Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.     

Use of Hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. paratuberculosis did not significantly alter the number of apoptotic cells.     

Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL-10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti-TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP-infected monocytes but not Maa-infected monocytes. Addition of anti-TLR2 antibody enhanced phagosome acidification and phagosome-lysosome fusion in MAP-containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa-infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL-10 on phagosome acidification and organism killing, as IL-10 production was not affected by addition of anti-TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP-infected bovine mononuclear phagocytes.     

Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences

The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.     

Genomic polymorphisms for Mycobacterium avium subsp. paratuberculosis diagnostics

Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP(A)s) and those only present in the latter (LSP(P)s). We examined the distribution of 3 LSP(A)s and 17 LSP(P)s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP(A)8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP(P)s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.     

Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis.

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.     

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.     

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.     

Disparate Host Immunity to Mycobacterium avium subsp. paratuberculosis Antigens in Calves Inoculated with M. avium subsp. paratuberculosis,M. avium subsp. avium,M. kansasii,and M. bovis

The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO⁺ CD25⁺ T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.     

Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis

Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.     

In vitro cellular immune responses to recombinant antigens of Mycobacterium avium subsp. paratuberculosis

Five recombinant antigens (Ags; 85A, 85B, 85C, superoxide dismutase [SOD], and 35-kDa protein) were purified from Mycobacterium avium subsp. paratuberculosis and evaluated for their ability to stimulate peripheral blood mononuclear cells (PMBCs) from fecal-culture-positive cows (low and medium shedders) and culture-negative healthy cows. Recombinant Ags 85A, 85B, and 85C induced significant lymphocyte proliferation as well as the production of gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha (TNF-alpha), but not IL-4, from low and medium shedders. The 85 antigen complex did not stimulate PMBC proliferation from culture-negative healthy cows. The 35-kDa protein also induced significant lymphocyte proliferation as well as the production of IFN-gamma and IL-4 from low and medium shedders. CD4(+) T cells and CD25(+) (IL-2R) T cells were stimulated the most by 85A and 85B, while the 35-kDa protein primarily stimulated CD21(+) B cells involved in humoral immune responses. Interestingly, SOD was less immunostimulatory than other antigens but strongly induced gammadelta(+) T cells, which are thought to be important in the early stages of infection, such as pathogen entry. These data provide important insight into how improved vaccines against mycobacterial infections might be constructed.     

Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.     

Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins

Johne''s disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis.     

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.     

Experimental paratuberculosis in calves following inoculation with a rabbit isolate of Mycobacterium avium subsp. paratuberculosis

The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease.     

Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis

Five pigmented isolates of Mycobacterium avium subsp. paratuberculosis were examined by pulsed-field gel electrophoresis (PFGE), IS900 restriction fragment length polymorphism (IS900-RFLP), and IS1311 polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with SnaBI, designated 9, 10, and 11, and with SpeI, designated 7, 8, and 9, which generated three multiplex profiles designated [9-7], [10-8], and [11-9]. All of the pigmented isolates had the same IS900-RFLP BstEII and PvuII profiles. The IS900-RFLP BstEII profile was new, but the IS900-RFLP PvuII profile corresponded to PvuII type 6 of a sheep strain described by Cousins and colleagues (D. V. Cousins, S. N. Williams, A. Hope, and G. J. Eamens, Aust. Vet. J. 78:184-190, 2000). IS1311-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of M. avium subsp. paratuberculosis from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.     

Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis

The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that are not represented in any other mycobacterial species tested (n = 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol. 40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of these M. avium subsp. paratuberculosis-specific sequences in Escherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. avium subsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.