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PubMed2001

Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria.

The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium (M.) tuberculosis and Mycobacterium avium ssp. avium (M. avium), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis (M. ptb), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium, as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae. Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA-gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)-dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC-dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to approximately pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium. In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of approximately 5.2. After infection with M. smegmatis and M. gordonae, acidification was initially (1-5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.

Animals↗
PubMedDate not supplied

A modeling study on the sustainability of a certification-and-monitoring program for paratuberculosis in cattle.

Certification-and-monitoring programs for paratuberculosis are based on repetitive herd testing to establish a herd's health status. The available tests have poor sensitivity. Infected but undetected herds may remain among certified "paratuberculosis-free" herds. The objective was to determine if truly free herds acquire a certified status and keep it over time when infected but undetected herds remain. The Dutch program was used as a basis to construct a mechanistic deterministic model of the evolution over 25 years of the number of herds per health status. Three health states for herds were defined: not detected as infected in the certification process to obtain a free status; not detected as infected by any of the repetitive tests for monitoring the certified free status; detected as infected. Among undetected herds, two types were defined: truly free versus undetected but infected. Transitions between states were due to the purchase of an infected animal, infection via the environment, clearance via culling or sales, detection of an infected animal, and certification. A sensitivity analysis was carried out. We showed that--for a 100% specific test only--most of the truly free herds at the beginning of the program got a certified free status and kept it over time. Most infected herds were either detected as infected or cleared. The number of certified truly free herds increased with a decrease in the animal-level prevalence or in the risk of purchasing an infected cattle, for example by restricting purchases to cattle from herds at the highest level of certification.

Animal Husbandry↗
PubMed2000

Classification of lesions observed in natural cases of paratuberculosis in goats.

Sixty-eight adult goats with clinical or sub-clinical paratuberculosis, from naturally infected flocks, were subjected post mortem to a pathological examination that focused on the intestinal lymphoid tissue. The lesions were divided into four categories. Focal lesions, found in 16 goats, consisted of small granulomata in the ileocaecal Peyer's patches or related lamina propria. Diffuse multibacillary lesions (34 goats) consisted of a granulomatous enteritis, affecting different intestinal sites. Numerous macrophages containing many mycobacteria were present, resulting in macroscopical changes in the normal gut morphology; in the ileum of nine of the 34 animals, however, these changes were confined to the apex of the villi and the intestinal wall was not thickened. In diffuse lymphocytic lesions (10 goats), the lymphocyte was the main inflammatory cell, with some macrophages (containing few if any mycobacteria). In diffuse mixed lesions (eight goats) the infiltrate consisted of numerous lymphocytes and macrophages, with small numbers of mycobacteria. The three types of diffuse lesion were often associated with necrosis in the lymph vessels of the mucosa, mesentery and lymph nodes, and with greater thickening of the jejunum than of the ileum. Mycobacterium avium subspecies paratuberculosis was cultured from 69% of goats with diffuse lesions and from 44.4% of those with focal lesions.

Animals↗
PubMed2001

Lymphocytic neuritis of the ileum in sheep with naturally acquired and experimental paratuberculosis.

In a comparison of natural and experimentally induced ovine paratuberculosis, aggregations of mononuclear inflammatory cells were detected around nerves in the ileal submucosa in eight of 12 sheep with natural disease and in five of 14 animals with unequivocal experimental paratuberculosis. Such lesions were not seen in 10 other sheep that, despite attempted experimental infection, remained free from the disease, as judged clinically, histopathologically and by PCR assay. The lesions described resembled those observed in human leprosy.

Animals↗
PubMed1999

Evolutionary bottlenecks in the agents of tuberculosis, leprosy, and paratuberculosis.

Parasitic mycobacteria cause important human and animal diseases including tuberculosis, leprosy, and paratuberculosis. Several methods demonstrate a high degree of sequence conservation in three parasitic mycobacterial species (Mycobacterium tuberculosis, M. leprae, and M. avium subspecies paratuberculosis). Each of these species has completely conserved deoxyribonucleic acid (DNA) sequence in an internal transcribed spacer. In contrast, several species of environmental mycobacteria (M. intracellulare, M. kansasii, M. gordonae, and M. scrofulaceum) have substantial strain-to-strain variation in this region. These data suggest that each of the parasitic species has gone through a recent evolutionary bottleneck. Comparisons of tandem-repeat DNA from ancient and modern mycobacterial strains may allow this hypothesis to be tested directly.

Animals↗
PubMed1992

Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn's disease.

The possible role of infection with Mycobacterium paratuberculosis (MAP) for the etiopathogenesis of Crohn's disease (CD) has been a matter of long-term controversy. In addition to similarities with the pathology of ruminant paratuberculosis, DNA fingerprinting confirmed the organism isolated from gut tissue, but the specificity of the immune repertoire has not as yet been evaluated. We report here on a serological study of 29 patients with CD, 20 patients with ulcerative colitis and 18 healthy control subjects, using three antigens attributed with species-specificity and selective immunogenicity following MAP infection. Antibodies binding to the 38-kD band of MAP extract were demonstrable by the Western blot technique in 57% of CD patients. Antibody levels to the 24-kD (p24BCD) cathodic bands, determined by competition ELISA using a monospecific murine antiserum, and to the 18-kD protease-resistant purified bacterioferritin, detected by standard ELISA, were significantly elevated in 53% of CD patients. However, these three antibody specificities tested in individual CD patients did not show any correlation with each other. Thus, 18% of patients were positive for all three specificities, whilst 84% had antibodies to at least one of the specific antigens. Although the exact proportion of affected patients is yet to be defined, the serological results obtained support the view that MAP infection may play an etiological role in Crohn's disease.

Antibodies, Bacterial↗
PubMed1986

Immunodiffusion analysis shows that Mycobacterium paratuberculosis and other mycobactin-dependent mycobacteria are variants of Mycobacterium avium.

Antigenic analysis by immunodiffusion has been applied to 74 strains of mycobactin-dependent mycobacteria. Thirty-eight strains were of Mycobacterium paratuberculosis from cases of Johne's disease of cattle or goats. The remaining cultures were obtained from a variety of animals and included the wood pigeon bacillus. Rabbit antisera were raised to some of the strains and these, together with antisera to M. avium and M. intracellulare, were used to examine sonicate preparations of all the cultures. All were found to be antigenically identical with M. avium and none were found to belong to M. intracellulare. A predominance of the cultures from Johne's disease belonged to the potential brunense subspecies of M. avium, and the remainder together with the majority of the other mycobactin-dependent strains belonged to the type subspecies. In view of these findings the separate species status of M. paratuberculosis is refuted and some difficulty remains in the nomenclature of strains giving rise to Johne's disease.

Animals↗
PubMed1990

Some diagnostic features of the pathogenesis of bovine paratuberculosis (Johne's disease) and serum biochemical changes after oral reinfection.

Ten bull-calves were infected with 10(8) viable cells of Mycobacterium paratuberculosis per os. During the 400-day period of observation faecal and blood samples were taken from animals at 30-day intervals. Faecal samples were examined microscopically, blood samples by the CFT, AGID and LST tests. Intradermal allergic tests were carried out at PI (post infection) days 92, 217, 336, using mammalian, avian and johnin PPD. In the period of study, these efficiency indices showed fluctuations characteristic of the given tests. In the period between PI day 160 and 400 fifteen biochemical parameters were measured monthly, TRP, ALP, TRIG and CHOL were reduced by day 400, pointing to disorders of digestion and absorption. Increased activities of CK, ALD, LDH, alpha-HBDH and ALT indicated skeletal muscle and/or liver damage in the first place. Serum CK, ALD activities and TRIG and TRP concentrations may serve as useful complementary values to the specific diagnosis of paratuberculosis, particularly in the advanced stage of the disease.

Animals↗
PubMed1991

Mycobacterial antigen detection by immunohistochemistry in goat paratuberculosis.

An immunohistochemical method (PAP) for a microscopic diagnosis of paratuberculosis in goats is described as an alternative method to the Ziehl-Neelsen technique. Mycobacterial antigens are only found in enteric and mesenteric lymph node lesions. This method is the most sensible, particularly in cases in which there are no paratuberculosis lesions and the Ziehl-Neelsen technique shows no acid-fast bacilli (AFB).

Animals↗
PubMed2006

Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.

A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.

Animals↗
PubMed2001

Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.

Amino Acid Sequence↗
PubMed2005

Injury caused by self-inoculation with a vaccine of a Freund's complete adjuvant nature (Gudair) used for control of ovine paratuberculosis.

OBJECTIVE: To document the occurrence and consequences of accidental self-inoculation of vaccinators (producers, farm employees, contractors) with the recently registered Gudair vaccine for the control of ovine paratuberculosis in Australia. DESIGN AND PROCEDURE: A survey of the first 50 primary producers permitted to use the vaccine in sheep and a description of six cases of accidental self-inoculation for which medical attention was sought, and which occurred after the vaccine became widely available. RESULTS: The survey recorded that, of 37 respondents vaccinating 155,523 sheep, there were 21 incidents of exposure to the vaccine, an overall rate of one incident per 7406 vaccinations. In five of these incidents there was only superficial skin contact with vaccine; in 16 there was needle penetration without vaccine injection. There were no reports of self-inoculation with vaccine. Six cases of self-inoculation with Gudair vaccine that required medical intervention are described. Of these five were in males and one in a female; four involved injection of vaccine into the leg and single cases involved a foot or hand. Most cases required surgical removal of the injected vaccine to allow wound repair; three required extensive surgery and open drainage. Even with surgery recovery took as long as 9 months. Possible risk factors for self-inoculation and the resulting outcome are discussed. CONCLUSIONS: Gudair ovine paratuberculosis vaccine can cause prolonged granulomatous inflammation if inadvertently injected into human tissue. After.self-inoculation, early surgical debridement of the damaged tissue and drainage to remove the vaccine material are advised to avoid progression to extensive necrosis.

Animals↗
PubMed1997

Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective?

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.

Animals↗
PubMed2005

Detection of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic.

We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis, albeit at a low level for viable cells.

Base Sequence↗
PubMed2005

Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment.

In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.

Animals↗
PubMed2006

Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms.

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.

Biofilms↗
PubMed1994

Occurrence, in Crohn's disease, of antibodies directed against a species-specific recombinant polypeptide of Mycobacterium paratuberculosis.

Sera from patients with Crohn's disease and control were analyzed by an enzyme-linked immunosorbent assay based on the Mycobacterium paratuberculosis-specific recombinant polypeptide a362. Anti-a362 immunoglobulin G (IgG) (P < 0.05) and IgA (P < 0.001) titers were higher in patients with Crohn's disease than in controls. A monomodal Gaussian distribution of anti-a362 IgA levels were found for controls, and a bimodal distribution was found for patients with Crohn's disease. An M. paratuberculosis etiology is suggested for the 36% of patients with Crohn's disease who had an anti-a362 IgA level higher than that of controls.

Antibodies, Bacterial↗
PubMed2006

Mycobacterium avium subsp. paratuberculosis PtpA is an endogenous tyrosine phosphatase secreted during infection.

Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.

Actinomyces↗