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.     

Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates

A combination of representational difference analysis and comparative DNA sequencing revealed that four type I (sheep) isolates of Mycobacterium avium subsp. paratuberculosis were differentiated from nine type II (bovine) isolates by the presence of an 11-bp insertion in a novel M. avium subsp. paratuberculosis-specific region of genomic DNA. Further, our studies show that M. avium subsp. paratuberculosis type I isolates contain three type-specific loci that are missing in M. avium subsp. paratuberculosis type II but are present in M. avium subsp. avium. Taken together, the results are consistent with the hypothesis that M. avium subsp. paratuberculosis type I strains are an evolutionary intermediate between M. avium subsp. avium and M. avium subsp. paratuberculosis type II isolates or share a subset of M. avium subsp. avium type-specific loci through horizontal transfer.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity,strain sharing,and identification of unique targets for diagnosis

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.     

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.     

Amplified fragment length polymorphism reveals genomic variability among Mycobacterium avium subsp. paratuberculosis isolates

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.     

Sequence polymorphisms in a surface PPE protein distinguish types I, II, and III of Mycobacterium avium subsp. paratuberculosis

In the last 2 decades, a variety of different molecular typing methods have been developed to differentiate strains of Mycobacterium avium subsp. paratuberculosis. The most successful techniques are based on insertion sequences, repetitive loci, comparative genomics, or single nucleotide polymorphisms. In the present study, we chose to examine whether a single M. avium subsp. paratuberculosis gene could serve as a means of differentiation of a variety of isolates. The MAP1506 gene locus encodes a member of the polymorphic PPE protein family that has putative roles relevant to M. avium subsp. paratuberculosis pathogenicity. The MAP1506 locus was sequenced from a collection of 58 M. avium subsp. paratuberculosis isolates from different sources, hosts, and typing profiles. Following sequence alignment and analysis, it was found that bovine (type II) strains of M. avium subsp. paratuberculosis consistently differed from ovine (type I) and intermediate (type III) strains in seven and eight nucleotides, respectively. Polymorphic regions of the MAP1506 locus were selected for analysis by denaturing gradient gel electrophoresis, allowing visual discrimination of the three subtypes of M. avium subsp. paratuberculosis isolates. This is the first report describing the use of PCR and denaturing gradient gel electrophoresis on a single gene as a method to distinguish types I, II, and III of M. avium subsp. paratuberculosis.     

Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools-necessary to understand disease processes and to identify subclinical infection-are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne's disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10(6)-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10(3) M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.     

Development and characterization of monoclonal antibodies and aptamers against major antigens of Mycobacterium avium subsp. paratuberculosis

Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.     

Fibronectin attachment protein is necessary for efficient attachment and invasion of epithelial cells by Mycobacterium avium subsp. paratuberculosis

Attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by two epithelial cell lines were enhanced by soluble fibronectin (FN). Peptide blocking of the FN attachment protein (FAP-P) inhibited the internalization of M. avium subsp. paratuberculosis. Disruption of FAP-P expression significantly reduced attachment and ingestion of M. avium subsp. paratuberculosis by T-24 and Caco-2 cells. The results indicate that the interaction between FN and FAP-P facilitates attachment and internalization of M. avium subsp. paratuberculosis by epithelial cells.     

AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay.

Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA.     

Fibronectin attachment protein homologue mediates fibronectin binding by Mycobacterium avium subsp. paratuberculosis

Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis.     

Mitogen activated protein kinase p38 pathway is an important component of the anti-inflammatory response in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes

We investigated the role of cell signaling through the mitogen-activated protein kinase-p38 (MAPK p38) pathway on the antimicrobial functions and cytokine expression by bovine monocytes after ingestion of Mycobacterium avium subsp. paratuberculosis. We evaluated the dynamic secretion of interleukin (IL)-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) as well as phagosome acidification and organism killing at several time points after in vitro infection of bovine monocytes with M. avium subsp. paratuberculosis. Monocytes treated with M. avium subsp. paratuberculosis had a significant increase in IL-10 expression at 2, 4, and 6h post-infection and an increase expression of TNF-alpha at 2, 4, 6, and 24h post-infection. In contrast, IL-12 expression did not increase at any time point post-infection. Moreover, MAPK p38 was rapidly phosphorylated at 10 and 60 min after M. avium subsp. paratuberculosis ingestion. Chemical inhibition of the MAPK p38 signaling pathway (SB203580) resulted in decreased expression of IL-10 and increased expression of IL-12 at 6h post-infection. Chemically blocking the MAPK p38 pathway also increased acidification of phagosomes as well as increasing the capacity of macrophages to kill organisms. Taken together, these results indicated that selective activation of MAPK p38 may be a major mechanism exploited by M. avium subsp. paratuberculosis to circumvent the antimycobacterial effects of mononuclear phagocytes.     

Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.     

Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp. paratuberculosis

Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.     

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

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.     

Definitive differentiation between single and mixed mycobacterial infections in red deer (Cervus elaphus) by a combination of duplex amplification of p34 and f57 sequences and Hpy188I enzymatic restriction of duplex amplicons

Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.     

Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.     

Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures

A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrold's Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johne's disease.     

A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.