Immunogenicity and cytoadherence of recombinant heparin binding haemagglutinin (HBHA) of Mycobacterium avium subsp. paratuberculosis: functional promiscuity or a role in virulence?

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. Recently, an association of MAP bacilli with Crohn's disease in humans has been proposed. Due to genetic similarities and serological cross-reactivity of the M. avium complex with other mycobacteria, functional analysis of species-specific proteins may allow new insights into the pathogenesis of mycobacterial diseases. We report production and molecular characterization of the recombinant HBHA from the MAP complex bacilli. The HBHA was expressed in Escherichia coli and Mycobacterium smegmatis using efficient expression vector systems. The recombinant HBHA was found to be immunogenic and therefore induced antibody responses in cattle against the MAP bacilli with a possible cross reactivity with M. bovis infection. The MAP complex HBHA was thus found to be a target of the host humoral responses in Johne's disease. The recombinant HBHA protein was also found to be adherent to the Caco2 cell lines in-vitro, a significant observation to understand possible virulence mechanisms. Since M. tuberculosis HBHA was earlier shown to be involved in dissemination of the tubercle bacilli, the immunogenicity and cytoadherent nature of this MAP protein possibly suggests functional promiscuity.     

New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.     

Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotein

Johne's disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by the sequential linkage of the ORFs of the approximately 17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C-terminus with approximately 14.6-kDa N-terminal portion of Map3527. Mice immunized with Map74F had a significant IgG1 response but not IgG2a. In immunized animals, the IgG1/IgG2a ratio increased until 4 weeks after MAP challenge. The ratio decreased from 8 weeks indicating a shift to a Th1 response. Antigen specific IFN-gamma response, CD3+ and CD4+ T cells increased significantly in immunized mice. Following challenge, MAP burden was significantly lower in liver, spleen and mesenteric lymph nodes of immunized animals compared to control animals indicating protection against MAP infection. This was further evident by the improved liver and spleen pathology of the immunized animals, which had fewer granulomas and lower numbers of acid-fast bacilli. Results of this study indicated that immunization of mice with Map74F protected mice against MAP infection.     

Mycobacterium avium subsp. paratuberculosis infection in a patient with HIV,Germany

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne disease in ruminants, has been incriminated as the cause of Crohn disease in humans. We report the first case of human infection with MAP in a patient with HIV; infection was confirmed by obtaining isolates from several different specimen types.     

Mycobacteria causing human cervical lymphadenitis in pastoral communities in the Karamoja region of Uganda

Mycobacteria from lymph node biopsies of patients with cervical lymphadenitis reporting for tuberculosis treatment in Matany and Moroto Hospitals in the transhumant areas of Karamoja, Uganda were isolated and characterized. The AccuProbe culture identification kits for Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC) and M. avium were used to identify the isolates. Spoligotyping, IS901 PCR and IS1311 and IS1245 restriction fragment length polymorphism (RFLP) were used to characterize the isolates. Of the 43 biopsies, ten M. avium, seven M. tuberculosis, three M. bovis, and two M. intracellulare were isolated. Two isolates could not be identified with AccuProbe and from 19 samples no mycobacteria could be isolated. Three isolates with the Beijing spoligotype were identified from the seven M. tuberculosis isolates. The spoligopatterns of the M. bovis isolates had previously been detected in cattle in Uganda. Isolation of members of the MAC group reflects the complex interaction between the transhumant communities, water sources and their cattle. None of the M. avium isolates harboured IS901, and all showed several bands on IS1311 and IS1245 RFLP, in accordance with M. avium subsp. hominissuis. Composite dendrograms of IS1311 and IS1245 RFLP showed that the isolates were similar and identical patterns were found. The isolation of M. bovis confirms the human infection with zoonotic mycobacteria in areas where consumption of raw milk and meat is routine. Isolation of environmental mycobacteria also confirms their increasing role in human disease and the occupational risk of infection in the transhumant ecosystem in the absence of safe drinking water and environmental contamination.     

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.     

Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium

BACKGROUND: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced. METHODS: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals. CONCLUSIONS: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis.     

Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), results in serious economic losses worldwide especially in cattle, sheep and goats. To control the impact of JD on the animal industry, an effective vaccine with minimal adverse effects is urgently required. In order to develop an effective vaccine, we used allelic exchange to construct three mutant MAP strains, leuD, mpt64 and secA2. The mutants were attenuated in a murine model and induced cytokine responses in J774A.1 cell. The leuD mutant was the most obviously attenuated of the three constructed mutant strains. Our preliminary vaccine trial in mice demonstrated different levels of protection were induced by these mutants based on the acid-fast bacilli burden in livers and spleens at 8 and 12 weeks postchallenge. In addition, vaccination with leuD mutant induced a high level of IFN-γ production and significant protective efficacy in both the reduction of inflammation and clearance of acid-fast bacilli, as compared with the mock vaccinated group.     

The epidemiology of atypical mycobacterial diseases in northern England: a space-time clustering and Generalized Linear Modelling approach

The incidence of infection by mycobacteria, other than tubercle bacilli (MOTT) is increasing in the United Kingdom, Europe and the United States. These diseases increase morbidity and are an increasing public health concern. However, the epidemiology of disease due to these species is not well characterized. We used space-time clustering approaches and Generalized Linear Modelling to investigate the potential predictors of disease in cases of infection by organisms of the Mycobacterium avium complex (MAC) and M. malmoense recorded in the north of England during 2000-2005. There was significant spatial and temporal clustering in juvenile cases of infection by MAC but not for cases of infection in adults by either species. There were no significant predictors of infection by M. malmoense or juvenile cases of M. avium. Incidence of disease caused by M. avium in adults was significantly related to health deprivation and weakly related to rainfall. We consider possible reasons for the difference in epidemiology in infection by M. avium in adults and juveniles.     

Optimization of Serum EVELISA for Milk Testing of Johne's Disease

Abstract Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.     

Epidemiological evidence for Mycobacterium avium subspecies paratuberculosis as a cause of Crohn's disease

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants including cattle, sheep, goats, and farmed deer. Recently, this bacterium has received an increasingly wide interest because of a rapidly growing body of scientific evidence which suggests that human infection with this microorganism may be causing some, and possibly all, cases of Crohn's disease. Recent studies have shown that a high percentage of people with Crohn's disease are infected with M. avium subsp. paratuberculosis; whether the association of this bacterium and Crohn's disease is causal or coincidental is not known. Crohn's disease is a gastrointestinal disease in humans with similar histopathological findings to those observed in the paucibacillary form of Johne's disease in cattle. The search for risk factors in Crohn's disease has been frustrating. However, epidemiologists have gathered enough information that points to an association between M. avium subsp. paratuberculosis and Crohn's disease. This paper reviews epidemiological models of disease causation, the major philosophical doctrines about causation, the established epidemiological criteria for causation, and the currently known epidemiological evidence of M. avium subsp. paratuberculosis as a possible cause of Crohn's disease.     

Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.     

National atypical mycobacteria survey, 2000.

Infections with atypical mycobacteria in Australia during 2000 occurred at a rate of 1.8 cases per 100,000 population. The main sites of disease were the respiratory tract, soft tissue, and the lymphatics. The Mycobacterium avium complex was the most common group of mycobacteria isolated from respiratory, lymphatic sites, and blood. The rapidly growing mycobacteria, predominantly the M. fortuitum-M. abscessus-M. chelonae group were the most common soft tissue infections. Atypical mycobacteria were isolated from significant numbers of sputum 'smear positive' patients, requiring further tests to exclude M. tuberculosis. Geographical differences were observed for some Mycobacterium species, notably the isolation of M. haemophilum from Western Australia, and M. ulcerans from Victoria and Queensland. Newer molecular techniques, while improving precision and accuracy of identification, raise additional questions about the ecology of the atypical mycobacteria and their role in disease.     

Detection of viable Mycobacterium avium subsp. paratuberculosis using luciferase reporter systems

Plasmid- and phage-based firefly luciferase reporter constructs were evaluated as rapid detection systems for viable Mycobacterium avium subsp. paratuberculosis (MAP). A MAP strain bearing a luciferase-encoding plasmid was detectable at 100 cells/mL in skim milk and 1000 cells/mL in whole milk. Three luciferase-encoding mycobacteriophage were evaluated for detection of wild-type MAP. The best of these, phAE85, allowed detection of >1000 cells/mL within 24-48 h. Membrane filtration did not improve the sensitivity of detection for either plasmid or phage reporters. Luciferase reporters show promise for rapid detection of viable MAP.     

Mycobacterium avium Subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons

We report on a coinfection of Mycobacterium avium subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons from one flock, from which squabs were occasionally consumed by humans. Triplex quantitative real-time PCR and culture methods were used for M. a. avium detection in livers and culture method was used for the detection of Salmonella sp. in samples of liver and caecum of 33 examined birds. M. a. avium was detected in a total of 31 (93.9%) and Salmonella Typhimurium in a total of 11 (33.3%) pigeons. Coinfection with both pathogens was found in 10 (30.3%), infection with Salmonella Typhimurium alone in 1 (3.0%), and infection with M. a. avium alone in 21 (63.7%) pigeons. Neither pathogen was detected in one pigeon. There was no difference in clinical symptoms exhibited by pigeons infected by M. a. avium and/or Salmonella Typhimurium. All Salmonella Typhimurium isolates were sensitive to all 15 antimicrobials tested. According to these results we emphasize good heat treatment of consumed squabs.     

Increase in Mycobacterium avium complex isolations among patients admitted to a general hospital.

In early 1979, an official of an Illinois hospital reported an increase in the number of patients from whom Mycobacterium avium complex recently had been recovered. Over the preceding 3 years specimens from a total of 51 patients were culture positive for M. avium complex: 7 in 1976, 8 in 1977, and 36 in 1978. Nine of 10 serotyped isolated were serotype 8. The increase was not attributable to an increase in the number of mycobacterial cultures performed. No other area hospitals had similar increases in rates of recovery of M. avium complex. Patients with M. avium complex were significantly more likely than patients with other mycobacteria to have been residents of the city where the hospital is located. The distribution of abnormalities in patients'' chest films differed significantly between patients with M. avium complex in 1978 and patients with M. avium complex in 1976-77; in 1978, patients although equally likely to have infiltrates, nodules, or cavities, were more likely to have no abnormalities or abnormalities consistent with chronic obstructive pulmonary disease, and less likely to have other abnormalities. The data suggest that the increased rate of recovery of M. avium complex from patients could not be attributed to ascertainment bias or laboratory variation but may be due to an increase in the incidence of disease or colonization among persons living in the community where the hospital is located.     

Molecular analysis and MIRU-VNTR typing of Mycobacterium avium subsp. avium, ‘hominissuis’ and silvaticum strains of veterinary origin

Besides Mycobacterium avium subsp. paratuberculosis (MAP), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), and ‘M. avium subsp. hominissuis’ (MAH) are equally important members of M. avium complex, with worldwide distribution and zoonotic potential. Genotypic discrimination is a prerequisite to epidemiological studies which can facilitate disease prevention through revealing infection sources and transmission routes. The primary aim of this study was to identify the genetic diversity within 135 MAA, 62 MAS, and 84 MAH strains isolated from wild and domestic mammals, reptiles and birds.Strains were tested for the presence of large sequence polymorphism LSP^A17 and were submitted to Mycobacterial interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) analysis at 8 loci, including MIRU1, 2, 3, and 4, VNTR25, 32, and 259, and MATR9. In 12 strains hsp65 sequence code type was also determined.LSP^A17 was present only in 19.9% of the strains. All LSP^A17 positive strains belonged to subspecies MAH. The discriminatory power of the MIRU-VNTR loci set used reached 0.9228. Altogether 54 different genotypes were detected. Within MAH, MAA, and MAS strains 33, 16, and 5 different genotypes were observed. The described genotypes were not restricted to geographic regions or host species, but proved to be subspecies specific.Our knowledge about MAS is limited due to isolation and identification difficulties. This is the first study including a large number of MAS field strains. Our results demonstrate the high diversity of MAH and MAA strains and the relative uniformity of MAS strains.     

Environmental risk factors for infection with Mycobacterium avium complex

Infection with Mycobacterium avium complex is acquired from the environment, but risk factors for M. avium complex infection and disease are poorly understood. To identify risk factors for infection, the authors performed a 1998-2000 cross-sectional study in western Palm Beach County, Florida, using a population-based random household survey. M. avium complex infection was identified by use of the M. avium sensitin skin test. Of 447 participants, 147 (32.9%) had a positive test reaction, 186 (41.6%) had a negative test reaction, and, for 114 (25.5%), test results were indeterminate. Among the 333 participants with positive or negative M. avium sensitin skin tests, age-adjusted independent predictors of M. avium complex infection in a multivariate model included Black race (odds ratio = 3.8, 95% confidence interval: 2.2, 6.6), birth outside the United States (odds ratio = 2.1, 95% confidence interval: 1.1, 3.9), and more than 6 years' cumulative occupational exposure to soil (odds ratio = 2.7, 95% confidence interval: 1.3, 6.0). Exposure to water, food, or pets was not associated with infection. Results indicate that soil is a reservoir for M. avium complex associated with human infection and that persons whose occupations involve prolonged soil exposure are at increased risk of M. avium complex infection.     

Reduced tissue colonization of Mycobacterium avium subsp. paratuberculosis in neonatal calves vaccinated with a cocktail of recombinant proteins

An increasing prevalence of paratuberculosis supports the need for new efficacious vaccines as an essential management tool. Two separate studies were performed in neonatal calves to evaluate the effectiveness of pooled recombinant Mycobacterium avium subsp. paratuberculosis (MAP) proteins (MAP1087, MAP1204, MAP1272c, MAP2077c) as a potential vaccine. In the first study vaccinated calves were immunized with 400 µg protein cocktail per dose, whereas the second study compared doses of 400 µg and 800 µg of protein cocktail, followed by challenge with live MAP for both vaccinated and nonvaccinated control calves 28 days post-vaccination. At the end of 12 months, tissue colonization with MAP was significantly reduced for the vaccinated calves compared to control animals. A higher dose of vaccine improved protection, with further reductions of MAP burden. Antigen-specific IFN-γ responses and serum antibody responses were similar regardless of vaccination, indicating exposure to MAP invoked conventional host immune responses. Host immunity differed due to vaccination, resulting in increased percentages of CD4+ T cells and B cells after stimulation of PBMCs with antigen. Interestingly, gene expression in PBMCs was similar for both control and vaccinated calves except for significant increases in IFN-γ, IL-12, and IL-17 expression observed in vaccinated calves. Vaccination with a cocktail of immunogenic recombinant MAP proteins was efficacious in reducing the level of infection and fecal shedding of neonatal calves and may be a potential tool for curtailing the spread of Johne’s disease.