Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne’s disease by in situ hybridization

Abstract

M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn’s disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn’s disease and sarcoidosis.

Introduction

M. avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease (JD), an incurable chronic enteritis disease in domestic and wild ruminants Chiodini, 1989, Greig et al., 1999. The organism is extremely slow growing, mycobactin-dependent and highly resistant to the host immune response (Chiodini, 1989). Therefore, infected animals may harbor the organism for years before they develop disease. The disease occurs worldwide with enormous economic losses Chiodini, 1989, Whipple, 1992. Its control or eradication is limited by the lack of sensitive diagnostic tests to identify and eliminate infected animals at the subclinical stage of the disease Chiodini, 1989, El-Zaatari et al., 1997. Although primarily considered as the cause of a ruminant disease, the cell wall deficient (CWD) form of this or a closely related organism has also been implicated as a possible cause of idiopathic diseases with histologic features similar to JD such as sarcoidosis and Crohn’s disease (CD) in humans (Aluwihare, 1971, Dvorak and Dickersin, 1979, Stanford et al., 1988, Moss et al., 1992, Sanderson et al., 1992, Cartun et al., 1993, Wall et al., 1993, Dell’Isola et al., 1994, Lisby et al., 1994). Evidence has been based on emergence of CWD forms in cultures of tissue specimens, their identification as M. paratuberculosis and the detection of M. paratuberculosis DNA (or closely related MAC organism) in tissues by PCR (Moss et al., 1992, Sanderson et al., 1992, Wall et al., 1993, Dell’Isola et al., 1994, Lisby et al., 1994, El-Zaatari et al., 1996). Therefore, specific CWD forms of mycobacteria may play an important role in the pathogenesis of these diseases.

Immunohistochemical methods and electron microscopic surveys have not revealed CWD organisms in tissues from patients with the above diseases (Aluwihare, 1971, Dvorak and Dickersin, 1979, Kobyashi et al., 1989, Cartun et al., 1993, Fidler, 1994, Clarke, 1997). At present, there is no method to identify CWD forms in tissue sections using conventional light microscopy as these organisms have no discernable structural elements that distinguish them from host tissue components (Mattman et al., 1960, Dvorak and Dickersin, 1979, Cartun et al., 1993). CWD cells replicate extremely slowly in culture, do not stain with the Zeil–Neelsen method, and specific PCR assays do not discriminate between nucleic acids of the two forms. In situ hybridization (ISH) provides a way to specifically detect microbes in tissue sections and has been adapted for detection of infectious agents that are difficult to visualize by conventional methods such as viruses Han et al., 1992, Lewis and Wells, 1992 and chlamydiae (Campbell et al., 1993). ISH may prove invaluable in demonstrating a putative etiologic agent where the organism resides as a CWD form. It may also be useful in detecting residual organisms after treatment of mycobacterial infection (Imaeda, 1984). Formation of CWD cells enhances their ease of detection by ISH, as it is difficult to lyse mycobacterial bacilli without destroying host tissue structures. In this communication, we describe a specific ISH-method that is designed specifically to differentiate between the CWD as opposed to the acid-fast form of M. paratuberculosis in infected tissues.