal positive control molecule to be used in single PCR to improve the reliability of the amplification results, (ii) evaluate the extraction protocol for M. paratuberculosis, described by Challans et al. (1994), regarding the suitability for routine diagnostic work, and (iii) investigate the possibility to use fluorescent PCR instead of nested PCR for the diagnosis of paratuberculosis, to improve the sensitivity without increasing the risk for cross-contamination.
Section snippets
Strains, culture and DNA extraction
Ileum mucosa samples from Swedish cattle were collected at the National Veterinary Institute, Uppsala, Sweden, during 1994–1999. The fresh tissue samples were treated with NaOH and oxalic acid, and cultured as previously described (Beerwerth, 1967, Jørgensen, 1982). Specimens found to be paratuberculosis infected were stored at −20°C until used in this study.
M. paratuberculosis field strain 411/94 and M. paratuberculosis strain ATCC 19698 were grown on slopes of modified Löwenstein–Jensen
Sensitivity of PCR
The sensitivity of single PCR, fluorescent PCR and nested PCR was assessed by using a serial dilution of M. paratuberculosis DNA. The amplicons from single and nested PCR were analysed by agarose gel electrophoresis followed by ethidium bromide staining. The amplicons from single PCR were also analysed by polyacrylamide gel electrophoresis and detected on-line with the ALFexpress, followed by computerised analysis of the fragments.
Single PCR amplification of M. paratuberculosis DNA equivalent
Discussion
Eradication of M. paratuberculosis is hampered by the lack of accurate and sensitive diagnostic methods. The major problem is to trace subclinically infected animals. These animals are difficult to identify by faecal culture as the shedding of M. paratuberculosis vary with the stage of infection (Chiodini et al., 1984, Cocito et al., 1994). Serological tests are of limited value because the humoral response is most active in the late stage of the infection. Cross-reactions with related bacteria
Acknowledgements
We thank Leena Lohikari and Anna-Lena Andersson for technical assistance. This study was financially supported by grants from the Swedish Council for Forestry and Agricultural Research.
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Progress in molecular typing of Mycobacterium avium subspecies paratuberculosis
2012, Research in Veterinary ScienceCitation Excerpt :As an example, IS900 was used for the identification and characterization of M. a. paratuberculosis strains (Turenne et al., 2008). It has also been targeted in several rapid typing techniques such as fluorescent PCR with oligonucleotides fluorochrome-labelled (Rowbotham et al., 1995), multiplex PCR (Al-Shamali et al., 1997; Bull et al., 2000; Irenge et al., 2009), nested PCR (Englund et al., 1999, 2001; Erume et al., 2001; Ikonomopoulos et al., 2004; Möbius et al., 2008a), in situ PCR (Sanna et al., 2000) and real time PCR (Englund et al., 2001; Fang et al., 2002). Other techniques used to target IS900 are PCR targeting flaking regions of the IS900 (Collins et al., 2002), PCR-hybridization assay of specific IS900 integration sites (Ozbek et al., 2003) and immunomagnetic separation combined with IS900 PCR targeting (Djonne et al., 2003).
Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation
2002, Veterinary MicrobiologyCitation Excerpt :The pellets were washed once in sterile water and once in hybridisation buffer (100 mM Tris–HCl (pH 7.4), 150 mM NaCl, 50 mM EDTA) and thereafter resuspended in 500 μl hybridisation buffer and 50 μl proteinase K (20 mg/ml). The mycobacterial DNA was released by bead beating with 300 μl zirconium beads (0.1 mm in diameter, Biospec Products) followed by overnight incubation at 50 °C (Englund et al., 2001). The tubes were centrifuged at 1000×g for 1 min and the supernatants were withdrawn and subjected to sequence capture as described above.
An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis
2002, FEMS Microbiology Letters