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.     

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes

Johne''s disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne''s disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne''s disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.     

How does Mycobacterium avium subsp. paratuberculosis resist intracellular degradation?

Paratuberculosis is a chronic, progressive disease of mainly ruminants caused by the facultative intracellular bacterium, Mycobacterium avium subsp. paratuberculosis. Infection usually occurs in young animals through oral uptake of food contaminated with the organisms. The ingested bacteria are transcytosed through M-cells overlying the Peyer's patches and are released in the stroma, where they are taken up by macrophages. Inside the macrophage, the mycobacteria resist enzymatic and toxic degradation and multiply until the infected macrophage ruptures. The thick, lipid-rich cell envelope is mainly responsible for micobacterial resistance. In addition to its barrier effect, which provides protections, the mycobacterial cell wall also contains several biologically active components that down-regulate the bactericidal function of macrophages. The basic survival strategy of pathogenic mycobacteria can be viewed at three levels: selective use of relatively safe entry pathways that do not trigger oxidative attack, modification of the intracellular trafficking of mycobacteria-containing phagosomes, and modulation of the cooperation between the innate and specific immunity. In doing so, pathogenic mycobacteria are successful intracellular organisms that survive and multiply inside macrophages. Current understanding about the survival strategies of M. a. paratuberculosis and its implications in the epidemiology, diagnosis, and control of the disease are discussed.     

Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n = 9) and naturally infected cows in the subclinical (n = 10) and clinical (n = 13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-γ) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.     

Immunological response to Mycobacterium avium subsp. paratuberculosis in chickens

Enzyme-linked immunosorbent assay (ELISA) and culture are 2 common diagnostic tests for detecting Mycobacterium avium subsp. paratuberculosis (Map) in Johne's disease, but they are not as sensitive as polymerase chain reaction (PCR). However, inhibitors can coextract with the target DNA and cause interference in PCR. Development of an immune capture assay followed by PCR amplification can alleviate this problem. In this study, we were able to induce an immune response in chickens using heat or formalin inactivated Map. The purified immunoglobulin (Ig)Y has a molecular weight of 160 kDa. The titers were at 1:6400 and 1:12 800 at weeks 5 to 6 and 8 to 9, respectively, as determined by the IDEXX modified ELISA kit for Johne's disease. The IgY produced from inactivated bacterial cells had no effect on its ability to recognize live Map cells as illustrated by immunofluorescence assay and immune capture PCR results.     

From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

Johne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP’s ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP’s ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP’s interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages.     

Host responses to Mycobacterium avium subsp. paratuberculosis: a complex arsenal

The immune system is not always successful in recognizing and destroying pathogens it may encounter. Host immunity to mycobacteria is characterized by a very complex series of events, designed to clear the infection. The first line of defense is uptake and processing of the pathogen by macrophages, followed by the initiation of cell-mediated immunity. The secretion of pro-inflammatory cytokines such as IFN-gamma is credited with containment of mycobacterial infections. Yet it is clear that activated T-cells may contain but fail to clear the infection in some hosts. Further, it is recognized that if infection progresses to a more clinical state, the production of pro-inflammatory cytokines is suppressed and expression of anti-inflammatory cytokines is increased. It is unclear what defines a host that can successfully contain the infection versus one that succumbs to severe immunopathologic disease. This review will address some of the key elements in host immunity to mycobacterial pathogens, with an emphasis on Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), in an attempt to understand the dialogue between immune cells and their mediators during infection and what causes this discourse to go awry.     

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.     

Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows

Between 1982 and 2000, fecal samples were obtained from 786 cows that were shedding Mycobacterium avium subsp. paratuberculosis (Map). These cows were resident on 93 Pennsylvania dairies (mean herd size, 64 milk cows) that had no or minimal previous testing for Map. Feces were cultured on four tubes of Herrold's egg yolk medium and the distribution of mean Map colony forming units (CFU) was evaluated. Most cows were light (< 10 CFU/tube, 51.4%) or high (> 50 CFU/tube, 30.8%) fecal shedders with fewer cows in the moderate category (10-50 CFU/tube). Of the 786 cows, 192 (24.4%) had colonies in only one of four tubes. In the multivariable negative binomial model, there were significant associations between mean CFU/tube and prevalence, herd size, and season and an interaction between herd size and season. The linear mixed model of continuous tube counts with a random herd effect yielded similar findings with associations with herd size as a continuous variable, season, and an interaction between categorized prevalence and continuous herd size. Variability in CFU/tube was greatest among cows in the same herd, intermediate for replicate tubes from the same cow, and smallest among cows in different herds. Reduction in the number of replicate tubes from four would have reduced the sensitivity of fecal culture for Map by approximately 6% (for three tubes) to 12% (for two tubes).     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis at a regional scale in Germany

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne's disease in dairy cattle. Genotyping of MAP is useful to gain a better understanding of the origin of infection, to evaluate regional control programs, to improve diagnostics, and to develop vaccines. In this study 91 MAP isolates mainly from symptomatic dairy cattle in Rhineland-Palatinate (RP, Germany), its neighbor federal states, and Luxembourg were genotyped using Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR). MIRU-VNTR and MLSSR produced 11 and 6 different genotypes among the 91 isolates, respectively. The combined analysis of both methods produced 25 genotypes with an index of discrimination (D) of 0.93 (95% CI: 0.91-0.95). The results revealed the genetic diversity of MAP and the dominance of two MAP genotypes commonly found in Europe, showed the usefulness of MAP genotyping in studies at a regional scale, and provided useful information for control initiatives in RP.     

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.     

Intrauterine and transmammary transmission of Mycobacterium avium subsp paratuberculosis in sheep

OBJECTIVE: To investigate intrauterine infection of foetuses with Mycobacterium avium subsp paratuberculosis and the presence of infection in mammary secretions of sheep. DESIGN: A study of 142 late-pregnant ewes and their foetuses from two heavily infected flocks. PROCEDURE: Infection of ewes was determined at necropsy by histopathology and culture of tissues and mammary secretions. Antemortem tests (clinical assessment, faecal culture and serology) were also applied. Foetuses from 59 infected ewes and 47 apparently uninfected ewes were examined by culture and histopathology. RESULTS: Five of five ewes with clinical ovine Johne's disease had infected foetuses. Only one of 54 subclinically affected ewes, and none of 47 uninfected ewes had an infected foetus. M a paratuberculosis was cultured from mammary secretions or mammary glands of only two of 76 ewes, both of which were clinical cases and had infected foetuses. CONCLUSION: Although intrauterine or transmammary transmission of Mycobacterium avium subsp paratuberculosis may occur frequently in clinically affected sheep, these are less common in subclinically infected ewes. Therefore these modes of transmission are unlikely to compromise existing control programs for ovine Johne's disease on most farms, especially if programs include the immediate culling of clinically affected sheep.     

Different immune response of pigs to Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis infection

Mycobacterium avium subsp. avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) are the most common mycobacterial species isolated from granulomatous lesions in swine in countries with controlled bovine tuberculosis. This study is focused on the immunological aspect of MAA and MAH infection in pigs. We detected induction of humoral and cell-mediated immunity in experimentally infected pigs. Specific antibodies were analyzed in serum by ELISA and the IFN-γ release assay was used for evaluation of cell-mediated immunity. While MAA induced a significant increase of both types of immune responses, MAH-infected pigs had an unvarying level of specific antibodies and showed low cell-mediated immunity with high individual variability. The subsequent in vitro experiment confirmed the lower immunogenicity of the MAH strain in comparison to MAA. MAH-infected porcine monocyte-derived macrophages showed a weaker induction of pro-inflammatory mediators in comparison to MAA, which included mRNA for IL-1β, TNF-α, IL-23p19, IL-18 and chemokines CCL-3, CCL-5, CXCL-8 and CXCL-10. Additionally, qualitative proteomic analysis revealed 28 proteins exclusively in MAA and 7 proteins unique to MAH. In conclusion, closely related M. avium subspecies MAA and MAH showed different capacities to stimulate the porcine immune system. From a diagnostic point of view, the IFN-γ release assay showed higher sensitivity than the detection of specific antibodies by ELISA and seems to be an effective tool for discrimination of MAA-infected pigs. In the case of MAH infection, the IFN-γ release assay could fail because of the low immunogenic capacity of the MAH strain.     

Diagnostic detection methods for Mycobacterium avium subsp. paratuberculosis in white-tailed deer

This study compares the results and suitability of serological testing, microscopic examination, deoxyribonucleic acid (DNA) detection, and bacterial culture for detecting Mycobacterium avium subsp. paratuberculosis (Map) infection in asymptomatic farmed white-tailed deer (WTD) (Odocoileus virginianus). Deer were classified as infected if culture slants from their feces, lymph nodes, or ileum were positive, or if a polymerase chain reaction (PCR) assay detected Map DNA in any of its tissues. Deer identified as positive by agar gel immunodiffusion (AGID) testing or enzyme-linked immunosorbent assay (ELISA) but not by bacterial culture, Ziehl-Neelsen staining, or PCR assay were classified as suspect. Culture of tissues classified 10/16 (62.5%), histopathologic examination 1/16 (6.3%), tissue smears 4/16 (25%), culture slant (CS)-PCR on feces 12/15 (80%), CS-PCR on tissue 13/16 (81.3%), and direct PCR on uncultured tissues 5/16 (31.3%) deer as infected. The ELISA classified 2/15 (13.3%) deer as positive and therefore suspect. The AGID test was negative for all deer. Fifteen of 16 deer were positive by 1 or more tests; only 1 deer was negative on all 11 assays. The CS-PCR gave superior results on antemortem fecal testing as well as postmortem tissue testing and can be recommended for improving the detection of Map in WTD at every stage of infection.     

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.     

Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assays use purified protein derivative of MAP (PPDj) which are complex antigen mixtures with low specificity. The objectives of this study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The study included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was an animal with ≥ 2 positive tests for ≥ 4 antigens, resulting in 13 cases and 13 non-cases. IFN-γ levels of cases were higher compared to IFN-γ levels of non-cases (P<0.05). The results of the IFN-γ assay using PPDj did not correlate well with the results using the novel antigens. PPDj produced elevated IFN-γ responses of samples from both the non-infected and the MAP infected herd, indicating unspecific IFN-γ responses and showed low consistency. Three latency proteins, LATP-1, LATP-2 and LATP-3 gave positive IFN-γ tests that correlated very well with the case definition suggesting high immunogenicity. Three tested antigens, LATP-2, MAP-1 and MAP-2 have no homologue in the M. avium subsp. avium or M. bovis genome and could be promising diagnostic antigens, especially LATP-2 correlated highly with the case definition.     

Review of Mycobacterium avium subsp. paratuberculosis antigen candidates with diagnostic potential

Mycobacterium avium subsp. paratuberculosis (MAP) is a slow growing bacterium that can infect ruminants and remain latent for years without development of any clinical signs or disease. Diagnosis is often based on detection of MAP antibodies in milk or serum samples or culture of bacteria from faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen candidates described to date with special emphasis on antigen candidates tested for CMI responses. Relevant information on 115 different MAP antigens was systematically extracted from literature and summarized in 6 tables of CMI antigens, secreted antigens, cell wall and membrane antigens, lipoprotein antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified for this application. Most of the novel diagnostic candidates were evaluated in few animals and it is recommended that an appropriate sample size is included for evaluation of antigen candidates in future studies.