ATP release by infected bovine monocytes increases the intracellular survival of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5'-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis-infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis.     

Binding of bovine growth hormone to. Mycobacterium avium ss paratuberculosis

Mycobacterium avium ss paratuberculosis causes a chronic progressive enteritis in cattle and other ruminants referred to as Johne's disease. It also has been suggested by some as possibly being associated with Crohn's disease in humans. In a previous study we observed that incubation of bovine monocytes with recombinant bovine growth hormone (bGH) altered the ingestion and intracellular growth of M. avium ss paratuberculosis in vitro. This led us to investigate whether bGH also has a direct effect on M. avium ss paratuberculosis. We observed that addition of bGH (5 microg/ml) had a direct inhibitory effect on the growth of M. avium ss paratuberculosis in Middlebrook 7H9 broth. In contrast, the growth of Mycobacterium smegmatis was unaffected, even at a bGH concentration of 50 microg/ml. Using 125I-bGH we observed high affinity binding (Kd = 1.32 nM) of bGH to M. avium ss paratuberculosis, with an estimated 204 binding sites per bacillus. To the best of our knowledge, this is the first report of a mammalian hormone binding to this important enteric pathogen.     

Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method.

The ability of Mycobacterium avium subsp. paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers. Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting. We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows. The data suggest that multiplication and death of M. avium subsp. paratuberculosis occur simultaneously in bovine monocytes infected in vitro. Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M. avium subsp. paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M. avium subsp. paratuberculosis. There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp. paratuberculosis by monocytes from the infected, immune responder cows. However, the observed numbers of viable M. avium subsp. paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.     

Antigenic relationship between Mycobacterium paratuberculosis and Mycobacterium avium

Four prototype strains of Mycobacterium paratuberculosis contained the type-specific glycopeptidolipid antigen of serovar 8 of the M avium complex. This glycolipid was distinguished by a 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl terminal unit. Of 59 low-passage, field isolates of M paratuberculosis, 2 contained this antigen, and these 2 isolates were indistinguishable from M avium serovar 8. However, most M paratuberculosis isolates had no characteristic surface glycopeptidolipid. Seemingly, M paratuberculosis, long regarded as a single species and the causative agent of bovine paratuberculosis, is not a homogeneous taxon. Most isolates obtained from infected ruminants may be antigenically defective, variants of M avium and, thereby, more successful pathogens.     

A critical role of interleukin-10 in the response of bovine macrophages to infection by Mycobacterium avium subsp paratuberculosis

OBJECTIVES: To evaluate the role of interleukin (IL)-10 in the inability of monocyte-derived bovine macrophages to kill Mycobacterium avium subsp paratuberculosis organisms in vitro. SAMPLE POPULATION: Monocytes were obtained from healthy adult Holstein dairy cows that had negative results when tested for infection with M avium subsp paratuberculosis. PROCEDURE: Monocyte-derived macrophages were incubated with M avium subsp paratuberculosis for 2, 6, 24, 72, or 96 hours with or without addition of saturating concentrations of a goat anti-human IL-10 that has been documented to neutralize bovine IL-10 activity. Variables assessed included ingestion and killing of M avium subsp paratuberculosis; expression of tumor necrosis factor (TNF)-alpha, IL-12, IL-8, major histocompatability (MHC) class II, vacuolar H+ ATPase, and B cell CLL/lymphoma 2 (BCL-2); production of nitric oxide; acidification of phagosomes; and apoptosis of macrophages. RESULTS: Neutralization of IL-10 enabled macrophages to kill 57% of M avium subsp paratuberculosis organisms within 96 hours. It also resulted in an increase in expression of TNF-alpha, IL-12, IL-8, MHC class II, and vacuolar H+ ATPase; decrease in expression of BCL-2; increase in acidification of phagosomes; apoptosis of macrophages; and production of nitric oxide. CONCLUSIONS AND CLINICAL RELEVANCE: The capacity of M avium subsp paratuberculosis to induce IL-10 expression may be a major determinant of virulence for this organism.     

Bacteriological investigations about paratuberculosis in dairy herds in Switzerland

In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.     

A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis

A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.     

Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis

Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.     

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.     

Comparison of two enzyme-linked immunosorbent assays for serologic diagnosis of paratuberculosis (Johne's disease) in cattle using different subspecies strains of Mycobacterium avium.

Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests (z = 0.182).     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

HPLC does not differentiate Mycobacterium paratuberculosis from Mycobacterium avium

HPLC, which is gaining its place as identification tool in mycobacteriology laboratories, has been proposed to distinguish Mycobacterium paratuberculosis from Mycobacterium avium. We had reported no significant difference between M. avium and M. paratuberculosis reference strain ATCC 19698. Because of the advantages offered by such a method, we enlarged our observations to include more isolates of M. paratuberculosis. Within the double cluster of peaks obtained by both M. avium and M. paratuberculosis, we could not find a consistent difference typical of M. paratuberculosis. Therefore, the present study confirmed that M. avium and M. paratuberculosis could not be distinguished by HPLC, raising doubts of a straightforward use of HPLC to identify M. paratuberculosis.     

IS900/ERIC-PCR as a tool to distinguish Mycobacterium avium subsp. paratuberculosis from closely related mycobacteria

There is an increasing demand for fast and reliable methods to distinguish Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from closely related mycobacteria and also a need for rapid strain specific typing of clinical isolates for epidemiological reasons. In the present study, the potential of rep-PCR as a fingerprinting method for M. paratuberculosis was assessed and compared to conventional RFLP. A PCR assay was designed and optimised to obtain reproducible fingerprints of mycobacterial DNA with primers targeting the enterobacterial intergenic consensus (ERIC) sequence and the M. paratuberculosis specific insertion sequence IS900. Reproducible fingerprints were obtained with 60 strains of M. paratuberculosis, 16 strains of M. avium subsp. avium, 3 strains of M. intracellulare, and 11 other mycobacterial strains. A species-specific band pattern that was clearly distinguishable from that of other mycobacteria was obtained with M. paratuberculosis. The rep-PCR did not detect any differences among M. paratuberculosis strains of different RFLP types, and was therefore not considered as an alternative fingerprinting method. However, the species-specific band pattern make IS900/ERIC-PCR a suitable alternative for distinguishing M. paratuberculosis from other mycobacteria, especially in cases of IS900 PCR positive mycobacteria. The fingerprinting method reported was fast and easy to perform, and produced highly reproducible results.     

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.     

Gas chromatographic characterization of the relationship between some Mycobacterium avium and Mycobacterium paratuberculosis strains

Mycobacterium avium strain P-55 and M. avium strain DENT differ from M. avium strain 16909-338 on the basis of their fatty acid spectra (C14:0, C18:0 and tuberculostearic [TBS] acids) studied by multivariate statistical analyses. Strains P-55 and DENT are closer to M. paratuberculosis strain 5889 than to M. avium strain 16909-338, a finding which is in harmony with earlier immunological observations. The recently isolated M. paratuberculosis strain 385 has proved different from M. paratuberculosis strain 5889.     

Development of ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis with truncated 34 kDa proteins

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.     

Analysis of antigens in Mycobacterium paratubercuolsis.

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good.Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations.Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity.The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand

AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.     

The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis.

The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.