Cell Wall Deficient Forms Research Articles

L-arabinose induces the formation of viable non-proliferating spheroplasts in Vibrio cholerae.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold, but persists in cold waters under the form of viable but non-dividing coccoidal shaped cells. Additionally, V. cholerae is able to form non-proliferating spherical cells in response to cell wall damage. It was recently reported that L-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that L-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable and upon removal of L-arabinose they start expanding in volume, form branched structures and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of a high expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of L-arabinose by the V. cholerae homolog of the E. coli galactose transport and catabolic system. Taken together, our results suggest that the formation of non-growing spherical cells is a common response of Vibrios exposed to detrimental conditions. They also permit to define conditions preventing any physiological perturbation of V. cholerae when using L-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon.Importance Vibrios among other bacteria form transient cell wall deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated to antimicrobial tolerance, chronic infections and environmental dispersion.The effect of L-Ara on V. cholerae could provide an easily tractable model to study the ability of Vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of L-Ara is ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

Changed immune and miRNA response in RAW264.7 cells infected with cell wall deficient mycobacterium tuberculosis.

Cell wall deficient (CWD) forms of Mycobacterium tuberculosis (Mtb) confers a marked resistance to immune system of the host. However, there is limit data on the effect of intracellular CWD-Mtb infection on macrophages. In the study, effects of CWD-Mtb on cell viability, cytokine response and miRNA expression of macrophages were analyzed. Cell viability was reduced, levels of interleukin-1α(IL-1α), IL-1β, IL-6, IL-10 and interferon-γ (IFN-γ) were also significantly changed after infection of RAW264.7 cells with CWD-Mtb. A total of 105miRNAs were deregulated between CWD-Mtb and wild Mtb group, and among them, miR-29b was upregulated in CWD-Mtb group. Downregulation of miR-29b resulted in significant elevation level of IFN-γ mRNA. Involved signaling pathways of potential target genes of differentially expressed miRNAs mainly focused on Tcell receptor signaling pathway, MAPK signaling pathway, neurotrophin signaling pathway, and regulation of actin cytoskeleton. Taken together, the results showed that cytokine production of CWD-Mtb infected macrophages was altered and many miRNAs were involved in regulation of macrophage response to CWD-Mtb infection, which probably determined the differential outcome following different phenotype Mtb infection. These findings open up a new and interesting avenue for an improved understanding of pathogenesis of CWD-Mtb.

Essential cellular modules for the proliferation of the primitive cell

Bacteria appeared early in the evolution of cellular life on planet Earth, and therefore the universally essential genes or biological pathways found across bacterial domains may represent fundamental genetic or cellular systems used in early life. The essential genes and the minimal gene set required to support bacterial life have recently been experimentally and computationally identified. It is, however, still hard to estimate the ancient genes present in primitive cells compared to the essential genes in contemporary bacteria, because we do not know how ancestral primitive cells lived and proliferated, and therefore cannot directly evaluate the essentiality of the genes in ancestral primitive cells. The cell wall is normally essential for bacterial proliferation and cellular division of walled bacterial cells is normally highly controlled by the essential FtsZ cell division machinery. But, bacteria are capable of reverting to their cell wall deficient ancestral form, called the “L-form”. Unlike “normal” cells, L-forms divide by a simple physical mechanism based on the effects of membrane dynamics, suggesting a mode of primitive proliferation before the appearance of the cell wall. In this review, we summarize the experimental and computational investigations of minimal gene sets and discuss the minimal cellular modules required to support the proliferation of primitive cells, based on L-form proliferation.

Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination

Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features (“fried eggs” colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine.

Glycerol uptake is important for L-form formation and persistence in Staphylococcus aureus.

S. aureus is a significant human pathogen and has previously been shown to form cell wall deficient forms or L-forms in vitro and in vivo during infection. Despite many previous studies on S. aureus L-forms, the mechanisms of L-form formation in this organism remain unknown. Here we established the L-form model in S. aureus and constructed a transposon mutant library to identify genes involved in L-form formation. Screening of the library for mutants defective in L-form formation identified glpF involved in glycerol uptake being important for L-form formation in S. aureus. Consistent with this observation, glpF was found to be highly expressed in L-form S. aureus but hardly expressed in normal walled form. In addition, glpF mutant was found to be defective in antibiotic persistence. The defect in L-form formation and antibiotic persistence of the glpF mutant could be complemented by the wild type glpF gene. These findings provide new insight into the mechanisms of L-form formation and persistence in S. aureus and may have implications for development of new drugs targeting persisters for improved treatment.

The tuberculin skin test: How safe is safe? - the tuberculins contain unknown forms capable of reverting to cell-wall-deficient mycobacteria

Tuberculin is made from proteins derived from tubercle bacilli that have been “killed” by heating. Yet in both Zwadyk’s 1994 study and Bemer-Melchior’s 1999 investigations ‘heatkilled’ tuberculosis and its related mycobacteria, whether in tuberculin, vaccination or otherwise, have dormant, practically indestructible cell-wall-deficient forms which can revert back to virulent TB bacilli “killed” ― by neither heat nor sterilization. The ability and actual preference of mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis to form filterable, multi-shaped cell-wall-deficient (CWD) forms and spores in order to survive unfavorable conditions has in fact been known for some time. But the possibility of PPD tuberculins for human use containing such potentially virulent CWD forms, even after autoclaving, sterilizing and ultrafiltration, has not. Autoclaved ultra-filtrates of the various mycobacteria used to produce tuberculin skin tests, consisting of M. Tuberculosis, M. bovis, and M. avium were investigated. All samples were mixed with growth stimulant, incubated, and placed on a special nutrient medium with a 1% agar base. Within 2-10 days after incubation colonies of a variety of non-acid-fast forms were noted, yet all of these proved, through PCR real time with FAM probe to still have antigens in common with their classic tubercular parent-form, from which they originated. Moreover, in true cell-wall-deficient fashion, the isolates, upon guinea pig inoculation, did not immediately produce visible lesions, but nevertheless persisted. However, tissue homogenates of the infected animals, once placed on a growth-enhancing medium showed cell-wall-deficient mycobacterial forms interspersed with classical acid-fast rods. And a repeated passage of such tissue homogenates back into non-infected guinea pigs, not only induced small mycobacterial granulomas in their livers, but a distinct increase in acid-fast rods. Moreover, similar cell-wall-deficient mycobacterial forms with acid-fast rods occurred when embryonated chicken eggs were inoculated with PPD tuberculins as well. The autoclaved and supposedly “sterilized” purified protein derivative [PPD] used in tuberculin skin tests contain cell-wall-deficient forms capable of eventually reverting back to virulent acid-fast tuberculosis, both typical and atypical.

Etiologic link between sarcoidosis and Propionibacterium acnes

Propionibacterium acnes is the only microorganism isolated from sarcoid lesions by bacterial culture. Numerous P. acnes genomes are found in lymph node samples from Japanese and European patients with sarcoidosis, whereas a few genomes are found in some non-sarcoid samples. The high frequency and specificity of detecting P. acnes within sarcoid granulomas suggests that this indigenous bacterium causes granuloma formation in many patients with sarcoidosis. P. acnes is the most common commensal bacterium in the lungs and lymph nodes. Occasional detection of P. acnes in non-granulomatous areas of these organs from non-sarcoid patients suggests that host factors are more critical than agent factors in the etiology of sarcoidosis. A particular protein, i.e., trigger factor, from P. acnes causes a cellular immune response only in sarcoid patients. The P. acnes trigger-factor protein induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only in those with latent P. acnes infection in their lungs. Eradication of P. acnes by antibiotics prevents the development of granulomas in this experimental model. P. acnes can cause latent infection in the lung and lymph nodes and persists in a cell wall-deficient form. The dormant form is endogenously activated under certain conditions and proliferates at the site of latent infection. In patients with P. acnes hypersensitivity, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Proliferating bacteria may escape granulomatous isolation, spreading to other organs. Latent P. acnes infection in systemic organs can be reactivated by another triggering event, leading to systemic sarcoidosis.

Lyme disease: the next decade

Although Lyme disease remains a controversial illness, recent events have created an unprecedented opportunity to make progress against this serious tick-borne infection. Evidence presented during the legally mandated review of the restrictive Lyme guidelines of the Infectious Diseases Society of America (IDSA) has confirmed the potential for persistent infection with the Lyme spirochete, Borrelia burgdorferi, as well as the complicating role of tick-borne coinfections such as Babesia, Anaplasma, Ehrlichia, and Bartonella species associated with failure of short-course antibiotic therapy. Furthermore, renewed interest in the role of cell wall-deficient (CWD) forms in chronic bacterial infection and progress in understanding the molecular mechanisms of biofilms has focused attention on these processes in chronic Lyme disease. Recognition of the importance of CWD forms and biofilms in persistent B. burgdorferi infection should stimulate pharmaceutical research into new antimicrobial agents that target these mechanisms of chronic infection with the Lyme spirochete. Concurrent clinical implementation of proteomic screening offers a chance to correct significant deficiencies in Lyme testing. Advances in these areas have the potential to revolutionize the diagnosis and treatment of Lyme disease in the coming decade.

AIDS: “It’s the bacteria, stupid!”

Acid-fast tuberculous mycobacterial infections are common in AIDS and are regarded as secondary "opportunistic infections." According to the National Institute of Allergy and Infectious Diseases, TB is the major attributable cause of death in AIDS patients. Could such bacteria play a primary or causative role in AIDS? Certainly, In screening tests for HIV, there is frequent, up to 70%, cross-reactivity, between the gag and pol proteins of HIV and patients with mycobacterial infections such as tuberculosis. By 1972, five years before gays started dying in the U.S., Rolland wrote Genital Tuberculosis, a Forgotten Disease? And ironically, in 1979, on the eve of AIDS recognition, Gondzik and Jasiewicz showed that even in the laboratory, genitally infected tubercular male guinea pigs could infect healthy females through their semen by an HIV-compatible ratio of 1 in 6 or 17%, prompting him to warn his patients that not only was tuberculosis a sexually transmitted disease, but also the necessity of the application of suitable contraceptives, such as condoms, to avoid it. Gondzik's solution and date of publication are chilling; his findings significant. Since 1982 Cantwell et al found acid-fast bacteria closely related to tuberculosis (TB) and atypical tuberculosis in AIDS tissue. On the other hand molecular biologist and virologist Duesberg, who originally defined retroviral ultrastructure, has made it clear that HIV is not the cause of AIDS and that the so-called AIDS retrovirus has never been isolated in its pure state. Dr. Etienne de Harven, first to examine retroviruses under the electron, agrees. In 1993 HIV co-discoverer Luc Montagnier reported on cell-wall-deficient (CWD) bacteria which he called "mycoplasma" in AIDS tissue. He suspected these as a necessary "co-factor" for AIDS. Remarkably, Montagnier remained silent on Cantwell's reports of acid-fast bacteria which could simulate "mycoplasma" in AIDS tissue. Mattman makes clear that the differentiation between mycoplasma and CWD bacteria is difficult at best and cites Pachas's 1985 study wherein one mycoplasma was actually mistaken for a CWD form of a bacterium closely related to the mycobacteria. It is important to realize that the statement "HIV is the sole cause of AIDS" is just a hypothesis. There are unanswered questions and controversy concerning the role of HIV "as the sole cause of AIDS." And until they are resolved, a cure is not possible. This paper explores the possible role of acid-fast tuberculous mycobacteria as "primary agents" in AIDS.

Mycobacterium avium Subspecies paratuberculosis Bacteremia in Type 1 Diabetes Mellitus: An Infectious Trigger?

Mycobacterium avium subspecies paratuberculosis (MAP) is the established cause of paratuberculosis in ruminants (i.e., Johne disease). The bacterium is shed in the milk of infected cows and survives pasteurization. Recently, an association between MAP and Crohn disease has been suggested, wherein MAP has been found to persist in a cell wall–deficient form, escaping clearance by the host immune system.

Comparison of the indentation and elasticity of E. coli and its spheroplasts by AFM

Atomic force microscopy (AFM) provides a unique opportunity to study live individual bacteria at the nanometer scale. In addition to providing accurate morphological information, AFM can be exploited to investigate membrane protein localization and molecular interactions on the surface of living cells. A prerequisite for these studies is the development of robust procedures for sample preparation. While such procedures are established for intact bacteria, they are only beginning to emerge for bacterial spheroplasts. Spheroplasts are useful research models for studying mechanosensitive ion channels, membrane transport, lipopolysaccharide translocation, solute uptake, and the effects of antimicrobial agents on membranes. Furthermore, given the similarities between spheroplasts and cell wall-deficient (CWD) forms of pathogenic bacteria, spheroplast research could be relevant in biomedical research. In this paper, a new technique for immobilizing spheroplasts on mica pretreated with aminopropyltriethoxysilane (APTES) and glutaraldehyde is described. Using this mounting technique, the indentation and cell elasticity of glutaraldehyde-fixed and untreated spheroplasts of E. coli in liquid were measured. These values are compared to those of intact E. coli. Untreated spheroplasts were found to be much softer than the intact cells and the silicon nitride cantilevers used in this study.

Cell wall deficient forms of mycobacteria: a review

Cell wall deficient (CWD)-forms or L-forms of bacteria are characterized by a complete or partial loss of cell wall components and by the change of cellular shape. CWD-forms (spheroplasts) of bacteria are commonly prepared in vitro and are used for various practical applications. However, very little is known about the conditions required for the formation of CWD-forms of bacteria and mycobacteria in vivoand their significance in aetiology of various chronic diseases of humans (tuberculosis, sarcoidosis, and Crohn’s disease) and animals (paratuberculosis). It is quite difficult to detect CWD-forms of mycobacteria in biological material and the possibilities of their detection by microscopy, culture, DNA hybridization techniques and other methods are limited. This obviously leads to a relatively small amount of published data on the detection and the isolation of CWD-forms of mycobacteria both from human or veterinary biological material, which can lead to the conclusion of a non-infectious origin of the diseases. The present review also includes studies performed by authors from the formerSoviet Unionand likely represents the first complete summarization of their knowledge in this sphere. However, certain results may be viewed as somewhat controversial. Accordingly, more attention should be paid to the research of cell wall deficient forms, not only in association with chronic and latent mycobacterial infections.

Cell wall deficient forms: stealth pathogens

Cell wall deficient forms: stealth pathogens

Preliminary characterization of chemically generated Mycobacterium avium subsp. paratuberculosis cell wall deficient forms (Spheroplasts)

Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohn’s disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johne’s Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johne’s positive and Johne’s negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johne’s disease positive and negative bovine sera.

Recovery of Cell Wall-Deficient Organisms From Blood Does Not Distinguish Between Patients With Sarcoidosis and Control Subjects

To determine if cell wall-deficient forms (CWDF) of mycobacteria can be grown in culture of blood from subjects with sarcoidosis. A special multicenter study of sarcoidosis (A Case Control Etiologic Study of Sarcoidosis), supported by the National Heart, Lung, and Blood Institute. PATIENTS AND CONTROL SUBJECTS were recruited at 10 institutions in the United States. Control subjects (controls) were of the same gender and race, and within 5 years of age as matching patients with sarcoidosis (cases). Cultures were incubated from 347 blood specimens (197 cases, 150 controls). Two investigators trained to recognize CWDF mycobacteria examined material obtained from culture tubes after 3 weeks. Structures thought to be CWDF were seen with equal frequency in cases (38%) and controls (41%). Thirty-nine percent of cases and 37% of controls were read as negative for CWDF. This study fails to confirm earlier reports that CWDF mycobacteria can be grown from the blood of patients with sarcoidosis, but not from control subjects.

Is there a role for microorganisms in the pathogenesis of sarcoidosis?

Sarcoidosis is a granulomatous disease that has the immunopathological features of being antigen-driven. It is a complex disease that appears to arise from the interaction of one or more triggers with an immunologically predisposed host. Previous reports of familial clustering and varying prevalence of sarcoidosis in different populations could reflect differences in ethnic predisposition or differences in local environmental exposures. This review focuses specifically on these areas that have been the subjects of intensive investigation recently. Specific focus is provided on the issue of an infective trigger and highlights popular candidates. It is concluded that microbes are a likely trigger (but not as an infection) in a genetically predisposed individual and that this initial event culminates in the sarcoidosis granulomatous response.

Detection of mycobacterium avium subspecies paratuberculosis in Crohn’s diseased tissues by in situ hybridization

OBJECTIVES: Reports about the association between Crohn’s disease (CD) and cell wall-deficient (CWD) forms of Mycobacterium avium subspecies paratuberculosis ( M. paratuberculosis) are controversial. This may be due to the heterogeneous nature of CD where only about 50% of the patients show granulomatous inflammation. Detection of CWD forms of M. paratuberculosis in tissues from patients with CD would support its association with the disease. To help identify these forms in inflamed tissues, a previously developed and optimized nonradioactive in situ hybridization method was applied on well-defined tissue materials obtained from patients with CD, ulcerative colitis (UC), and controls. METHODS: Specimens from 37 patients with CD (15 with epitheloid cell granulomas and 22 without granulomas), 21 UC, and 22 noninflammatory bowel disease (IBD) patients were analyzed by the in situ hybridization method based on the digoxigenin-labeled M. paratuberculosis IS900 fragment, previously shown to be species specific. Samples were counterstained with hematoxylin and eosin to show the location of the positive signal. Positive controls made of beef cubes injected with CWD and acid-fast M. paratuberculosis and negative controls were included in each experiment to monitor for nonspecific hybridization or staining. RESULTS: Six of 15 (40%) patients with CD and granulomas showed positive signals in myofibroblasts and macrophages. Interestingly, no positive signals were observed within granulomas. Only 4.5% of 22 CD samples from patients with nongranulomatous disease, 9.5% of 21 UC, and remarkably, none of the 22 non-IBD patients were M. paratuberculosis positive. CONCLUSION: The demonstration of DNA from CWD forms of M. paratuberculosis in this limited number of CD tissues further supports and confirms previous reports of its association with the granulomatous type of the disease.

Detection of Mycobacterium avium subspecies paratuberculosis in Crohn's diseased tissues by in situ hybridization.

Reports about the association between Crohn's disease (CD) and cell wall-deficient (CWD) forms of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) are controversial. This may be due to the heterogeneous nature of CD where only about 50% of the patients show granulomatous inflammation. Detection of CWD forms of M. paratuberculosis in tissues from patients with CD would support its association with the disease. To help identify these forms in inflamed tissues, a previously developed and optimized nonradioactive in situ hybridization method was applied on well-defined tissue materials obtained from patients with CD, ulcerative colitis (UC), and controls. Specimens from 37 patients with CD (15 with epitheloid cell granulomas and 22 without granulomas), 21 UC, and 22 noninflammatory bowel disease (IBD) patients were analyzed by the in situ hybridization method based on the digoxigenin-labeled M. paratuberculosis IS900 fragment, previously shown to be species specific. Samples were counterstained with hematoxylin and eosin to show the location of the positive signal. Positive controls made of beef cubes injected with CWD and acid-fast M. paratuberculosis and negative controls were included in each experiment to monitor for nonspecific hybridization or staining. Six of 15 (40%) patients with CD and granulomas showed positive signals in myofibroblasts and macrophages. Interestingly, no positive signals were observed within granulomas. Only 4.5% of 22 CD samples from patients with nongranulomatous disease, 9.5% of 21 UC, and remarkably, none of the 22 non-IBD patients were M. paratuberculosis positive. The demonstration of DNA from CWD forms of M. paratuberculosis in this limited number of CD tissues further supports and confirms previous reports of its association with the granulomatous type of the disease.

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn’s disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections. M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn’s disease and sarcoidosis. The assay may also be valuable for studies on Johne’s diseased animals.

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

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.