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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.

Autofluorescent compounds present in intestinal tissue often hinder the ability to utilize multiple, spectrally different, fluorophores. In addition, fixatives and blocking solutions may contribute to background autofluorescence or nonspecific immunofluorescent labeling. During immunofluorescence protocol development, autofluorescent pigments were observed in frozen bovine mid-ileal intestinal tissue sections. Coagulant fixatives, normal serum blocking, histochemical stains Sudan Black B (SBB) and 3,3′-diaminobenzidine (DAB), and spectral separation using imaging software were compared for their ability to reduce autofluorescence, as well as their effect on immunofluorescent labeling. Fluorescent pigments of frozen bovine mid-ileal intestinal tissue sections, most likely caused by eosinophils and lipofuscin, were masked successfully with a combination of DAB and SBB. Little to no statistical differences were observed for all other methods investigated; however, tissue fixed with 1:1 acetone methanol and 10% horse serum diluted in 0.05 M Tris buffer demonstrated lower mean fluorescence intensities. Spectral separation of specific immunofluorescent labeling from background autofluorescence is a simple method for removing unwanted fluorescence; however, successful separation is dependent on tissue and labeling quality.

Culture of Mycobacterium aviumsubsp. paratuberculosis(MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. To ensure efficient and reproducible PCR results from a difficult sample matrix such as feces, there are many obstacles that a DNA extraction method must overcome, including the presence of inhibitors and the thick waxy cell wall of MAP. In the current study, 6 commercial DNA extraction kits were evaluated using fecal samples from naturally infected cattle shedding various amounts of MAP. Upon extraction, DNA purity and yield were measured, and real-time PCR was performed for detection of the insertion sequence (IS)900 and ISMAP02 targets. The kits evaluated showed significant differences in the purity and yield of DNA obtained. The best results were observed with kits E and A, having identified 94% (16/17) and 76% (13/17) of the positive samples by IS900 PCR, respectively. Both of these kits utilized bead beating in a lysis solution for cell disruption, followed by spin column technology (kit E) or magnetic bead–based technology (kit A) for nucleic acid isolation and purification. Two kits (A and F) demonstrated improved performance when used in conjunction with the respective manufacturer’s PCR test. The present study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.

ABSTRACTThe protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium aviumsubsp. paratuberculosisthat contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. aviumsubsp. paratuberculosis.

Colostrum samples experimentally inoculated with Mycobacterium aviumsubsp. paratuberculosis(MAP; strain K-10) at increasing concentrations between 1 × 101and 1 × 109cells/ml were tested for recovery of MAP DNA using a nested ISMAP02 target polymerase chain reaction initially developed for detecting MAP DNA in fecal samples. The following detection rates were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1 × 107and 1 × 109cells/ml, 75% between 1 × 103and 1 × 106cells/ml, and 50% between 1 × 101and 1 × 102cells/ml replicates. Detection rates achieved for the colostrum sample replicates inoculated with sonicated MAP cell suspension were 75% for 1 × 109cells/ml, 100% between 1 × 107and 1 × 108cells/ml, 75% for 1 × 106cells/ml, 0 for 1 × 104cells/ml, and 25% between 1 × 101and 1 × 103cells/ml. When negative control colostrum samples were tested, 16 of 18 (89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the MAP DNA detection rates of the present assay improved with increasing concentrations of MAP in the colostrum sample replicates, although MAP DNA was also detected in 2 of 18 (11%) negative control samples, suggesting an undefined technical problem with the assay or, perhaps, sample contamination during preparation. Overall, the present findings suggest a potential role of the proposed polymerase chain reaction assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.

With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.

ABSTRACTWith the genome sequence of Mycobacterium aviumsubsp. paratuberculosisdetermined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. aviumsubsp. paratuberculosisin host serum. The power of this approach is that it enables a direct comparison of M. aviumsubsp. paratuberculosisproteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. aviumsubsp. paratuberculosis. Also included were previously reported or known M. aviumsubsp. paratuberculosisantigens to serve as a frame of reference. Sera from healthy control cattle (n= 3) and cattle infected with either M. aviumsubsp. aviumand Mycobacterium boviswere exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. aviumsubsp. aviumproteins that was higher than the degree of cross-reactivity with the more distantly related M. bovisproteins. Finally, sera from naturally infected cattle (n= 3) as well as cattle experimentally infected with M. aviumsubsp. paratuberculosis(n= 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. aviumsubsp. paratuberculosis.

ABSTRACTSpecific antibodies, available in unlimited quantities, have not been produced against Mycobacterium aviumsubsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. aviumsubsp. paratuberculosiswere produced from BALB/c mice immunized with a whole-cell extract of M. aviumsubsp. 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. aviumsubsp. paratuberculosislambda 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. aviumsubsp. 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. aviumsubsp. paratuberculosisas well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.

ABSTRACTThe Mycobacterium aviumsubsp. paratuberculosis35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johne's disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.

Measurement of secreted interferon (IFN)-γ has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium aviumsubsp. paratuberculosis.Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-γ assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosiswere administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-γ were observed. Intradermal injection resulted in significant increases in secreted IFN-γ for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosisantigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-γ was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-γ was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-γ, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosisinfection in naturally exposed cattle.

The aim of the study reported here was to estimate the sensitivity and specificity of the gamma interferon (IFN-γ) ELISA for paratuberculosis in sheep using receiver-operating characteristic analysis. Bacteriologic culture of tissues was used to define the reference positive population (n= 33). Two reference negative populations were used: culture-negative sheep from infected flocks (n= 77), and sheep from noninfected flocks (n= 358). We also evaluated the accuracy of 2 Mycobacterium aviumsubspecies paratuberculosis(MAP) antigen preparations, a whole-cell sonicate (MpS) and a johnin purified-protein derivative (PPDj). The source of the reference negative sheep used in the analysis affected overall accuracy of the IFN-γ ELISA. The area under the curve was 0.683 (95% confidence interval 0.574–0.787), using culture-negative sheep from infected flocks, was 0.831 (0.764–0.889), using sheep from noninfected flocks for the MpS, and was 0.809 (0.726–0.881) and 0.897 (0.862–0.925) for the PPDj, respectively. Using the MpS, the cut point that classified the most sheep correctly was an optical density reading of 0.20, for sensitivity of 40.7% (19.4–57.6) and specificity of 88.7% (77.0–95.7) or 97.6% (93.04–99.5), depending on the reference negative population used. Using the PPDj, the cut point that classified the most sheep correctly was 0.25 for sensitivity of 66.7% (47.2–82.7) and specificity of 93.5% (85.5–97.9) or 98.3% (96.4–99.4), respectively. The PPDj was more accurate at identifying MAP-infected sheep than was the MpS (P= 0.034).

This study was designed to evaluate if the immunosuppression typically observed during the immediate periparturient period (3 weeks before and after calving) in dairy cows influences the effectiveness of diagnostic tests for the detection of Johne's disease; and, if providing additional energy to the cows during this period would minimize any immunosuppressive effects. Twelve dairy cows naturally infected with Mycobacterium paratuberculosiswere fitted with rumen cannulas in late gestation and assigned to treatment groups: control, n= 6; or stuffed, n= 6. Cows in the control group were allowed to consume feed ad libitum. Cows assigned to the stuffed treatment group were also fed ad libitum but received additional total mixed ration by manually stuffing their rumens with refused feed to maintain a dry matter intake of 2% body weight/day before calving and 2.5% body weight/day after calving. Parturition had a significant impact on immune function with significant reductions in M. paratuberculosis-specific antibodies detected in the serum and milk regardless of treatment group. Similarly, in vitro immunoglobulin production was decreased at calving for both treatment groups. In addition, stuffing cows modulated cell-mediated immune function by reducing antigen-specific lymphocyte proliferation and interferon-γproduction after calving. Shedding of M. paratuberculosisin the milk was apparent in 58% (7/12) of cows after parturition with no difference noted between control and stuffed animals. Parturition had no major effect on fecal shedding of cows regardless of treatment. These data suggest that parturition had a significant effect on immune function parameters including diagnostic tests for paratuberculosis. Furthermore, providing additional energy to cows with Johne's disease did not preclude immunosuppressive effects during the periparturient period.

ABSTRACTThe development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium aviumsubsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. aviumcomplex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. aviumsubsp. paratuberculosisthat 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. aviumsubsp. paratuberculosis-specific sequences in Escherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coliexpression 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. aviumsubsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. aviumsubsp. paratuberculosisand 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. aviumsubsp. paratuberculosisproteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. aviumsubsp. paratuberculosisantigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.

ABSTRACTThe objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium aviumsubsp. paratuberculosisisolated in the United States and to identify M. aviumsubsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. aviumsubsp. paratuberculosisisolates recovered from animals (n= 203) and patients with Crohn's disease (n= 7) from diverse geographic localities. Six hundred bacterial cultures, including M. aviumsubsp. paratuberculosis(n= 303), non-M. aviumsubsp. paratuberculosismycobacteria (n= 129), and other nonmycobacterial species (n= 168), were analyzed to evaluate the specificity of two IS900integration loci and a newly described M. aviumsubsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. aviumsubsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. aviumsubsp. paratuberculosisisolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. aviumsubsp. paratuberculosisisolates 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. aviumsubsp. paratuberculosisstrains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. aviumsubsp. paratuberculosisisolates recovered from human and ovine sources.

The causative agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C(18)-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of M. avium subsp. paratuberculosis from ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 microg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 microg of vancomycin, 30 microg of amphotericin B, and 20 microg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several M. avium subsp. paratuberculosis isolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of M. avium subsp. paratuberculosis; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 microg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered M. avium subsp. paratuberculosis from 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered M. avium subsp. paratuberculosis from 26 and 16 specimens, respectively. The average times to positive were 7.4 +/- 8.3, 29.9 +/- 2.6, and 24 +/- 0 days, respectively. The contamination rates were 4.8, 22.6, and 20.0%, respectively.

ABSTRACTThe causative agent of Johne's disease is Mycobacterium aviumsubsp. paratuberculosis. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C18-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of M. aviumsubsp. paratuberculosisfrom ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 µg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 µg of vancomycin, 30 µg of amphotericin B, and 20 µg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several M. aviumsubsp. paratuberculosisisolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of M. aviumsubsp. paratuberculosis; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 µg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered M. aviumsubsp. paratuberculosisfrom 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered M. aviumsubsp. paratuberculosisfrom 26 and 16 specimens, respectively. The average times to positive were 7.4 ± 8.3, 29.9 ± 2.6, and 24 ± 0 days, respectively. The contamination rates were 4.8, 22.6, and 20.0%, respectively.

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne s disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for y-interferon (γ-IFN) production. Cells from subclinical cows produced significantly higher levels of γ-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of γ-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of γ-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that γ-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobac-terium paratuberculosisand cell-wall proteins of M. paratuberculosisand were evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis(LMp) were inoculated intraperitoneally or intravenously at 109/ml. Heat-killed M. paratuberculosis(HKMp) was prepared by treatment of bacteria at 85 C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosisand conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. Antibody titers in the terminal blood sample were higher for HKMp and KLH-CWPMp than for LMp rabbits (1:1,024 vs. 1:64). The KLH-CWPMp antibody did not cross-react with M. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues was much higher for the KLH-CWPMp polyclonal antibody. Immunoreactivity of the antibody resulted in staining of bacteria in the cytoplasm of macrophages, mononuclear giant cells, and extracellular bacteria in both intestine and lymph node.

Mycobacterium aviumsubspeciesparatuberculosis(M. paratuberculosis) is the etiologic agent of paratuberculosis (Johne's disease), a chronic granulomatous enteritis in ruminants. Currently, there is a need for improved diagnostic tests because of the lack of methods for accurate, rapid and reliable detection ofM. paratuberculosisinfection. AM. paratuberculosisgene (hspX) was cloned, sequenced, and a 30 bp species-specific oligonucleotide was synthesized. As an internal control to identify mycobacterial strains, a 33 bpMycobacteriumgenus-specific oligonucleotide was synthesized based on the conserved 5 terminus of the mycobacterialrecAgene. Dioligonucleotide hybridization (dOH) analysis identified 28/28 (100%) mycobacterial strains and specifically identified 14/14 (100%) reference (ATCC 19698), bovine, ovine and human isolates ofM. paratuberculosis.TheM. paratuberculosis-specific oligonucleotide distinguishedM. paratuberculosisisolates from related mycobacteria, including all closely related members of theMycobacterium aviumcomplex (MAC) tested in this study. The members of MAC tested in this study includedMycobacterium aviumsubspeciesavium(M. avium),M. paratuberculosis,Mycobacterium aviumsubspeciessilvaticum(M. silvaticum) andMycobacterium intracellularestrains. Hybridization was not observed with DNA extracted from a selected group of other bacterial pathogens. The experiments indicate that the dOH analysis is a useful diagnostic tool to detect mycobacterial infection, specificallyM. paratuberculosis.The dOH method could be a good alternative to existing assays and will be adapted for specific identification ofM. paratuberculosisfrom faecal samples, mixed bacteriologic cultures, tissue specimens and whole blood.

A new method (NADC) for isolation of Mycobacterium paratuberculosisfrom fecal samples is described and evaluated using fecal samples from a known paratuberculosis-infected herd of cattle. The NADC method includes centrifugation of the total fecal sample supernatant and use of a 2-step decontamination protocol. The growth rate of M. paratuberculosisand contamination rate of cultures when using this method are compared to 3 other published methods: sedimentation, centrifugation, and Cornell. Sensitivity was lowest for the Cornell method precluding detection of some low shedders; however, contamination was not observed for this method. Contamination was the most severe in samples processed by the centrifugation method but was also high for the sedimentation method, resulting in unreadable culture tubes for some fecal samples. The NADC method was 10-fold more sensitive for detection of M. paratuberculosiscolonies and contamination was significantly reduced compared to other 3 methods.

Pasteurella multocidatoxin depresses weight gain in rats and pigs. It also affects tissues with rapidly dividing cells. In the present study, we investigated the role of this protein toxin on chondrocyte growth in vivo. Rats were divided into a single- or multiple-dose group and were given, respectively, either a single injection (0.15 or 0.6 μg/kg toxin subcutaneously) or multiple injections (0.01-0.2 μg/kg subcutaneously) of toxin. Bone (humerus) and other selected tissues were stained for bromodeoxyuridine immunoreactivity (BrDU-IR) in order to gauge cell proliferation. Physeal area was measured in rats from the multiple-dose group. Serum from single-and multiple-dose groups were tested for tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) activity using a bioassay system. Decreased weight gain, feed intake, and feed efficiency were observed in single- and multiple-dose groups of rats. Decreased BrDU-IR indices were present in the resting and proliferative zone chondrocytes of the humeral physis in rats from the multiple-dose group, as was decreased physeal area. Increased serum IL-6 bioactivity was present in rats after 24 hours, and no changes in TNF-α bioactivity were seen in any group. No alterations in BrDU-IR were seen in rats fed restricted (80% of control) diets. These studies show that sublethal doses of toxin decrease weight gain and affect growth of long bones through suppression of chondrocyte proliferation. These effects may be mediated by direct binding of the toxin to target cells or IL-6 but are not associated with altered feed intake or TNF-induced cachexia.

Cell-mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-γ ELISA) and the Johnin skin test, have the potential to detect animals infected with Mycobacterium aviumsubspecies paratuberculosis(MAP) early in the course of the disease. While these CMI tests tend to be relatively specific in noninfected flocks, in MAP-infected flocks, these tests often identify animals that cannot be confirmed infected by any other reference test, including necropsy and culture. The aim of this study was to determine if antigen exposure by inhalation or oral ingestion of killed MAP organisms would cause a detectable CMI response in sheep. Forty-eight lambs 4 months of age were randomly divided into a control group, an orally exposed group (dosed with 1 × 1010autoclaved MAP organisms 3 times), and an inhalation-exposed group (dosed once with 1 × 105dead organisms). Lambs were skin tested and/or bled pre-exposure and 1, 2, 3, 4, and 12 months postexposure. No significant difference was seen with either the oral- or inhalation-exposed groups of lambs versus controls with either the IFN-γ ELISA or the skin test at any time pre- or postexposure. These results suggest that infection/invasion of MAP organisms must occur in order to have a positive skin test or IFN-γ ELISA beyond the false-positive rate. Simple exposure is not enough to elicit a detectable CMI response.

ABSTRACTJohne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium aviumsubspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. aviumsubsp. paratuberculosisand the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosisprotein array to identify seroreactive and diagnostic M. aviumsubsp. paratuberculosisantigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. aviumsubsp. paratuberculosisand M. tuberculosisshowed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosisprotein array probed with sera from M. aviumsubsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosisproteins, with 58% of them having =70% identity to M. aviumsubsp. paratuberculosisorthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosisantigens were orthologs of previously reported M. aviumsubsp. paratuberculosisantigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. aviumsubsp. paratuberculosisrecombinant proteins, representing reactive and nonreactive M. tuberculosisorthologs, further confirmed that the M. tuberculosisarray has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. aviumsubsp. paratuberculosisproteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.