Mycobactin Dependence Research Articles

High prevalence of subclinical paratuberculosis in buffaloes (Bubalus bubalis) in Maranhão, Brazil.

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.

FIRST ISOLATION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS FROM WILD GUANACOS (LAMA GUANICOE) ON TIERRA DEL FUEGO ISLAND

The aim of this study was to search for Mycobacterium avium subsp. paratuberculosis (Map) infection in a free-ranging wild animal species in a region where Johnes's disease has yet to be reported and to classify Map isolates using a genomic typing method. Fecal samples were obtained from 501 wild guanacos (Lama guanicoe) from Tierra del Fuego Island, Chile, in August 2006. Samples were cultured using Herrold's egg yolk medium with and without mycobactin J. After 9 mo of incubation, suspected Map colonies showing mycobactin dependence were confirmed by real-time polymerase chain reaction (PCR) based on IS900 and F57. Isolates were further tested using IS1311 PCR with restriction endonuclease analysis in order to type the guanaco Map strains. Twenty-one of 501 (4.2%) animals were fecal culture-positive for Map; identity was confirmed by real-time PCR and isolates were classified as cattle-type. Most culture-positive animals were located in four contiguous geographic areas, and the infection was most commonly found among adult animals. Prevalence was higher in females (5.9%) than males (3.1%) but the difference was not statistically significant. This represents the first isolation of Map from a free-ranging wildlife species in Chile. It expands the geographic range of paratuberculosis and the diversity of wildlife species that can become infected with Map.

Factors Affecting Isolation and Identification of Mycobacterium avium subsp. paratuberculosis from Fecal and Tissue Samples in a Liquid Culture System

Culture of Mycobacterium avium subsp. paratuberculosis is the definitive diagnostic test for Johne's disease, a chronic granulomatous enteropathy of animals. Compared to solid media, the identification of all strains of the organism in liquid media can be more difficult because the appearance of colonies and mycobactin dependence are not observable, and the growth of other organisms needs to be distinguished, commonly by PCR. Factors affecting the isolation rate of S strains and the contamination rate in modified Middlebrook 7H9 broth (Bactec 12B) and 7H10 agar were studied using 11,598 fecal samples and 2,577 tissue samples from sheep from 1,421 farms over 10 years. Minimization of contamination in Bactec cultures required the avoidance of the carryover of fecal particles from the first sedimentation step in the double-incubation centrifugation method, and contamination was reduced significantly by incubating the sample in a solution containing vancomycin, amphotericin B, and nalidixic acid for 3 days compared to 2 days. The growth of irrelevant microorganisms confounded the identification of M. avium subsp. paratuberculosis in liquid culture by inhibiting IS900 PCR and in solid medium culture by inhibiting the growth of M. avium subsp. paratuberculosis or obscuring colonies. The contamination of samples was clustered in certain laboratory submissions and was reduced by including ampicillin in Bactec medium without affecting the odds of isolation of M. avium subsp. paratuberculosis. The long-term contamination rate for fecal cultures was about 7%, and that for tissue cultures was <0.2%. Liquid medium was more sensitive than solid medium culture for M. avium subsp. paratuberculosis. The applicability of these findings for C strains is discussed.

The complete genome sequence ofMycobacterium aviumsubspeciesparatuberculosis

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map; and Map-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

Pigmented paratuberculosis granulomata in the liver of sheep

Pigmented paratuberculosis granulomata in the liver of a sheep affected with Johne’s disease are described. The granulomata were visible on the surface of the liver, irregular in shape, patchily distributed in the parenchyma and contained yellowish-orange pigment. Microscopically, the granuloma was composed of epithelioid cells containing small poorly basophilic granules (H&E), and surrounded by a tiny fibrous capsule. Intestinal lesions were widespread from the ileum to the rectum and included thickening, corrugation and staining of the mucosa with a yellowish-orange pigment. Histologically, epithelioid cells containing pigment granules infiltrated the enteric mucosa and submucosa. The mesenteric lymph nodes were swollen and contained white irregular spots, which were visualized microscopically as aggregates of epithelioid cells. Clumps of short, slender acid-fast bacilli were isolated in pure culture on Herrold’s egg-yolk agar medium from various lesions including the granulomata. The organism was identified as Mycobacterium avium subsp. paratuberculosis on the basis of slow growth and strict mycobactin dependence. The nature of the yellowish-orange pigment was not identified but it was possibly produced by the Mycobacterium spp.

Mycobacterium avium and Mycobacterium intracellulare infection in mammals.

Mycobacterium avium subsp. avium and M. intracellulare are ubiquitous organisms in the environment. The reservoir of M. avium subsp. avium is generally accepted to be environmental, in particular, water and soil are sources of the organism. In contrast to M. avium infection in wild and domestic birds, M. avium infection in mammals occurs only sporadically and is rarely transmissible. Generalised disease is usually uncommon, owing to the non-progressive, chronic character of the infection. However, some cases of disseminated disease have been reported, e.g. in captive non-domestic hoofed animals as well as in immunosuppressed dogs and cats. The majority of M. avium and M. intracellulare infections in livestock are detected at slaughter and the diagnosis is confirmed by bacteriological procedures. Condemnation of affected portions of the carcass can result in significant economic losses, although gross lesions are mostly restricted to lymph nodes close to the alimentary tract. Successful treatment with antibiotics in combination with surgery has been reported in some affected domestic cats, but is not considered to be effective or economical in other species. In the past, differentiation of M. avium bacteria from the closely related M. avium subsp. paratuberculosis was based on the mycobactin dependence and prolonged incubation period of the latter. More recently, amplification of the genomic insertion sequence IS900 has proved to be a powerful tool for identification of M. avium subsp. paratuberculosis. The potential zoonotic importance of M. avium infections has been indicated, but requires clarification.

Lack of mycobactin dependence of mycobacteria isolated on Middlebrook 7H11 from clinical cases of ovine paratuberculosis

Mycobacterium avium subsp. paratuberculosis is distinguished from other mycobacterial species by its dependence on the iron-chelator factor mycobactin and its prolonged incubation period when grown in vitro. Traditionally, a very low rate of isolation has been considered characteristic of sheep strains of this mycobacterium species. In the present study, a comparison was made of the performance of two media, Löwenstein-Jensen and Middlebrook 7H11/OADC both with and without mycobactin, on the isolation of M. a. paratuberculosis from ovine cases of the disease. A high isolation rate in both media was observed. Moreover, our results indicated that mycobactin dependence of sheep strains of M. a. paratuberculosis is medium related, being dependent on Löwenstein-Jensen and independent on Middlebrook 7H11/OADC.

Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue.

Mycobacterium paratuberculosis is known to cause Johne's disease, a granulomatous ileitis in ruminants, and may be involved in some cases of Crohn's disease. Like M. paratuberculosis, the wood pigeon strain of Mycobacterium avium may also show mycobactin dependence on primary isolation that is attenuated on further subculturing. A wood pigeon strain, M. avium restriction fragment length polymorphism (RFLP) type A/I, is also capable of causing granulomatous ileitis in experimental animal models but is not known to cause disease in humans. M. avium RFLP type A is associated with disease in immunocompromised hosts. Three DNA probes, pMB22 and the two subclones pMB22/S4 and pMB/S12, were found to be capable of distinguishing among M. paratuberculosis, M. avium type A, and M. avium type A/I (wood pigeon strain) on the basis of RFLPs. These DNA probes were used to identify two mycobacterial isolates (M. paratuberculosis and M. avium type A/I, wood pigeon strain) derived from the intestinal tissues of two patients with Crohn's disease. In addition, the wood pigeon strain of M. avium was identified from a patient with ulcerative colitis, and M. avium RFLP type A was identified from a patient with colonic carcinoma. This is the first time that M. avium A/I (wood pigeon strain) is known to have been isolated from human tissue. There are too few isolates to speculate about the etiological significance of mycobacteria and inflammatory bowel disease, but it is reasonable to conjecture that M. paratuberculosis may be responsible for some cases of Crohn's disease and that the wood pigeon strain of M. avium may also be an inflammatory bowel disease pathogen in humans.

Mycobacterium paratuberculosis factors that influence mycobactin dependence

Mycobacterium paratuberculosis does not produce any detectable mycobactin, an iron-binding compound that is synthesized by most Mycobacterium spp. and necessary for the growth of all mycobacteria. This study examined the influence of various culture conditions on mycobactin dependence in M. paratuberculosis. Using a radiometric growth assay, we found the minimal concentration of mycobactin J necessary for growth of M. paratuberculosis to be 0.006 μM, whereas 1.2 μM (1 μg/ml) was required for optimal growth. In media without mycobactin at iron concentrations ≤100 μM, growth of M. paratuberculosis occurred at pH 5.0, but not pH 6.8. Iron concentrations >100 μM did not significantly increase growth at pH 5.0, but at pH 6.8 the growth rate increased with increasing amounts of iron reaching a rate equal to control cultures containing mycobactin. Mycobacterium paratuberculosis appeared to lose mycobactin dependence when subcultured; however, this was subsequently shown to be a result of mycobactin carried over from primary medium. Removal of this contaminating cell-wall-associated mycobactin reestablished mycobactin dependence. We conclude that mycobactin dependence must be carefully determined because it is a key test used in identification of M. paratuberculosis and may be easily influenced by media pH, iron concentration, and mycobactin carryover from primary media.

Characterization of Mycobacterium paratuberculosis by gas-liquid and thin-layer chromatography and rapid demonstration of mycobactin dependence using radiometric methods.

Thirty-six Mycobacterium paratuberculosis isolates of bovine, caprine, and ovine origins were evaluated by using gas-liquid chromatography (GLC), thin-layer chromatography (TLC), and BACTEC 7H12 Middlebrook TB medium (12A; Johnston Laboratories, Inc., Towson, Md.) in an effort to more rapidly differentiate this group of organisms from other mycobacteria. Bacterial suspensions (0.1 ml) were inoculated by syringe into 7H12 broth containing 2 micrograms of mycobactin P per ml and control broth without mycobactin P. Cultures were incubated at 37 degrees C and read daily with a BACTEC Model 301 (Johnston Laboratories). After 8 days of incubation, the growth index readings for the test broths containing mycobactin P were twice those of the control broths without mycobactin P. Sixty-five isolates of mycobacteria other than M. paratuberculosis were also examined. No difference was noted between the growth index readings of control and mycobactin-containing broths. Except for Mycobacterium avium-Mycobacterium intracellulare, TLC studies differentiated M. paratuberculosis from the other mycobacterial species tested. The GLC data reveal that all M. paratuberculosis isolates had a distinctive peak (14A) which was not found among M. avium-M. intracellulare complex organisms. These data indicate that 7H12 radiometric broth was able to rapidly demonstrate the mycobactin dependence of M. paratuberculosis and GLC and TLC procedures were capable of rapidly differentiating this organism from the other mycobacteria studied.

Mycobacterial infection in goats: Diagnosis and pathogenicity of the organism

Abstract Counterimmunoelectrophoresis of serum samples and intradermal tuberculin testing were compared with cultural results for the diagnosis of mycobacterial infection in a goat herd. Only eight of 47 culture-positive animals were not identified by a combination of the other two methods. Typical isolates resembled Mycobacterium paratuberculosis in cultural characteristics and mycobactin dependence but serologically resembled Mycobacterium avium . One isolate was inoculated into guinea-pigs, mice, chickens and calves and, although the infection in guinea-pigs, mice and chickens resembled that of M. paratuberculosis , it did not cause Johne's-like disease in calves.

Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium.

Fifty-three strains of M. avium and related species all produced one or more exochelins, the extracellular iron-binding compounds of the mycobacteria, when grown iron deficiently. Only those strains which could grow without the addition of mycobactin (i.e., mycobactin independent) produced mycobactin, the intracellular iron-binding compound of the mycobacteria. Exochelins varied from 20 to 2,000 micrograms per g of cell dry weight; mycobactins were between 1 and 10 mg per g of cell dry weight. M. paratuberculosis (13 strains) and 13 strains of M. avium, both species dependent upon mycobactin for growth, failed to produce spectrophotometrically detectable amounts of mycobactin (less than 0.2 microgram per g of cell dry weight), although mycobactin could be recognized in one strain of M. avium grown with an additional supply of salicylate and examined by a radiolabeling technique. On repeated subculture three of the mycobactin-dependent strains of M. avium, but none of those of M. paratuberculosis, lost their mycobactin dependence and on reexamination were found to produce their own mycobactin at 0.3 mg per g of cell dry weight. It is concluded that mycobactin biosynthesis is probably strongly repressed in the mycobactin-dependent strains rather than being a genetic deletion. The exochelins, when examined by high-pressure thin-layer chromatography were revealed as being multiples of similar compounds, with up to 20 individual iron-binding compounds being recognizable with some strains. It is argued that the exochelins represent the single most important means of iron acquisition in mycobacteria growing in vitro and in vivo, and their elaboration by the fastidious M. paratuberculosis and related species explains how these organisms are able to grow in vivo in the absence of an external supply of mycobactin.

The pathogenicity of Mycobacterium avium and related mycobacteria for experimental animals.

The pathogenicity of 40 strains of Mycobacterium avium, M. paratuberculosis, M. intracellulare and M. lepraemurium was investigated in chickens, rabbits, guinea-pigs, mice and calves. Mycobactin dependence and serological type were also determined. There was no evidence that mycobactin dependence was related to pathogenicity. Antigenic similarities were demonstrated between M. avium and M. paratuberculosis, and one isolate had the pathogenic characteristics of both species.

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.

The dependence of some strains of Mycobacterium avium on mycobactin for initial and subsequent growth.

This paper demonstrates that various degrees of mycobactin dependence exist within the M. avium group of mycobacteria and that this characteristic is not confined to M. paratuberculosis. The strains employed came from a wide range of animal species and all were virulent for chickens. Mycobactin dependence was observed in the M. avium serotypes 1, 2 and 3. The importance of employing a medium containing mycobactin for primary isolations observation may be of interest in relation to the primary isolation of other species of mycobacteria.

Growth and metabolic characteristics of Mycobacterium paratuberculosis.

The cultural characteristics of newly isolated strains of Mycobacterium paratuberculosis on a variety of media were studied. The mycobactin dependence ascribed to this species of Mycobacterium was found to be easily circumvented by incorporation of 1% ferric ammonium citrate in serum or egg yolk media, but the earliest and most abundant growth occurred on serum medium that contained mycobactin and sodium pyruvate. In the presence of ferric ammonium citrate, respiration did not decrease during 5 days; but in the presence of all other agents studied, respiration decreased after the first day.

Growth and Metabolic Characteristics of Mycobacterium paratuberculosis

The cultural characteristics of newly isolated strains of Mycobacterium paratuberculosis on a variety of media were studied. The mycobactin dependence ascribed to this species of Mycobacterium was found to be easily circumvented by incorporation of 1% ferric ammonium citrate in serum or egg yolk media, but the earliest and most abundant growth occurred on serum medium that contained mycobactin and sodium pyruvate. In the presence of ferric ammonium citrate, respiration did not decrease during 5 days; but in the presence of all other agents studied, respiration decreased after the first day.