Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA
Introduction
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an obligate pathogen that causes an infiltrative enteritis and wasting syndrome, Johne's disease (JD) in ruminants. JD has a significant economic impact on ruminant livestock industries worldwide Ott et al., 1999, Johnson Ifearulundu et al., 1999. M. paratuberculosis resides within macrophages in the cellular infiltrate in the gut wall and in the draining mesenteric lymph nodes (Momotani et al., 1988). Like other pathogenic mycobacteria, they have the ability to survive and replicate within non-activated macrophages, circumventing host defence mechanisms including the oxidative burst activity of the activated macrophage Momotani et al., 1988, Bannantine and Stabel, 2000, Ott et al., 1999, Laneelle and Daffe, 1991. The mechanisms allowing survival and persistence of M. paratuberculosis within the hostile environment of the macrophage are not well understood. Current suggestions are predominantly based on experiments with other mycobacterial species such as Mycobacterium tuberculosis (M. tuberculosis) or Mycobacterium avium subspecies avium (M. avium). The latter is 98% genetically similar to M. paratuberculosis (Bannantine et al., 2002). Mycobacteria are believed to prevent fusion of the phagosome with lysosomes because of incomplete maturation of the phagosome (Via et al., 1997).
For the major mycobacterial diseases of tuberculosis and leprosy, small animal models and in vitro cultured cell lines are routinely used in the laboratory to study host–pathogen interactions Aldwell et al., 2001, Gomes et al., 2001, Triccas et al., 2001. While these model systems have undoubtedly assisted in identifying some genes involved in bacterial persistence and disease, they have failed to identify specific pathogen-related factors responsible for the disease state within the natural host.
In this paper, we describe a method to extract and purify high quality RNA from M. paratuberculosis in clinically infected ruminants. In most bacterial diseases, experimental reproduction of disease in the natural host and preparation of significant quantities of fresh clinical bacterial material can be difficult. Experimental JD infection can be achieved in a range of animal species (Beard et al., 2001) and it is possible to obtain fresh samples of large segments of intestinal tract at various stages of infection. Messenger RNA has a short half-life therefore it is essential to stabilise RNA as soon as the environment in which the bacteria reside is disturbed. RNA is also highly susceptible to degradation by external influences such as RNase enzymes or hydroxide. The RNA to be stabilised in this experiment was located within M. paratuberculosis cells infecting host intestinal macrophages. Therefore, the stabilisation agent must successfully and rapidly permeate the eukaryotic tissue and mycobacterial cell wall, while protecting the RNA from various types of degradation. In this paper, two stabilising agents were assessed, namely guanidinium thiocyanate (GTC) and RNAlater solutions. The potential of this method is illustrated by analysis of the M. paratuberculosis katG gene. The katG gene is shown to be upregulated in M. paratuberculosis isolated from the diseased animal compared to M. paratuberculosis grown in vitro. This is the first reported example of analysis of selectively purified mycobacterial RNA isolated from a clinically affected ruminant.
Section snippets
Bacterial strains
CLIJ623 is a wild-type Australian bovine isolate of M. paratuberculosis grown in vitro in Watson–Reid medium (WRM) (Watson, 1935). In vivo-derived M. paratuberculosis samples CLIJ619, CLIJ290, CLIJ294 and CLIJ523 are mycobacteria preparations isolated from one Friesian cow naturally infected with M. paratuberculosis, two goats experimentally infected with CLIJ623 and one goat experimentally infected with an ovine M. paratuberculosis isolate, respectively (Stewart et al., 2002).
RNA extraction and purification
M.
Preparation of RNA for real-time PCR
A sample of 10 μg of RNA was treated with DNaseI (3 units) Ambion, Austin, TX) in the buffer provided by the manufacturer at a concentration of 0.1 μg/μl and incubated at 37 °C for 10 min. The DNaseI enzyme was inactivated by treatment with a DNase inactivation solution as described by the manufacturer. Reverse transcription was performed on 1 μg RNA sample using Multiscribe reverse transcriptase enzyme (Applied Biosystems, UK) according to the manufacturer's protocol in a total volume of 50
Selective harvesting of mycobacterial cells from ileum scrapings
The Ziehl-Neelsen stain confirmed the presence of acid fast, rod-shaped bacilli in the mycobacterial pellet. The yield of RNA from in vivo-derived samples varied for each individual host, however up to 10 μg of M. paratuberculosis RNA was successfully purified from each of the 10-cm sections of ileal tissue. Agarose gel electrophoresis analysis of RNA identified bacterial 16s and 23s ribosomal bands (Fig. 1). To demonstrate that the recovered bacterial RNA was predominantly that of M.
Discussion
The identification of bacterial genes that are differentially expressed in the disease causing state is a main goal of whole genome expression studies with pathogenic bacteria. Currently, the limiting factor in this type of work is the ability to extract sufficient quantities of high quality bacterial RNA from diseased tissue. This report describes methodology for a selective and effective purification of M. paratuberculosis from the gut of JD-infected animals. Intact RNA was successfully
References (34)
- et al.
In vitro control of Mycobacterium bovis by macrophages
Tuberculosis (Edinburgh)
(2001) - et al.
HspX is present within Mycobacterium paratuberculosis-infected macrophages and is recognized by sera from some infected cattle
Vet. Microbiol.
(2000) - et al.
A method to isolate RNA from gram-positive bacteria and mycobacteria
Anal. Biochem.
(1994) - et al.
A method for purification and characterisation of Mycobacterium avium subsp. paratuberculosis from the intestinal mucosa of sheep with Johne's disease
Vet. Microbiol.
(1998) - et al.
Role of iron in experimental Mycobacterium avium infection
J. Clin. Virol.
(2001) - et al.
Mycobacterial cell wall and pathogenicity: a lipodologist's view
Res. Microbiol.
(1991) - et al.
Quantitative real-time PCR for the measurement of feline cytokine mRNA
Vet. Immunol. Immunopathol.
(1999) - et al.
Herd-level economic losses associated with Johne's disease on US dairy operations
Prev. Vet. Med.
(1999) - et al.
Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities
Vet. Microbiol.
(1995) - et al.
Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7
J. Biol. Chem.
(1997)
Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences
J. Clin. Microbiol.
Molecular characterization of Mycobacterium paratuberculosis isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900
J. Clin. Microbiol.
Experimental paratuberculosis in calves following inoculation with a rabbit isolate of Mycobacterium avium subsp. paratuberculosis
J. Clin. Microbiol.
Systematics, differentiation, and detection of bacterial infections—the family Mycobacteriaceae
Immun. Infekt.
Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host
Infect. Immun.
The envelope of mycobacteria
Annu. Rev. Biochem.
Characterization of Mycobacterium paratuberculosis of bovine, caprine, and ovine origin by gas–liquid chromatographic analysis of fatty acids in whole-cell extracts
Am. J. Vet. Res.
Cited by (14)
Analysis of DNA extraction methods for detection of Treponema pallidum: A comparison of three methods
2022, Journal of Microbiological MethodsCitation Excerpt :However, the use of the in-house sonication method has shown very promising results (Bello et al., 2020). Furthermore, Granger et al. (2004) reported that the use of sonication would minimize the risk of cross-contamination between samples (Pchelintsev et al., 2016). Dai et al. (2016) concluded that the sonication-based technique would be considered more promising and efficient for homogeneous cell decomposition in a short period.
Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication
2020, Brazilian Journal of Infectious DiseasesCitation Excerpt :Also, many laboratories use in-house methods for this step, mainly to curb costs.11–13 The DNA extraction method that uses the cellular disruption by sonication has also been used in several laboratories,14–16 but there is still doubt about the efficacy of DNA extracted directly from sputum clinical samples (which have a wide variety of microorganisms, many impurities, and dependence on good collection) for use in PCR, as the protocol does not include a purification step.17,18 Therefore, this study aimed to compare a sonication technique as the method for obtaining DNA from sputum samples with the commercial method (silica resin - Detect-TB kit, marketed by Labtest / MG-Brazil).
Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis
2015, Journal of Microbiological MethodsCitation Excerpt :Two variations to this method were also attempted by adding 1% Triton X-100 or 0.1% NP40 to the H2O before the boiling step. A previous study by Granger et al. (2004) reported sonication to enrich mycobacterial cells in tissue samples. Given this, we adapted Granger's method in an attempt to increase the quantity and quality of DNA obtained from bovine tissue samples.
How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection?
2011, Journal of Microbiological MethodsCitation Excerpt :Using the bead beater resulted in 65–95% higher yield compared to pre-treatment with cell extraction buffers, although total DNA extraction procedures did result in more inhibitors of PCR (Cook and Britt, 2007; Ravva and Stanker, 2005). However, one study found that a cell extraction method for “enriching” mycobacterial RNA was successful for RT-PCR (Granger et al., 2004). M. paratuberculosis is not usually seen in stained human biopsies, although it can be detected by in situ hybridization (Sechi et al., 2001).
The recovery of Mycobacterium avium subspecies paratuberculosis from the intestine of infected ruminants for proteomic evaluation
2008, Journal of Microbiological MethodsMycobacterium avium subsp. paratuberculosis-specific mpt operon expressed in M. bovis BCG as vaccine candidate
2008, Veterinary Microbiology