Elsevier

Veterinary Microbiology

Volume 81, Issue 3, 8 August 2001, Pages 257-271
Veterinary Microbiology

Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene

https://doi.org/10.1016/S0378-1135(01)00348-0Get rights and content

Abstract

The aim of this study was to determine if fluorescent PCR could be used instead of nested PCR, for the detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in clinical specimens, to improve the sensitivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratuberculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagnostic work. The sensitivity of each PCR system was assessed by analysing a serial dilution of M. paratuberculosis DNA. To improve the reliability of the PCR and to facilitate the interpretation of the PCR results, a positive internal control molecule (“mimic”) was developed and used for single and fluorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extraction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on seventeen ileum mucosa specimens, previously found positive for M. paratuberculosis by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis organisms, illustrating the effect of inhibitory substances in combination with small amounts of M. paratuberculosis DNA. We conclude that the extraction method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimens, but that the method is useful for identifying positive samples. Fluorescent PCR was a suitable alternative to both single PCR and nested PCR for the detection of M. paratuberculosis.

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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