Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA

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Abstract

RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.

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

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