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Clin Microbiol Rev. Jul 2001; 14(3): 489–512.
PMCID: PMC88986

Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine

Abstract

Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.

Bacteria of the genus Mycobacterium are gram-positive, acid-fast organisms that include a number of significant human and animal pathogens. Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiological agent of a severe gastroenteritis in ruminants, known as Johne's disease. H. A. Johne and L. Frothingham initially reported the disease in Germany in 1894. However, it was not until 1910 that F. W. Trowt successfully fulfilled Koch's postulates by growing M. paratuberculosis in the laboratory and reproducing the disease in experimentally infected cattle (46, 148).

Johne's disease is prevalent in domestic animals worldwide and has significant impact on the global economy (290). Its influence in the United States alone is staggering, causing an estimated loss of $1.5 billion to the agriculture industry every year (279). It is considered to be one of the most serious diseases affecting dairy cattle (200). However, accurately assessing losses in productivity and profit at the level of an individual herd is difficult, making it likely that the impact of this disease is underestimated nationwide (165, 223). Nonetheless, a Johne's disease regression model estimates this loss to be from $40 to $227 per cow inventoried per year, based on the percentage of culled cows with clinical signs (219).

Isolation of M. paratuberculosis from intestinal tissue of Crohn's disease patients has led to concern that it may be pathogenic for humans (204). This issue is still controversial, with several reports documenting either the presence or absence of this bacterium in Crohn's disease patients (36, 43, 58, 110, 167, 257). Given the difficulty that investigators have had in isolating a putative infectious agent for this disease from human tissues and the lack of suitable animal models, it is not surprising that Koch's postulates have not been fulfilled for Crohn's disease. Thus, a causal relationship between M. paratuberculosis and Crohn's disease has not been demonstrated. Similarly, chemotherapy with antimycobacterial agents has given mixed results in Crohn's disease patients (see references 61 and 151 for reviews). Finally, it has been hypothesized that only a subset of Crohn's disease cases have an infectious etiology (53). The reader is referred to a recent review by Hermon-Taylor et al. (151) for a thoughtful analysis of the involvement of M. paratuberculosis in Crohn's disease.

Another aspect of this controversy has surfaced recently with a report that in milk pasteurization trials, a clinical strain of M. paratuberculosis was more thermally tolerant than either Mycobacterium bovis or Coxiella burnetii, the current milk pasteurization standard microorganism (285). While some reports have indicated that high-temperature short-time pasteurization does not effectively kill M. paratuberculosis in milk (135, 136), others have demonstrated killing by turbulent-flow conditions (277). Thus, the pathogenic role of M. paratuberculosis in human disease and the identification of potential sources of infection are topics of intense debate.

Neonatal and juvenile animals are at the highest risk for acquiring an infection of M. paratuberculosis (56, 61). Young animals are most commonly infected through the fecal-oral route. This occurs either by ingesting the organism through contaminated milk or food products or by accidental ingestion of the microorganism from contaminated surfaces (61). M. paratuberculosis, similar to other pathogenic mycobacteria, targets the mucosa-associated lymphoid tissues of the host (189). It preferentially targets the mucosa-associated lymphoid tissues of the upper gastrointestinal tract, where it is endocytosed by the M cells of the ileal Peyer's patches and subsequently phagocytosed by subepithelial and intraepithelial macrophages (121, 189, 208). M. paratuberculosis bacilli probably remain in the phagosome, where they multiply intracellularly (168). Cytokine production and the initiation of a cellular immune response by the host causes the appearance of an intestinal granuloma, and a cellular response is initiated in the nearby lymph nodes in an attempt to clear the infection (56, 61, 189). This inflammatory process leads to the clinical manifestations of a corrugated intestinal epithelium and the corresponding characteristic malnutrition syndrome associated with Johne's disease.

CHARACTERISTICS OF M. PARATUBERCULOSIS

Taxonomic and Phylogenetic Analysis

The mycobacterial species M. avium is currently subdivided into three subspecies, M. avium subsp. avium (M. avium), M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (M. silvaticum). The subspecies designation of M. paratuberculosis is based on DNA-DNA hybridization studies (159, 259, 298, 328) and numerical taxonomy analysis (298). Although commonly grouped with M. avium in the Mycobacterium avium-intracellulare complex, M. intracellulare is a genetically distinct species (7, 256). At the subspecies level, M. paratuberculosis can be differentiated phenotypically from M. avium and M. silvaticum by its dependence on mycobactin (298) and genotypically by the presence of multiple copies of an insertion element, IS900 (67, 137).

Analysis of the rRNA genes (rDNA) of mycobacteria has resulted in the division of this genus into two separate clusters. These correspond to the traditional fast-growing mycobacteria, represented by nonpathogenic environmental isolates, and the slow-growing mycobacteria, containing most of the overt pathogens (251, 280, 311). The rDNA gene copy number also reflects this division between fast- and slow-growing mycobacteria; fast-growing mycobacteria contain two sets of rRNA genes, whereas the slow growers, including M. paratuberculosis and M. avium, contain only one copy (19, 47, 48, 286). Like other mycobacteria, the rDNA genes are found in a single operon in M. paratuberculosis (120, 185, 186, 286). Interestingly, as in Streptomyces spp. (237, 287), this operon does not contain a tRNA gene, a feature found in the Mycobacterium smegmatis rrnB ribosomal operon (131).

An internal transcribed spacer region of approximately 280 bp separates the 16S and 23S rDNA genes, and a smaller internal transcribed spacer of 91 bp separates the 23S rDNA and 5S rDNA genes (186). Both M. paratuberculosis and M. avium have an unusual insertion of approximately 16 nucleotides in the 23S rRNA gene, which is also found in the 23S rRNA genes of other mycobacteria (186, 303). This region appears to be variable among mycobacteria. Sequence comparison shows a single mismatch between M. paratuberculosis and M. avium, whereas this sequence contains an additional 4 bp in M. phlei (303).

The dnaJ gene encodes a highly conserved heat shock protein and has been used in other bacterial genera for phylogenetic mapping (123, 329). This gene was sequenced from 19 mycobacterial species, and the phylogenetic relationship inferred from this data was in close agreement with the traditional classification based on 16S rRNA sequencing for slowly growing mycobacteria. As expected, the M. avium and M. paratuberculosis dnaJ genes were 99% homologous, substantiating the previously established close relationship between these two mycobacterial species (292).

The size of the M. paratuberculosis genome has been estimated to be 4.4 to 4.7 Mb (61). Compared to other mycobacteria, this is similar to the M. tuberculosis genome of 4.41 Mb (65) and the M. bovis genome of 4.4 Mb (Sanger Center; http://www.sanger.ac.uk) but slightly larger than the estimated size of the genome of M. leprae (3.3 Mb; sequencing recently completed) (Sanger Center). However, the M. paratuberculosis genome sequence is nearly complete and appears to be larger than expected at approximately 5 Mb (University of Minnesota; http://www.cbc.umn.edu/ResearchProjects/Ptb/) similar to that of the M. avium genome (The Institute for Genomic Research; sequencing recently completed http://www.tigr.org). M. paratuberculosis DNA has a base composition of 66 to 67% G+C, similar to M. tuberculosis and M. bovis (65 and 64%, respectively) (Sanger Center).

Extrachromosomal plasmid DNA was first reported in Mycobacterium fortuitum (178). This 5.0-kb plasmid was designated pAL5000 and has been used extensively for the construction of other recombinant plasmids in the molecular genetic studies of many other mycobacterial species. Naturally occurring plasmids have also been observed in clinical and environmental isolates of M. avium, M. intracellulare, and M. scrofulaceum (106). However, no endogenous plasmid DNA has yet been reported in any isolates of M. paratuberculosis.

Major Antigens

The search for M. paratuberculosis-specific antigens for diagnostic testing or preventive therapy has led to the discovery of several immunoreactive proteins (Table (Table1).1). Many of these proteins have homology to other mycobacterial antigens, such as the GroES and GroEL proteins. The GroES antigens are highly antigenic, small (ca. 100-amino-acid), highly conserved heat shock proteins. Recently, the GroES antigens of M. avium and M. paratuberculosis have been cloned and sequenced (60). Not surprisingly, the M. avium and M. paratuberculosis GroES coding sequences and deduced amino acid sequences are 100% identical to each other and are over 90% identical to the M. tuberculosis and M. leprae sequences, differing by only 3 amino acids. The homologous GroES protein from M. tuberculosis has two T-cell epitopes, which are both conserved in M. avium and M. paratuberculosis (60).

TABLE 1
Known immunogenic proteins of M. paratuberculosis

Other immunoreactive proteins of M. paratuberculosis include a 32-kDa secreted protein with fibronectin binding properties implicated in protective immunity (4, 100) and a 34-kDa cell wall antigenic protein homologous to a similar protein in M. leprae (87, 88, 127, 264). This 34-kDa protein carries two species-specific B-cell epitopes that have been exploited in the histopathological diagnosis of Johne's disease (62). Cameron et al. (33) described a seroreactive 34-kDa serine protease expressed in vivo by M. paratuberculosis. This antigen is different from the 34-kDa antigen described above. Another strongly immunoreactive protein of 35 kDa has also been identified in M. avium complex isolates, including M. paratuberculosis (102). Sequencing data for this antigen are not available, and its relation to any of the above antigens has not been established. Nonetheless, it reacted with sera from cattle in both the subclinical and clinical stages of Johne's disease but was nonreactive with sera from cattle vaccinated with M. bovis BCG (102).

A more thoroughly characterized protein of 65 kDa from M. paratuberculosis is a member of the GroEL family of heat shock proteins (100, 101). Like the GroES proteins, the GroEL antigens from other mycobacteria are highly immunogenic (260, 296, 297). As expected, the M. paratuberculosis GroEL protein is homologous to similar proteins of M. tuberculosis (93%), M. leprae (89%), and M. avium (98%). Adsorbed antisera from infected animals failed to consistently recognize this antigen, however, precluding its use in the serodiagnosis of Johne's disease. Similarly, a DNA vaccine constructed from the M. avium 65-kDa antigen fused to the green fluorescent protein failed to confer protective immunity to vaccinated mice (308).

The alkyl hydroperoxide reductases C and D (AhpC and AhpD) are the most recently characterized immunogenic proteins of M. paratuberculosis (222). Unlike other mycobacteria, large amounts of these antigens are produced by M. paratuberculosis when the bacilli are grown without exposure to oxidative stress. AhpC is the larger of the two proteins and appears to exist as a homodimer in its native form since it migrates at both 45 and 24 kDa under denaturing conditions. In contrast, AhpD is a smaller monomer, with a molecular mass of about 19 kDa. Antiserum from rabbits immunized against AhpC and AhpD reacted only with M. paratuberculosis proteins and not with proteins from other mycobacterial species, indicating that antibodies against these proteins are not cross-reactive. Furthermore, peripheral blood monocytes from goats experimentally infected with M. paratuberculosis were capable of inducing gamma interferon (IFN-γ) responses after stimulation with AhpC and AhpD, confirming their immunogenicity (222). In conclusion, these proteins are potentially useful for developing future vaccines and diagnostic assays or monitoring disease progression.

Several putative M. paratuberculosis-specific antigenic proteins have been described in the literature (Table (Table1).1). These include a cellular antigen of 34.5 kDa, (214), a 42-kDa protein of unknown function (320), and a 44.3-kDa antigen (214). Unfortunately, the above-mentioned 34.5-kDa protein has not been compared to either the 34-kDa serine protease described by Cameron et al. (33) or the 35-kDa protein described by El-Zaatari et al. (102). Similarly, a comparative analysis of the 42- and 44.3-kDa antigens has not been reported.

Transcriptional Control

Mycobacteria, like other prokaryotes, have a plethora of DNA-dependent RNA polymerase sigma factors (130). The M. tuberculosis genome project annotates 14 genes encoding either confirmed or putative sigma factors (65). Sigma factors provide the core RNA polymerase with various recognition specificities, allowing the transcription of regular housekeeping, stress, and virulence-associated genes (193, 267). Interestingly, a point mutation in the principal sigma factor, sigA, was demonstrated to attenuate virulent M. bovis (73). In general, mycobacterial promoters function poorly in Escherichia coli and have a greater G+C content, reflecting their genome composition. A rather highly conserved −10 region and an extended TGN motif (12, 13) characterize an important set of mycobacterial promoters, while other promoters seem to possess a more highly conserved −35 region (212).

In the sole analysis of M. paratuberculosis promoters, Bannantine et al. (9) studied eight promoter regions of M. paratuberculosis and found significant differences from those of E. coli. In this study, gene fusions to the β-galactosidase reporter gene were analyzed in both E. coli and M. smegmatis. As expected, these fusions displayed lower or negligible levels of β-galactosidase activity in E. coli than in M. smegmatis. Primer extension and sequence analysis of upstream regions demonstrated −35 and −10 consensus sequences of TGMCGT (where M represents C or A) and GGGCCS (where S represents G or C), respectively. These M. paratuberculosis promoters are more closely related to each other than they are to other mycobacterial promoters. Interestingly, the −35 hexamer is more highly conserved than the −10 hexamer while the latter has a consensus that departs significantly from the proposed extended TGN motif mentioned above. The only other M. paratuberculosis promoter described is the PAN promoter (213), which differs from the consensus described by Bannantine et al. (9) and also lacks a TGN motif. It is possible that M. paratuberculosis, one of the slowest-growing mycobacteria, may possess promoters that depart significantly from those of other mycobacteria. However, Bannantine et al. (9) may have identified a unique set of promoters that do not use the TGN motif. In this context, the various studies described above used different vectors, reporter genes, and selection or screening strategies that may have led to the identification of different types of promoters. More studies are necessary to ascertain whether the main M. paratuberculosis sigma factor uses promoters with TGN motifs or the special consensus identified by Bannantine et al. (9). In general, mycobacterial translation signals show a significant number of GTG start codons and a characteristic GC codon bias (212). Regarding M. paratuberculosis ribosome binding sites (RBS), all the corresponding mRNAs from the lacZ fusions analyzed by Bannantine et al. (9) possess a putative RBS complementary to the 3′ sequence of the 16S rRNA from M. leprae. In this study, a putative initiation codon and an open reading frame (ORF) followed the RBS sequences. The start codons were either ATG (5 of 8) or GTG.

Homologous Recombination

Allelic replacement is based on recombination between homologous sequences and is useful to generate mutations in a gene of interest. In M. tuberculosis, allelic exchange has presented major difficulties, which only recently have been overcome. Initially, the small number of homologous recombination events obtained in M. tuberculosis was attributed to a defect in the homologous recombination system, possibly related to the presence of an intein in the recA gene (86). Recent work demonstrated that homologous recombination seems to be functional in M. tuberculosis, but problems occur due to the high frequency of illegitimate recombination observed in that pathogen (118, 227). To circumvent these problems, a series of vectors with thermosensitive replicons (11, 233), sucrose selection (231233), long concatomeric molecules (8), and pretreatment of DNA with restricition endonucleases (154, 224, 225) have been developed. These approaches have been successful in the more closely related mycobacterial species M. intracellulare (154, 195), suggesting that M. paratuberculosis may be amenable to them as well. Additionally, a study of the IS900 insertion sites in M. paratuberculosis indicated that homologous recombination events may have occurred between IS900 insertion elements present at different loci (29).

MOLECULAR EPIDEMIOLOGY

Restriction Fragment Length Polymorphism Analysis

Because M. paratuberculosis and M. avium are closely related genetically, they are difficult to differentiate. The application of molecular genetic methods in epidemiological studies allowed separation of clinical isolates of M. avium and M. paratuberculosis (64, 198, 309, 317). These analyses were instrumental in reassigning an isolate previously identified as M. paratuberculosis strain 18 as an M. avium strain (67, 105, 315, 316).

Restriction fragment length polymorphism (RFLP) analysis also has been used epidemiologically to ascertain a possible common clonal origin for M. paratuberculosis strains isolated from unrelated animal and human sources (54, 66, 89, 115, 228, 315). This technique can also separate sheep and cattle field isolates of M. paratuberculosis (15, 69), suggesting that M. paratuberculosis strains may be undergoing host adaptation. Interestingly, M. paratuberculosis isolates from other ruminants give similar RFLP profiles to cattle isolates (299). A single case study documented the isolation of two different strains of M. paratuberculosis, each with a unique RFLP profile, from the same animal, implying the possibility of a coinfection (228). An effort to standardize RFLP analysis for clinical isolates of M. paratuberculosis combined previously reported methods (69, 228) and resulted in 28 different RFLP types from 1,008 strains tested. These 28 RFLP types were derived from combining 13 RFLP (PstI) types and 20 RFLP (BstEII) types (229). Recently, a multiplex PCR based on specific IS900 loci in the M. paratuberculosis genome was cross-referenced to these RFLP types (29), indicating that future typing of M. paratuberculosis isolates may not require bacterial culture, making large-scale epidemiological studies of Johne's disease possible.

IS900

Insertion sequence elements are small, mobile genetic elements containing genes related to transposition functions. IS900 was the first example of an insertion sequence element to be found in mycobacteria (68, 137); it is a member of the IS110 family of insertion sequences (191). An interesting aspect of the insertion sequences belonging to this family is their lack of both terminal inverted repeats and direct repeats commonly associated with other transposable DNA elements (191). Four insertion sequences in this family are from mycobacteria, IS900 (M. paratuberculosis) (137), IS901 (M. avium) (177), IS902 (M. silvaticum) (210) , and IS1110 (M. avium) (152). Two new insertion elements, IS1547, recently discovered in M. tuberculosis (107), and IS1626, from M. avium and M. intracellulare (244), are also related to the IS110 family. All of these insertion elements, except for IS1626, which has been found only in 1 to 3 copies, are found in their respective genomes at 10 to 20 copies per chromosome (68, 107, 137, 177, 210).

IS1626 is the most closely related insertion sequence to IS900 (Table (Table2).2). These two insertion elements have 82% homology, with the greatest variability occurring at the 5′ end of their nucleotide sequences. IS1626 has a mechanism of transposition and consensus sequence remarkably similar to IS900. Furthermore, like IS900, IS1626 inserts itself with a defined orientation with respect to its consensus sequence (244).

TABLE 2
Comparison of the putative mycobacterial insertion element transposase genes belonging to the IS110 family

In contrast, the IS900 and IS901 transposase genes have a DNA homology of only 60% and an amino acid homology of 49% (177). Similarly, the homology between the IS900 and IS902 transposase genes is 60% at the nucleotide level and 50% at the amino acid level (210). The transposase from IS900 appears to be quite distinct from the other mycobacterial insertion elements belonging to the IS110 family, since only short regions of homology are found at the 3′ end of both the DNA and amino acid sequences.

The IS900 transposase gene is functional, since an artificial transposon derived from IS900 could stably integrate into the chromosomes of M. smegmatis (91, 103), M. vaccae, and M. bovis BCG (91). Furthermore, the method of transposition appears to involve both simple insertions and cointegrates, suggesting that IS900 transposes by a replicative mechanism (91). IS900 contains two ORFs encoded on opposite strands. The transposase protein, p43, is encoded by ORF1 and can be expressed in vitro (300). This protein is approximately 43 kDa but migrates at an apparent molecular mass of 44.5 to 45 kDa, perhaps due to its high basic character (predicted pI 10.4), which may retard its migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (300). Western blot analysis shows that antibodies against this protein react with both a 45-kDa protein and an apparent cleavage product of approximately 28 kDa. This antibody reacts with only the 28-kDa protein from clinical strains of M. paratuberculosis. Neither M. avium nor M. silvaticum extracts demonstrated positively staining bands at these molecular masses confirming that p43 is specific to IS900-bearing mycobacteria.

The PAN promoter and an adjacent ORF called ORF2 were discovered by a search for M. paratuberculosis subspecies-specific genes and expression signals. The PAN promoter is located adjacent to but outside of IS900. Its proximity to ORF2 suggested that PAN is able to drive the expression of ORF2 (213). The PAN promoter is present in a single copy in many pathogenic strains of mycobacteria but is absent from M. marinum, M. terrae, M. gordonae, and several fast-growing mycobacteria (133). The PAN region of M. paratuberculosis has 70% similarity to M. tuberculosis, M. bovis and M. bovis BCG regions, and the −10 and −35 promoter regions are completely conserved (247). Although it is tempting to speculate that the corresponding genes driven by the PAN promoter may have a similar function, no analysis of these regions has been performed to date.

ORF2 of IS900 was designated as the hed (host expression-dependent) gene (95). The putative Hed protein encoded by IS900 contains a hydrophobic sequence of 9 amino acids, which has significant homology to eight other prokaryotic and eukaryotic proteins involved in the transport of various essential growth requirements (94). Of the five prokaryotic transport proteins containing this conserved 9-amino-acid motif, three are involved in iron transport, leading to speculation that Hed may provide M. paratuberculosis with an alternate iron transport mechanism (94). Another possible role for Hed as a repressor of the native mycobactin pathway in M. paratuberculosis is implied by the observation that M. smegmatis transformants carrying a multicopy plasmid with IS900 demonstrate a significantly decreased growth rate in broth cultures. This phenotype can be restored to wild-type levels by the supplementation with ferric mycobactin J (217). Clearly, these studies present intriguing hypotheses of iron utilization in M. paratuberculosis which merit further research.

The expression of hed requires that IS900 be inserted into the chromosomal DNA in a directional manner (Fig. (Fig.1).1). This directionality is accomplished by IS900 inserting itself between the putative RBS and start codon of a given gene in the chromosome, thus aligning the hed initiation codon next to a functional RBS from the target gene and downstream of an active promoter (29, 94) (Fig. (Fig.1).1). The target gene RBS (AGGAG) is in fact the complement of the IS900 consensus sequence, CATGN4–6 *CNCCTT (where * denotes the site of insertion) (29, 94, 137). Since no termination codon is present in the hed sequence, the corresponding translational products of different IS900 elements in the genome are of different lengths. Because the hed gene carries its own RBS at the 3′ terminus, the translation of the target gene may be restored by this replacement RBS (95) (Fig. (Fig.1).1). This hypothesis was confirmed recently by Bull et al., (29), who analyzed genomic regions immediately adjacent to 14 different IS900 insertions and found that IS900 was inserted in the hed orientation with respect to its consensus sequence in all loci. In five of these loci, these insertions resulted in the inactivation of the corresponding target genes, possibly altering gene expression. For 13 of these loci, 94 to 100% homology to the M. avium genome was found. However, one locus was absent from the M. avium genome and several other mycobacterial species, including M. silvaticum. This DNA region in the M. paratuberculosis genome appears to encode a transcriptional regulator and a polyketide synthase. Of the loci that had homology to accessions in GenBank, genes encoding a sigma factor, a nitrate reductase, and a methyl transferase were identified. ORFs of unknown functions were found in the other loci. Of the 14 loci, 7 were present in a panel of 81 M. paratuberculosis isolates tested, and 52 of these M. paratuberculosis strains contained all 14 IS900 insertion loci.

FIG. 1
IS900 inserts in a target gene of M. paratuberculosis in an orientation-specific manner. A putative target gene of M. paratuberculosis is illustrated as a hatched line, with the corresponding upstream and downstream sequences of the genome shown as open ...

IS1311

Only one other insertion element, IS1311, has been described in the M. paratuberculosis chromosome (74, 253). This insertion element is not unique to M. paratuberculosis, since it is also found in M. avium and M. intracellulare at approximately 7 to 10 copies throughout the genome (74, 321). IS1311 is a member of the IS256 family of insertion elements and differs from IS900 in that it contains terminal inverted repeats (191). Although this has not been analyzed in IS1311, other members of the IS256 family also generate an 8 to 9-bp direct target repeat. Of the 33 members in the IS256 family of insertion sequences, 8 others are from mycobacteria (Table (Table3).3). These are IS1081 (M. bovis) (70), IS1245 (M. avium) (143), IS1395 (M. xenopi) (238), IS1407 (M. celatum) (239), IS1408 (M. branderi) (191), IS1511 (M. gordonae) (191), IS1512 ( M. gordonae) (191), and IS6120 (M. smegmatis) (144). Significant similarity is seen throughout the entire nucleotide and amino acid sequences of IS1311 and IS1245 (Table (Table3).3). Conversely, the other mycobacterial members of the IS256 family only have short (24 to 105-nucleotide) regions of similarity to the IS1311 transposase gene. A slightly higher similarity (45 to 68%) is noted at the amino acid level (Table (Table3).3). IS1311 is somewhat smaller than IS900 (1,317 and 1,451 bp, respectively) and appears to contain a single ORF encoding a putative transposase. IS1311 and IS900 are not homologous at either the nucleotide or amino acid level. Analysis of M. paratuberculosis and M. avium IS1311 sequences shows a slight difference of five point mutations (321). In addition, some copies of IS1311 in the cattle strains of M. paratuberculosis contain an additional point mutation that can be used to distinguish them from the sheep strains (321).

TABLE 3
Comparison of the putative mycobacterial insertion element transposase genes belonging to the IS256 family

IS1110, IS1626, and Other Related Insertion Elements

The presence of other insertion elements in the M. avium genome that are closely related to IS900 implies that M. paratuberculosis may also be harboring other undiscovered insertion elements. These insertion sequences, IS1110 and IS1626, are not found in all strains of M. avium, suggesting that they are recent acquisitions by the M. avium genome (152, 244). Of interest is another insertion element from M. avium and M. intracellulare, IS1613, whose sequence has been submitted to GenBank (accession number AJ011838) and which displays 83% homology to IS900 by BLAST analysis.

PATHOGENESIS AND ANIMAL MODELS

Interaction of M. paratuberculosis with Macrophages

Survival within macrophages is a hallmark of M. paratuberculosis. Recent evidence indicates that simultaneous intracellular multiplication and killing of M. paratuberculosis occurs, reflecting an initial T-helper 1 (Th1) cellular immune response of the host (331). The nutritional and hormonal status of an animal may also influence its susceptibility to M. paratuberculosis infections. Reduced dietary calcium (Ca2+) protects beige mice from M. paratuberculosis infections (276), but a corresponding increase in endogenous 1,25-(OH)2D3 (vitamin D) levels reverses the beneficial effects of low Ca2+ levels (276, 278). Additionally, transient exposure of monocytes to growth hormone or prolactin enhances intracellular multiplication of M. paratuberculosis in primary bovine monocytes (109). Because the levels of these hormones also fluctuate during parturition and lactation, it is possible that similar alterations in the hormonal milieu might also influence the intracellular multiplication of M. paratuberculosis in bovine mononuclear phagocytes in vivo.

Other mycobacteria, including M. avium and M. tuberculosis, circumvent macrophage antigen processing by inhibiting phagolysosomal fusion (59, 221) and acidification (221, 283). In general, mycobacteria are relatively resistant to the bactericidal mechanisms of professional phagocytes. Resistance to reactive oxygen intermediates has been linked to catalase and peroxidase activities (38, 192), and alkyl hydroperoxidase reductase (AhpC) is implicated in resistance to both reactive oxygen and reactive nitrogen intermediates (41, 192). The gene products involved in these processes are candidate virulence determinants, which are likely to play an important role in disease. Treatment of M. paratuberculosis-infected bovine monocytes with IFN-γ induces the release of nitric oxide (NO) (330). However, the levels of NO produced by these monocytes are far below those needed to kill M. paratuberculosis in a cell-free system. This observation implies that the amount of NO produced by recombinant IFN-γ (rIFN-γ)-activated bovine monocytes is insufficient to kill intracellular M. paratuberculosis in vitro.

Regarding the use of macrophage-derived cell lines, both the activated murine J774.16 (272) and bovine BoMac (273) macrophages can restrict the growth of M. paratuberculosis. Surprisingly, no studies have been reported in the literature quantifying the ability of M. paratuberculosis to replicate and survive in cultures of primary human macrophages or human intestinal epithelial cell lines, important in the context of its potential role as the etiological agent of Crohn's disease.

Role of Cytokines in M. paratuberculosis Infections

M. paratuberculosis also influences cytokine production in infected animals. Both serum IFN-γ levels (274) and IFN-γ gene expression in ileal tissue and cecal lymph nodes (291) were higher in subclinically infected animals than in animals exhibiting clinical symptoms of Johne's disease. However, Adams et al. (3) did not find significant differences in mRNA IFN-γ expression from peripheral blood monocytes in subclinically infected cattle, suggesting that cytokine production is a local phenomenon restricted to infected tissues. Bovine macrophages can be induced to release interleukin 1 (IL-1) (1, 176), IL-6 (1, 2), and tumor necrosis factor alpha (TNF-α) (1, 2) upon stimulation with M. paratuberculosis antigens. These cytokines are also associated with granuloma formation and cachexia in other disease syndromes (42, 170, 184, 211). In particular, IL-1 seems to play an important role in protecting experimentally infected mice against M. paratuberculosis infection (169). These investigators show that in vivo administration of a monoclonal antibody against the IL-1 receptor hinders the elimination of M. paratuberculosis from these animals.

Cytokines appear to affect the ability of M. paratuberculosis to survive within the macrophages. In the J774.16 murine macrophage cell line, preincubation with moderate levels (10 to 1,000 IU) of TNF-α prior to infection with M. paratuberculosis significantly increases the number of viable bacteria recovered whereas high doses (4,000 IU) of TNF-α reduce bacterial numbers (272). Pretreatment of bovine monocytes with IFN-γ slightly increases phagocytosis of M. paratuberculosis and inhibits its intracellular growth (331). Furthermore, continuous incubation of bovine monocytes with IFN-γ or human granulocyte-macrophage colony-stimulating factor significantly restricts the intracellular growth of M. paratuberculosis (333). Together, these studies demonstrate that local tissue cytokine concentrations are important in determining the outcome of a mycobacterial infection.

Role of Iron Uptake in M. paratuberculosis Infections

Mammalian hosts actively restrict iron supply to bacterial pathogens. Cytokines may function by causing macrophages to actively limit their intracellular iron concentration in an attempt to restrict bacterial growth (172, 312). Activated macrophages downregulate transferrin receptors, reducing iron levels (32). Nramp1, an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages, has been implicated in controlling mycobacterial replication by actively removing iron or divalent cations from the phagosomal space, further decreasing the amount of iron available to intracellular bacteria (34, 129). To scavenge iron in limiting environments, most mycobacterial species produce the lipid-soluble siderophore mycobactin and the water-soluble siderophore exochelin (10, 269). Since M. paratuberculosis is a mycobactin auxotroph, it can synthesize exochelin only (10, 269). These two siderophores appear to be relevant in the pathogenicity of mycobacteria, as gene mutations in the biosynthetic pathway of mycobactin T in M. tuberculosis impairs its ability to replicate in a human macrophage cell line (93). How M. paratuberculosis survives intracellularly when producing only exochelins is unclear.

Genetic Systems for M. paratuberculosis

The genetic manipulation of M. paratuberculosis is notoriously difficult. Its lengthy generation time, resistance to enzymatic or chemical lysis, and difficulty in performing genetic exchange commonly used for other mycobacteria all contribute to this problem. Therefore, the establishment of a gene transfer system in M. paratuberculosis was a significant breakthrough in the molecular genetic analysis of this organism (112). Prior to this, the ability to directly manipulate DNA elements from the M. paratuberculosis genome without the use of surrogate bacterial hosts had not been possible. This study demonstrated for the first time that M. paratuberculosis could be transformed with both foreign plasmid and bacteriophage DNA, providing a molecular genetics-based method for genetic manipulation. It also established that the aminoglycoside phosphotransferase gene (aph) from Tn903, which confers kanamycin resistance, could function as a selectable marker and that the firefly luciferase gene could be used as a reporter for gene expression. Based on this study, the firefly luciferase was subsequently used in M. paratuberculosis to determine antimicrobial susceptibilites (324).

The ability to mutate genes randomly is another requirement to study the underlying molecular genetic basis of pathogenesis in an organism. Based on the previous study, which established that M. paratuberculosis is susceptible to the bacteriophage TM4 (112), a transposon mutagenesis system utilizing phAE94 (a temperature-sensitive derivative of TM4) was developed (149). This system uses phAE94 as a vector to deliver a transposable element (Tn5367) randomly into the bacterial genome (11, 149, 233). The aph gene present in Tn5367 provides a selectable marker to distinguish mutants carrying a transposon insertion. For this study, strain K-10, a virulent bovine M. paratuberculosis isolate with a small number of in vitro passages (112), was used to generate a transposon mutant bank with phAE94 and was shown to yield a collection of approximately 5,000 mutants. Theoretically, mutants from this bank that are attenuated in virulence can be isolated for further testing in animal or cell culture model systems (149). Recently, the usefulness of the phAE94 delivery vector system was confirmed using the M. paratuberculosis strains 989 and ATCC 19698 (TMC 1613), in which approximately 2,000 mutants were generated (35). The M. paratuberculosis strain K-10 mutant collection has been recently expanded by our laboratory to a representative bank of 13,500 independent mutants necessary for a 95% theoretical coverage of the genome with transposon insertions in all nonessential genes (N. B. Harris and R. G. Barletta, unpublished data).

Animal Models

The most relevant information on host-pathogen interactions is gathered by using the natural host in infection trials. Unfortunately for Johne's disease research, the natural ruminant hosts need to be maintained under containment for 2 to 3 years before they develop clinical paratuberculosis. Therefore, the use of ruminants for research is limited to the few institutions with facilities capable of meeting these needs. Consequently, several small-animal models, including chickens (181, 302), guinea pigs (114), hamsters (114, 148, 156), mice (39, 40, 114, 187), and rabbits (114, 148, 156, 205, 206, 245), have been developed for M. paratuberculosis infections. These small-animal models except mice (see below) are somewhat limited in their use for studying M. paratuberculosis infections because they do not consistently reproduce disease symptoms in experimentally infected animals. For example, oral inoculation of rabbits (205, 206) with approximately 108 CFU of M. paratuberculosis culminated in clinical and histopathological lesions in only 62 to 75% of the animals. In contrast, 100% of calves orally inoculated with 106 CFU of M. paratuberculosis developed disease (182). Possibly the inability of M. paratuberculosis to cause disease in other animal species may be attributed to the genetic background of the animals and/or the marginal virulence of M. paratuberculosis in these species.

More extensive work has been performed on the mouse model, with some promising developments. Early researchers observed that certain inbred and outbred strains of mice, such as the C57 black and Swiss white strains (39, 40, 187), were more susceptible to M. paratuberculosis infections than was the CBA strain (39). Unfortunately, some of the earlier work on M. paratuberculosis infections in mice is of limited value because the researchers did not report which mouse strain was used (114, 148).

Resistance to mycobacterial infections in mice, including M. paratuberculosis, is associated with the Bcg locus on mouse chromosome 1 (116, 265, 266). Two allelic forms of the Bcg gene, Bcgs and Bcgr, confer susceptibility or resistance to infection, respectively. As reviewed by Blackwell and Searle (24), the Bcg gene has been identified as nramp1 (which encodes natural resistance-associated macrophage protein). A point mutation at position 169 in the amino acid sequence of the protein substitutes glycine for aspartic acid and leads to the susceptible phenotype. The Nramp1 protein is an integral membrane protein expressed in the lysosomal compartment of monocytes and macrophages and appears to function as an iron transporter. How this function influences bacterial survival is still unknown. Both C57/B6 mice (40, 306, 307) and BALB/c mice (50, 293) have the Bcgs allele and are susceptible to M. paratuberculosis infections. Conversely, the C3H/HeJ strain is resistant to M. paratuberculosis and has the Bcgr genotype (293).

Studies using C57/B6 and BALB/c mice confirm that these animals are capable of harboring 104 to 107 CFU of M. paratuberculosis per g of tissue in the liver and spleen for 6 months (50, 52, 306). Corresponding lesions typical of a mycobacterial infection are observed histologically in the mesenteric lymph node, liver, and spleen, but the intestine (ileum, cecum, and colon) does not contain lesions or acid-fast bacilli. The pattern of infection seen in BALB/c mice is somewhat atypical compared to natural infections in cattle, where the development of granulomas with acid-fast bacilli in the intestinal mucosa is common. However, liver lesions have been found in cattle naturally infected with M. paratuberculosis (27), and one report exists of a disseminated M. paratuberculosis infection in a cow (155). Nonetheless, the BALB/c mouse model may be useful for evaluating potential chemotherapeutic treatments, since Chiodini et al. (52) were able to significantly reduce bacterial counts in tissues of infected mice by prolonged rifabutin treatment.

Typical Johne's disease intestinal lesions have been induced in beige, SCID beige, and athymic nude mice. These mice have mutations that render them deficient in one or more components of the cellular immune system and thus are more susceptible to M. paratuberculosis infections, among others. BALB/c mice immunosuppressed with either cyclophosphamide or prednisolone also establish an intestinal infection following oral M. paratuberculosis inoculation (113). Beige mice are derived from C57/B6 mice and are deficient in lysosomal granules, type 2 pneumocytes and mast cells, and natural killer (NK) cells (122). Phenotypically, these deficiencies make them more susceptible to pyogenic bacterial infections. Beige mice have been utilized extensively to study M. avium infections and experimental chemotherapy in AIDS patients (21, 22, 122). When inoculated intravenously with ca. 108 CFU of M. paratuberculosis, these mice develop a disseminated infection by 2 months postinoculation, with concurrent granulomatous lesions in the liver and spleen, which are maintained for up to 12 months. Approximately 102 CFU of viable bacteria per g of tissue can be recovered from the small intestine by 1 month. This number increases to ca. 104 CFU/g of tissue by 6 months and is maintained through 12 months postinfection (276, 318). The beige mouse model has been used to demonstrate the influence of dietary calcium (Ca2+) on M. paratuberculosis survival and cytokine secretion (275, 276, 278).

Severe combined immunodeficient beige (SCID beige) mice have a dual deficit which makes them attractive to study paratuberculosis (215, 216). Similar to SCID mice, SCID beige mice lack functional T and B cells, and, like the beige mice, they have decreased numbers and activity of NK cells (83, 190). Therefore, these mice provide the means of studing innate immunity in the absence of acquired immunity. As expected, SCID beige mice are also more susceptible than BALB/c or C57B/6 mice to M. paratuberculosis. The establishment of a progressive infection is possible in these mice, with gross pathological abnormalities, intestinal granuloma formation, and the clinical symptoms of weight loss and cachexia gradually increasing in severity over time (215). The route of inoculation (oral versus intraperitoneal) also significantly affects the onset and severity of the M. paratuberculosis infection in the SCID beige mice. Intraperitoneal injections at a dose of 105 CFU cause focal lesions and the detection of acid-fast bacilli in the liver by 4 weeks postinfection. By 12 weeks after infection, the average body weight of the intraperitoneally infected animals was significantly lower than that of controls, and all infected animals demonstrated generalized muscle wasting and depletion of abdominal fat, which progressed through the entire study (26 weeks). Conversely, oral inoculation with the same dose of M. paratuberculosis caused much milder symptoms. In this group, gross lesions were observed in only 1 of 18 infected animals by 26 weeks postinoculation. Similarly, histological lesions in the liver, spleen, and intestines were not observed until 26 weeks and then were found only in four of nine mice. Only one mouse had clinical symptoms by the termination of the study. Therefore, it can be concluded that the oral infection route causes a milder form of disease in these mice when low doses of M. paratuberculosis are used.

Athymic nude gnotobiotic mice have also been used in experiments examining M. paratuberculosis pathogenesis. Since these mice lack a mature thymus, they are congenitally immunodeficient (147). Intragastric inoculation of athymic nude mice with 1010 bacteria causes a progressive and severe enteritis, consistent with naturally occurring M. paratuberculosis infections seen in ruminants (113, 146, 147). Similar to the SCID beige mouse model, a drop in body weight and increase in mortality are observed over a period of months (113). Recovery of M. paratuberculosis from feces and tissues increases to approximately 104 to 105 CFU/g of feces and 106 to 108 CFU/g of tissue by 36 weeks postinfection. Similarly, a progressive intestinal granuloma formation and increased numbers of acid-fast bacilli are observed over the same period. These results are consistent with those obtained with SCID beige mice and confirm that a functional cellular immune response is necessary to contain M. paratuberculosis infections in the host.

Immunologically chimeric mice are a recently developed animal model in which bovine fetal tissue or peripheral blood cells are engrafted into SCID mice or SCID beige mice. SCID mice are preferentially used as recipients for these xenografts since their lack of functionally mature B and T lymphocytes prevents tissue rejection. However, once reconstituted with either bovine fetal hematopoietic tissues or peripheral blood leukocytes, these mice can both produce antibodies, indicative of a functional B-cell response, and reject subsequent bovine tissue allografts, demonstrating a functional T-cell response (25, 92, 138, 139). Therefore, these mice can mimic normal bovine immune function. In the sole study analyzing the bovine immune response to mycobacterial infections with this model, Smith et al. (268) concluded that both CD8+ and γ/δT cells help protect cattle from infection with M. bovis.

In summary, more experimentation is necessary to improve the animal models to study the various aspects of Johne's disease pathogenesis. However, the mouse models currently available offer interesting alternatives for the study of different aspects of this disease. The more accurate animal model for use in the study of Johne's disease would be the one that parallels the features of ruminant paratuberculosis pathogenesis, including the protracted manifestation of clinical signs. For these studies, it is important to identify not only the appropriate genetically defined strain of mice but also the dose and route of inoculation, optimal age for infection, and frequency of repeated challenges with virulent or attenuated microorganisms. For example, an experimental model resulting in higher multiplication of M. paratuberculosis in the liver and spleen than is the ileum would indicate that the infection has become systematic, which is not a relevant model of ruminant paratuberculosis. Conditions that lead to preferential bacterial multiplication in the intestinal tract should be thoroughly explored experimentally. Nonetheless, a systemic infection model may be suitable for a preliminary examination of candidate attenuated strains, if they show a considerable decrease in virulence compared with wild-type strains. Evidently an immunologically chimeric model would be more useful for studying the response of bovine immune cells to subunit or live vaccines.

DIAGNOSTICS

Identification of M. paratuberculosis Subspecies-Specific Genes

Identification of either subspecies-specific genes or unique modifications in conserved genes (such as insertions, deletions, or hypervariable regions) of M. paratuberculosis is necessary for the development of DNA-based diagnostic tests. Two candidates reported in the literature to date besides IS900 are the hspX gene (98) and a 620-bp DNA fragment designated F57 (61). The hspX gene encodes a putative heat shock-like protein possessing an arginyl-glycyl-aspartic acid (RGD) peptide motif. Proteins with this type of motif have been implicated in mediating cell attachment (240) and stimulating phagocytosis and are likely to be virulence determinants (26, 142). BLAST analysis of hspX against M. avium and other mycobacterial genomes show that hspX has approximately 60% homology to similar sequences present in other mycobacteria, indicating that it may be unique to M. paratuberculosis. A PCR panel assay using this gene, the 16S rRNA sequence, and insertion elements IS901 (M. avium), IS1245 (M. avium complex), and IS900 (M. paratuberculosis) has been developed recently and could differentiate between M. avium and M. paratuberculosis with an accuracy of 99.2% (99). However, two of the 12 M. paratuberculosis strains tested in this study were negative for hspX by PCR. Further analysis of these two strains was not reported, so it is unknown if they were devoid of the hspX gene or if the PCR failed to amplify the appropriate gene product. Although the functional analysis of hspX regarding macrophage adhesion and entry has yet to be performed, it does appear to have immediate merit as a diagnostic tool.

Poupart et al. (241), first published the isolation and use of F57 as a diagnostic probe for detecting M. paratuberculosis. This fragment does not hybridize with DNA from other mycobacterial species and is not related to any known M. paratuberculosis sequence including IS900 (241) and hspX (98). BLAST analysis of this DNA sequence further support these data, since only 56% homology is found to a DNA region from M. avium. This F57 fragment and the upstream sequence of the gene encoding a 34-kDa antigenic protein have been used recently to develop a diagnostic duplex PCR capable of differentiating M. bovis, M. avium, and M. paratuberculosis (63). Further characterization and functional analysis of this gene fragment is necessary to ascertain its role in M. paratuberculosis pathogenesis.

Although not specific for M. paratuberculosis, a set of genes located in a 6.5-kb region of the M. paratuberculosis genome have been cloned and sequenced (301). These genes are also found in M. sylvaticum, and they have a G+C content of 58%, which is significantly lower than the 67% average for the M. paratuberculosis genome. The five ORFs within this region are gsa, gsbA, gsbB, gsc, and gsd and are homologous to genes that encode functions related to extracellular polysaccharide or lipopolysaccharide biosynthesis from M. tuberculosis and other gram-negative bacteria (301). Finally, as the subspecies-specific antigens described earlier in this review are sequenced and characterized, more unique genes of the M. paratuberculosis genome will be discovered. The sequencing and analysis of the entire M. paratuberculosis genome, currently under way at the University of Minnesota (http://www.cbc.umn.edu /ResearchProjects/Ptb/), will also uncover more regions specific to this pathogen.

Molecular Genetic-Based Diagnostic Tests

The advent of diagnostic probes based on specific bacterial DNA sequences has allowed fastidious microorganisms, such as M. paratuberculosis, to be rapidly identified. This strategy requires the identification of a DNA sequence present only in M. paratuberculosis but not in M. avium or other closely related mycobacteria. Initially, this approach was based on Southern hybridization analysis, but soon thereafter PCR tests based on these principles were developed (305). The unique regions of the M. paratuberculosis genome most widely used for PCR are the 16S rRNA gene and the insertion element IS900 (see the following section for a detailed discussion of IS900-based PCR tests). Additionally, an experimental PCR test for hspX has been recently described (99).

In general, PCR analysis has been unable to match the sensitivity of fecal culture for identifying small numbers of bacteria (319, 332), but attempts to increase the sensitivity of this diagnostic test have yielded promising results (72, 202). In part, the lack of sensitivity of the PCR technique is due to the extreme difficulty of removing PCR inhibitors when preparing DNA from fecal extracts. Englund et al. (104) combined fecal culture and PCR in an attempt to circumvent this problem. In this study, plasmid DNA containing human β-actin was incorporated into DNA extracted from primary cultures of M. paratuberculosis-suspect colonies, after which a nested PCR was performed using primers against both β-actin and the IS900 element of M. paratuberculosis. Using this method, false-negative PCR results could be identified by the lack of amplification of both β-actin and the IS900 product. However, it was still judged to be less sensitive than the culture method alone, highlighting the difficulty in removing inhibitors from primary samples. PCR has proven more useful for detecting the presence of M. paratuberculosis in infected sheep, a significant benefit since recovery of M. paratuberculosis from ovine fecal or intestinal samples is notoriously difficult (54, 71, 89). This tool is also valuable for differentiating species of M. avium. The IS900 sequence is the most commonly chosen target gene and is used to distinguish M. paratuberculosis from M. avium (209) and M. silvaticum (202), with the limitations described below.

IS900 PCR

The first commercial diagnostic PCR test for Johne's disease based on the IS900 sequence was developed by Idexx Laboratories, Inc. (305). The primers reported in that paper by Vary et al. (305) (Vary primers) are widely used in variations of the IS900 PCR test (80, 99, 196, 203). Other primers that amplify a region spanning the Vary primers have also been reported (203). The location of these primer sets in relation to the IS900 sequence is illustrated in Fig. Fig.2.2. Using these primers, Cousins et al. (80) could amplify IS900-like PCR products from environmental mycobacteria, thus demonstrating that false-positive results are obtainable from strains other than M. paratuberculosis.

FIG. 2
Sequence comparison of the 5′ ends of IS1626 and IS900. The sequences of the primer sets for M. paratuberculosis IS900 published by Vary et al. (305) (Vary primers) and Millar et. al. (202) (Millar primers) are shown in boldface type. The Millar ...

Using a variation of the primers described by Millar et al. (202), Naser et al. (218) amplified PCR products from M. avium complex (MAC) strains originating from AIDS patients. Although these PCR products hybridized with an IS900-specific probe, it was not established whether these MAC isolates contained a sequence identical to IS900 or a novel sequence with high homology to IS900. This study supports recent findings that nearly one-third of all isolates of M. avium subsp. avium recovered from human sources have readily amplifiable fragments corresponding to the IS900 insertion element when a variety of previously published IS900 primer sets were used (V. Kapur, personal communication). In addition, a BLAST analysis of the 229-bp region amplified by the Vary primers revealed that both primers overlap regions of high homology between IS900 and IS1626, present in M. avium. In particular, 23 of 24 nucleotides of the forward primer and 20 of 24 nucleotides of the reverse primer are conserved in IS1626 (Fig. (Fig.2).2). All these data indicate that PCR results based on the IS900 sequence alone should be interpreted with caution and that the IS900 primers used may not be specific for M. avium subsp. paratuberculosis, since IS900 and/or IS900-like elements may be present in other closely related mycobacteria.

A variation of the IS900 PCR has recently been introduced by Bull et al. (29), in which they developed a multiplex PCR of IS900 loci (MPIL), based on flanking genomic DNA sequences at 14 different IS900 insertion sites throughout the M. paratuberculosis genome. Since this MPIL assay is dependent on DNA flanking IS900 and since these researchers also determined that seven different IS900 loci are conserved among all M. paratuberculosis strains they tested, it is feasible that a rapid diagnostic test could be developed based on multiple IS900 loci, which would eliminate the potential for false-positive results.

Combined PCR and Restriction Enzyme Digestion Tests

Restriction enzyme analysis has been coupled with PCR to differentiate M. avium and M. paratuberculosis and to identify different strains of M. paratuberculosis. The basis for this test involves point mutations within the ORF of a highly conserved gene, which results in the loss of a restriction enzyme recognition site. When a portion of the gene is amplified using PCR and subsequently digested with the appropriate restriction enzymes, fragments of different lengths are obtained as a result of the specific polymorphisms. Eriks et al. (105) first used this technique to successfully differentiate M. paratuberculosis from M. avium by using a region of the 65-kDa heat shock protein gene. This strategy was also used with point mutations in the IS1311 mobile transposable element to differentiate M. avium from M. paratuberculosis and to differentiate cattle strains of M. paratuberculosis from sheep strains (196, 321). This principle has also been applied to IS900 PCR (80). However, given the recent findings of IS900-like sequences described above in MAC isolates, it remains to be determined whether the restriction enzyme polymorphisms of IS900 can differentiate M. paratuberculosis from all closely related mycobacteria.

In general, the latest information regarding the specificity of IS900 suggests that the search for M. paratuberculosis-specific regions useful for diagnostic purposes is unfinished. Undoubtedly, the information that will be provided by analyzing the completed M. avium and M. paratuberculosis genomes is likely to yield more conclusive answers.

Diagnostic Serology Tests

During an M. paratuberculosis infection, the animal develops both humoral and a cell-mediated immune (CMI) responses, which can be correlated with the stage of disease and the type of observed lesion. Overall, M. paratuberculosis lesions are described histopathologically as granulomatous inflammatory reactions. This classification has been further subdivided into a spectrum of histological manifestations ranging from tuberculoid (discrete) granulomas to lepromatous (diffuse) lesions (46) and is based on a similar classification first described in leprosy (250). The tuberculoid form of the disease is associated with a strong CMI response early in the course of infection (56, 78, 236). This CMI response involves the recruitment and proliferation of CD4+ Th1 cells, CD8+ cells, and peripheral blood mononuclear cells that secrete high levels of cytokines, including IFN-γ (56), which can be detected serologically (14, 234, 274, 291). Paralleling this CMI response is a strong B-cell proliferative response to M. paratuberculosis antigens (310). The lepromatous form of paratuberculosis appears in the later stages of the disease and is characterized by a decreasing CMI response, an increasing humoral response, and high antibody titers (56, 234, 263). Moreover, a transition from subclinical infection to clinical disease is accompanied by a marked antigen-specific B-cell unresponsiveness (310).

Early diagnostic testing for M. paratuberculosis was limited to the intradermal (skin) test. This test evaluates the delayed-type hypersensitivity (DTH) reaction of an animal to injected M. paratuberculosis extracts and is an indication of the CMI response of the animal (46, 78). However, problems with antigenic cross-reactivity of environmental mycobacteria have recently precluded its use as a diagnostic tool for paratuberculosis (78). Because of the importance in determining the infection status of subclinically infected animals, research in this area has led to the development of a bioassay (326) and enzyme-linked immunosorbent assay (ELISA) (255) that detects elevated IFN-γ levels in response to M. bovis infections. This test was further modified for identifying M. paratuberculosis-infected animals (23). Evaluation of the IFN-γ test by using experimentally and naturally infected cattle and sheep demonstrated its utility for distinguishing animals in the initial stages of infection (23, 234, 236, 274). However, in a recent study by McDonald et al. (197), all noninfected control animals tested positive at least once during the course of their study, suggesting that the IFN-γ ELISA may require further optimization.

Three different tests are currently available for measuring antibodies against M. paratuberculosis in the serum of infected animals. These are the complement fixation (CF) test, the agar gel immunodiffusion (AGID) test, and ELISA. Since a strong humoral response does not occur until the later stage of Johne's disease, the sensitivity of these tests is the highest for animals with lepromatous lesions (57, 235, 262), those with clinical symptoms (96, 153, 289), or those that shed large numbers of bacteria (17, 18, 270, 289) (Table (Table4).4). Therefore, the main limitation of these antibody tests is their inability to accurately identify animals early in the course of an infection. Conversely, all of these tests are highly specific, with false-positive results occurring at low frequency.

TABLE 4
Summary of M. paratuberculosis serology studies in the last decade

Among the antibody tests, ELISA is more sensitive than either the AGID or CF test (61, 78). It performs similarly for cattle, sheep, and goats (30, 97) and can be used with comparable sensitivity for either milk or serum samples (288). Comparative studies of the CF and AGID tests and ELISA repeatedly show discrepancies in the ability of these tests to identify all infected animals (84, 85, 153, 199, 270). As suggested by Sugden et al. (284), this may be due to genetic variation of the individual animal or the lack of representation of the entire range of immunodominant antigens for M. paratuberculosis within a given test.

Many variations of the ELISA method have been used to detect antibodies against M. paratuberculosis (81, 160, 288, 327). One of the crucial components of this test is the antigen preparation used to capture antibodies from the test sera. Therefore, the method of antigen preparation directly affects both the sensitivity and specificity of the ELISA (81). This was shown by an inhibition study performed with three commercially available ELISA kits, in which the M. avium strain 18 protoplasmic antigen (PPA-3) and the lipoarabinomannan polysaccharide antigen (LAM) cross-reacted but the M. paratuberculosis strain VRI 316/102-2 crude protoplasmic antigen (CTL) and LAM did not (284). The cross-reactivity between the PPA-3 and LAM antigens indicated that these tests have at least some antigens in common, but the lack of cross-reactivity between the CTL and LAM ELISAs suggest the absence of shared antigens in these preparations. Therefore, it is possible that, individually, these commercially available ELISAs can identify only a subset of an infected population of animals.

Preabsorption of test sera with M. phlei antigens is another significant modification proven to increase ELISA sensitivity (1618, 75, 81, 85, 327). This step removes nonspecific antibodies against environmental mycobacteria that could potentially cross-react with M. paratuberculosis antigens. Antigen preparation of the M. phlei absorption antigens also influences the sensitivity of the ELISA (81). As expected, the most reproducible results are obtained when the same method is used to prepare both the test antigen and antigens used for preabsorption.

DISEASE CONTROL

Natural Reservoirs

Effective disease control programs depend on addressing potential sources of infection and routes of transmission. Studies have demonstrated that M. paratuberculosis is viable for up to 250 days in water, feces, and cattle slurry (20, 179, 188). Consequently, contamination of an animal's environment by manure from infected animals is the most common mode of transmission. Vertical transmission during pregnancy has also been implicated since M. paratuberculosis has been isolated from the uterus (173, 230), fetal tissues (183), and semen (180).

Another factor affecting the control of Johne's disease is its ability to infect many different animal species. The host range of M. paratuberculosis is not limited to ruminants, since isolated reports of paratuberculosis in swine (295) and rabbits (6, 140) appear in the literature. Additionally, spontaneous cases of Johne's disease have been found in many species of wild animals. In North America, M. paratuberculosis has been isolated from white-tailed deer (44, 261), mule deer (322, 323), bighorn sheep (322, 323), Rocky Mountain goats (322, 323), bison (28), and elk (323). Similarly, M. paratuberculosis has been found in wild red deer from the Italian Alps (220) and wild rabbits in Scotland (6, 140, 141).

Management of wild-animal herds for either profit or preservation of endangered species has exacerbated certain diseases that are normally uncommon in the wild-animal population, including Johne's disease. Paratuberculosis has been diagnosed in farmed deer (90, 108, 128, 242, 294) alpacas (249), and elk (161, 194, 252). Furthermore, it appears that an infection can persist in certain populations without causing overt disease (79), which prevents both the relocation of these animals to the wild and the introduction of other livestock onto land formerly inhabited by these herds. Of greater concern is the ability of wild animals to infect domestic livestock with M. paratuberculosis. An M. paratuberculosis strain originally isolated from a bighorn sheep was able to infect other species of wild animals as well as domestic ruminants, implicating wild-animal populations as a natural reservoir for this pathogen (322, 323). In these same studies, either direct contact with diseased animals or exposure to pen effluent from diseased animals caused healthy, noninfected elk and sheep to become infected with M. paratuberculosis, confirming that transmission of M. paratuberculosis can occur via both of these routes. These data are supported by a study by Cetinkaya et al. (37), which demonstrated that the presence of farmed deer on land concurrently inhabited by dairy cattle increased the risk of Johne's disease in the dairy cattle population.

Management

Since Johne's disease is so insidious within an animal population, management is considered to be the most useful tool for controlling paratuberculosis within domestic livestock herds. However, management control recommendations often fail because they do not take into account the unique circumstances of individual farms (254). Specific management practices are reviewed in detail elsewhere (46, 254) and demonstrate the necessity for good animal husbandry. The most important management practices that have been identified are overall cleanliness of the farm, manure handling, newborn-calf care, and restriction of contact between calves and mature animals (77, 132). Other identified risk factors include the breed of cattle raised on the farm, with Guernsey and Jersey dairy cattle being associated with a higher risk factor, and the presence of farmed deer (37). The association between a high prevalence of clinical paratuberculosis and acidic soils has also been implicated as a risk factor for Johne's disease (162164, 248). However, as discussed in a recent review on this topic (162), no experimental evidence exists to corroborate this relationship.

Another critical management tool is herd testing. Presently, ELISA serology and fecal culture are the most commonly used methods (254). Because these two tests measure different aspects of the disease (indirect antibody response to infection and shedding of live bacteria), a combination of the tests is more likely to detect infected animals (254). Therefore, infected animals can be removed (culled) from a herd, reducing the exposure of uninfected animals to the disease. Statistically, a significant drawback to testing entire herds lies in the poor positive predictive value of the tests, caused by the greater likelihood of a false-positive result in a truly negative herd when large numbers of animals are tested. In contrast, diagnostic testing of individual animals with a low pretest probability of infection is correlated with a poor negative predictive value (D. R. Smith, personal communication).

Vaccination

For the dairy industry, economic losses from Johne's disease are primarily due to premature disposal of animals and reduced milk production (76, 304, 325). Therefore, a vaccine that prevents animals from becoming infected would be an ideal goal. Vaccines for paratuberculosis have been commercially available for many years but unfortunately are not completely effective in preventing disease (46, 61, 175, 304, 314). Most studies conducted on paratuberculosis vaccines have been field trials in which a herd or geographical region has experienced a high burden of Johne's disease and a vaccination strategy was implemented in an attempt to reduce economic losses. In these studies, design parameters and/or experimental conditions were not fully described or controlled. Therefore, only general conclusions can be drawn regarding the efficacy of the vaccine in preventing disease. Nonetheless, Johne's disease vaccines appear to provide partial protection, since they reduce fecal shedding in cattle (46, 175, 176), the number of clinically affected cows (46, 304, 314), sheep (82), and goats (258), and the number of animals testing positive histologically or bacteriologically (Table (Table5)5) (304, 314). This partial protection has been confirmed in more controlled experimental trials using vaccinated and experimentally challenged ruminants. Like the field trials, these studies demonstrate a significant reduction in the number and severity of intestinal lesions, the number of positive fecal cultures, and the severity of clinical symptoms in vaccinated animals (145, 166, 281). These results contrast with former studies (125, 126, 182). Thus, further clinical trials using well-defined parameters are needed to confirm the more recent positive findings of the effect of vaccination.

TABLE 5
Summary of M. paratuberculosis vaccination studies in the last decade

From the controlled experimental vaccine trials performed with ruminants with Johne's disease, certain conclusions can be drawn. Stuart (282) used dairy calves inoculated subcutaneously with 5 mg (wet weight) (ca. 5 × 104 CFU) of a live attenuated vaccine at 1 week of age and exposed 1 month later, for a duration of 6 months, to infected calves actively shedding M. paratuberculosis. A subset of the vaccinated calves were revaccinated at 1, 21/2, and 4 years after exposure. All animals were monitored for 5 years, and surviving animals were sacrificed at the termination of the study. Statistically, fewer animals in the once-vaccinated group died of Johne's disease (8 of 28) than did the nonvaccinated animals (17 of 28) or the revaccinated animals (13 of 28). However, only five cows in this group were not infected with M. paratuberculosis after 5 years, compared to three cows for the revaccinated group and one animal for the nonvaccinated group. Thus, it appears that revaccination, or boosting, does not improve the ability of an animal to resist infection. Similarly, the vaccine does not prevent infection from occurring.

Vaccination and boosting was also tested in sheep (125). In this study, animals were vaccinated subcutaneously at 4 months of age with 5 mg (wet weight) of a heat-killed strain of M. paratuberculosis. Oral challenges of these animals were performed with a virulent bovine clinical strain of M. paratuberculosis 1 month after vaccination and again by incorporating M. paratuberculosis in the drinking water for 9 to 14 months after vaccination. Revaccination was performed in a subset of sheep at 11 months with either the same heat-killed vaccine or a fractionated extract containing peptides and glycolipids from three vaccine strains of M. paratuberculosis. Animals were sacrificed at approximately 20 months of age. At this time, all animals were infected with M. paratuberculosis and no significant differences in gross lesions and numbers of viable bacteria recovered from tissues were observed between the different experimental groups, substantiating the previous experiment that vaccination does not prevent subsequent infection by a virulent strain of M. paratuberculosis.

In the sole report on oral vaccination against Johne's disease in sheep, protective immunity was not elicited (126). Three-month-old animals were dosed with 5 × 106 CFU of M. paratuberculosis strain 316F weekly for 10 weeks and challenged 1 month later with 108 CFU of a clinical M. paratuberculosis isolate per week for an additional 10 weeks. At the termination of this study (1 month after the last challenge), vaccinated sheep displayed no differences from control animals in DTH responses or the number of viable bacilli recovered from either the mesenteric lymph nodes or small intestine. Mild lesions consistent with a M. paratuberculosis infection were observed in vaccinated, nonchallenged sheep, but viable bacilli were not recovered from these tissues. The main conclusion from this study was that oral vaccination was not effective in protecting against a challenge with a pathogenic strain. However, high doses of the vaccine strain were given over several weeks, conditions that that may not elicit the correct type of protective immune response. In this context, Andersen (5) induced a dose-dependent immunity in mice with a vaccine based on short-term culture filtrate proteins of M. tuberculosis. At high vaccine doses, a nonprotective humoral response occurred, but lower doses induced a Th1 response that was protective. Another problem of the oral vaccination trial in sheep is that the study was terminated after only 1 month postchallenge. Related studies have demonstrated a significant difference in the number and severity of lesions in vaccinated animals compared with nonvaccinated animals, but only after 6 months or more (145, 166, 282).

Protective immunity against mycobacterial diseases, especially paratuberculosis in ruminants, is poorly understood. It is assumed that vaccination stimulates a protective CMI response. M. paratuberculosis directly suppresses a murine T-cell DTH response to injections of sheep red blood cells (171), implying that this may be another method used by this bacterium to evade the host immune response. As stated above, a strong CMI response to M. paratuberculosis infections is associated with the formation of granulomatous tuberculoid lesions and containment of the disease by the host whereas a weak CMI response is associated with diffuse lepromatous lesions and disease progression. Furthermore, the strength of this response can be directly correlated with the presence and severity of tuberculoid lesions and the bacterial load in the tissues (31, 145, 236). In paratuberculosis, a strong DTH response, indicative of a strong CMI response, has been correlated with the ability of an animal to contain the infection (145, 313). This situation may be correlated with some observations in human tuberculosis, where a vaccine-induced low level of hypersensitivity was associated with protection but a persistent vaccine-associated hypersensitivity was not (111). Several groups have demonstrated the association of tuberculoid-type lesions with the elimination of an M. paratuberculosis infection in both sheep (145, 166, 236) and mice (50). Therefore, it can be hypothesized that the Johne's disease vaccines stimulate an initial strong cellular immune response, partially overcoming the suppressive effect of pathogenic M. paratuberculosis on the host immune system. To this end, vaccinated animals have higher IFN-γ and serum antibody responses than do naturally infected animals (145, 166).

In the United States, vaccination is still considered to be a controversial management tool (76). In general, vaccination can interfere with diagnostic testing, can cause observable granulomas at the vaccination site, and may present a health risk to veterinarians administering the vaccine due to accidental self-injection (226). However, a study in The Netherlands showed that vaccination against paratuberculosis was profitable for herd owners, reducing the number of clinically infected cattle by approximately 90% (304). It should be noted that in the United States, heat-killed M. avium strain 18 is used, while in Europe, a modified live vaccine is marketed. As reviewed previously (46, 61), both live attenuated and killed whole-cell (bacterin) vaccines are somewhat successful in controlling clinical paratuberculosis. Table Table55 lists studies from the last 10 years that have used either the live attenuated (82, 145, 166, 197, 207) or killed (174, 175, 197, 271, 304, 313, 314) M. paratuberculosis or M. avium vaccines. Comprehensive studies on the most effective strain(s) of M. paratuberculosis for use in vaccine production are also needed. Since various strains of M. bovis BCG differ in their ability to elicit experimentally induced immune reactions (134), it is possible that a similar phenomenon may occur with M. paratuberculosis. A number of M. paratuberculosis strains have been reported in the literature as being used for both experimental and commercial vaccine production (Table (Table5).5). These include strain 316F (61, 82, 166), strain 5889 Bergey (175), M. avium strain 18 (46, 271), field isolates (197), and other unidentified reference strains (304, 313, 314).

Research on M. tuberculosis secreted antigens suggests that a subunit vaccine may be developed against mycobacterial infections. For example, Horwitz et al. (158) have demonstrated the potential use of M. tuberculosis antigen 85B as a subunit vaccine against tuberculosis. A similar approach may be feasible for M. paratuberculosis, and a subunit vaccine of this kind would be readily compatible with current diagnostic tests. This type of vaccine may benefit from the use of an immunoadjuvant, such as the genetically detoxified derivative of either the Escherichia coli heat-labile enterotoxin or the closely related Vibrio cholerae cholera toxin. Mutant heat-labile enterotoxin and cholera toxin both elicit a protective humoral and cellular host immune response when used as a mucosal adjuvant in conjunction with killed bacteria (117), suggesting new possibilities for vaccine development for M. paratuberculosis.

It would be extremely useful to develop methods to differentiate vaccinated from infected animals. The time frame for a vaccinated animal to demonstrate detectable antibody levels can vary from 2 weeks to 6 months after vaccination (166, 271). Once vaccinated, animals appear to maintain these levels for at least 1 year (271) and up to 3 years (174, 175) afterward, which interferes with the assays currently used for diagnostic purposes and export requirements. Although the development of a live attenuated vaccine compatible with diagnostics is difficult, Cirillo et al. (55) have described a recombinant DNA technology approach to generate this type of vaccine. It is our conviction that either this type of a recombinant live attenuated vaccine or a subunit vaccine coupled with the proper use of adjuvants has the greater promise for a more effective vaccine against paratuberculosis.

Treatment

Presently, no drugs are approved for the treatment of Johne's disease in livestock, and the rare instances where antibiotic therapy is attempted is limited to the extralabel use of standard antimicrobial agents. Since treatment of paratuberculosis is expensive and unrewarding, it is only used in extreme efforts to prolong the life of very valuable animals for breeding purposes. The treatment regimens most commonly used for Johne's disease are either clofazimine (61), or isoniazid and either rifabutin or ethambutol, followed by a daily dose of isoniazid for the duration of the animal's life (124, 281). Although isoniazid is prescribed for the treatment of M. tuberculosis and M. bovis infections in cattle (243), both M. paratuberculosis and M. avium are resistant to isoniazid in vitro (157, 201, 324), and therefore it may not be effective in vivo.

M. paratuberculosis is susceptible to many antibiotics in vitro. These include D-cycloserine (45, 324), ethambutol (49, 246, 324), amikacin (49, 324), clarithromycin (246, 324), and rifabutin (324). The fluoroquinolone classes of antibiotics may also merit further investigation, since the experimental fluoroquinolone Bay y 3118 is highly effective against M. paratuberculosis (324). It has been suggested that antibiotic therapy does not result in a complete cure, possibly due to the inaccessibility of mycobacteria to the drugs in vivo (61). Alternatively, some of the drugs used in the in vivo studies (e.g., isoniazid) may not have been the most appropriate choices for treatment, since the corresponding in vitro drug susceptibility patterns of the M. paratuberculosis strains causing the infection were not reported. Progress in the development of antibiotics to treat paratuberculosis infections will require the rational design of studies that identify appropriate bacterial targets, utilize drugs that are capable of penetrating macrophages, and focus on cost-effective chemotherapies that are approved for use in food animals. Until these considerations are addressed, chemotherapy will not be a practical alternative for treatment of Johne's disease.

RESEARCH PRIORITIES AND CONCLUSION

The knowledge of how M. paratuberculosis causes disease still lags behind that for other pathogenic mycobacteria. Like other pathogens, the interactions between M. paratuberculosis and its host are complex. The progression of our understanding of the basic physiology of M. paratuberculosis is hampered by the difficulty of manipulating it in a laboratory setting. However, there is hope that as more of the M. paratuberculosis genome is sequenced and characterized, differences in the genetic structure and physiology between M. paratuberculosis and other mycobacteria will become evident. This will expand our understanding of what makes M. paratuberculosis unique and will further our repertoire of specific targets that can be exploited for diagnostic testing and treatment of Johne's disease. However, there are important gaps in this knowledge. Regarding the basic biology of the microorganism, it would be most important to determine the molecular basis of mycobactin dependency, which is the most prominent diagnostic feature of the subspecies paratuberculosis. Another important aspect would be to ascertain the role of M. paratuberculosis species-specific genes encoding virulence determinants associated with macrophage survival, host range, tissue specificity, and pathogenesis. In addition, to develop new live attenuated vaccines, it would be important to define mutations in virulence determinants or housekeeping functions, leading to an attenuated phenotype. In this context, a functional genomic approach based on the screening of mutant banks in combination with microarray technology offers the greatest promise.

These microbial studies will need to be complemented with studies using appropriate in vitro and in vivo systems. The in vitro models for Johne's disease would include the use of bovine cell lines, such as the Madin-Darby bovine kidney (MDBK) epithelial cell line (American Type Culture Collection), which could be useful to study the interaction of M. paratuberculosis with the mucosal epithelium, and the BoMac cell line, which could be used in the study of macrophage survival. Experiments performed with these cell lines may have to be validated with primary epithelial and macrophage cells. There are advantages to each of these approaches, as well as some distinctive differences between the two. Primary cells are untransformed, but they vary in phenotypic properties from one donor to the next. The cell lines will offer consistent results on a day-to-day basis, but since they are transformed, they may not accurately represent the mature primary cells in culture. In particular, epithelial cells are highly differentiated in vivo, unlike the MDBK cells. Therefore, the calf ileal loop assay, which has been successfully used to study Salmonella enterica serovar Typhimurium interactions with the cells of the mucosal epithelium (119), may be valuable for similar studies of M. paratuberculosis. In the immune response of the ruminant host, it would be important to elucidate the various immune cell interactions, such as the potential regulatory effects of γ/δ T cells on CD8+ cell function (51), found in greatest proportions in the neonatal calf (150). This issue is also important in the context of eliciting a protective immune response by vaccination and could have bearing on the future development of more effective live attenuated or subunit vaccines.

Since ruminant hosts require specialized containment facilities and are expensive to maintain, prior research needs to be performed to establish the more suitable small-animal model that best parallels Johne's disease. It seems that transgenic and immunologically chimeric mice offer the greatest possibilities for meaningful experiments. These models would be important to test potential vaccine strains and the elicitation of protective immunity prior to conducting controlled experiments in ruminants and, finally, large-scale field trials. The most rigorously designed parameters must be established prior to conducting these field trials so that accurate and meaningful data are obtained.

The potential role of M. paratuberculosis in the etiology of Crohn's disease, or its potential to serve as an opportunistic pathogen in patients with inflammatory bowel disease, deserves substantial future investigation. In this context, the recommendations of a panel from a workshop conducted at the National Institutes of Health (http://www.niaid.nth.gov/dmid /meetings/crohns) have established several research priorities to address potential M. paratuberculosis infections of humans. These guidelines parallel the recommendations on Johne's disease research provided above and include the establishment of cell or organ culture models, new small- and large-animal models of infections, and basic aspects of gene expression and differential genomics. In addition, the panel recommended to optimize diagnostics of M. paratuberculosis antigens, and drug susceptibility testing. Some progress has been made in this context, but substantial funding from this agency would be required to meet these goals in a timely fashion. In conclusion, progress on the understanding of M. paratuberculosis infections will require a combination of molecular genetic studies with appropriate in vitro and in vivo models to evaluate pathogenesis.

ACKNOWLEDGMENTS

Research in our laboratory is supported by NRI CGP-USDA grant 99-35204-7789 and BARD-USDA grant IS2564-95C. Support for N.B.H. was provided by the Department of Veterinary and Biomedical Sciences at the University of Nebraska, Lincoln.

We acknowledge Z. Feng for assistance with computer analysis and graphics preparation. Additionally, we thank our associates N. E. Cáceres, O. Chacon, Z. Feng, X. Liu, J. Zabaleta, and D. Zinniel for their helpful disscussions. J. Bannantine, H. Bercovier, L. E. Bermudez, J. D. Cirillo, C. Czuprynski, R. Donis, V. Kapur, R. A. Moxley, D. Smith, and D. J. Steffen are gratefully acknowledged for their critical review of the manuscript, and J. Hermon-Taylor and T. J. Bull are acknowledged for their insightful comments on M. paratuberculosis.

REFERENCES

1. Adams J L, Czuprynski C J. Mycobacterial cell wall components induce the production of TNF-α, IL-1, and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7. Microb Pathog. 1994;16:401–411. [PubMed]
2. Adams J L, Czuprynski C J. Ex vivo induction of TNF-α and IL-6 mRNA in bovine whole blood by Mycobacterium paratuberculosis and mycobacterial cell wall components. Microb Pathog. 1995;19:19–29. [PubMed]
3. Adams J L, Collins M T, Czuprynski C J. Polymerase chain reaction analysis of TNF-α and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis. Can J Vet Res. 1996;60:257–262. [PMC free article] [PubMed]
4. Andersen P, Askgaard D, Ljunqvist L, Bennedsen J, Heron I. Proteins released from Mycobacterium tuberculosis during growth. Infect Immun. 1991;59:1905–1910. [PMC free article] [PubMed]
5. Andersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun. 1994;62:2536–2544. [PMC free article] [PubMed]
6. Angus K W. Intestinal lesions resembling paratuberculosis in a wild rabbit (Oryctolagus cuniculus) J Comp Pathol. 1990;103:103–105. [PubMed]
7. Baess I. Deoxyribonucleic acid relationships between different serovars of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum. Acta Pathol Microbiol Immunol Scand. 1983;91:201–203. [PubMed]
8. Balasubramanian V, Pavelka M S, Jr, Bardarov S S, Martin J, Weisbrod T R, McAdam R A, Bloom B R, Jacobs W R., Jr Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol. 1996;178:273–279. [PMC free article] [PubMed]
9. Bannantine J P, Barletta R G, Thoen C O, Andrews R E., Jr Identification of Mycobacterium paratuberculosis gene expression signals. Microbiology. 1997;143:921–928. [PubMed]
10. Barclay R, Ratledge C. Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium. J Bacteriol. 1983;153:1138–1146. [PMC free article] [PubMed]
11. Bardarov S, Kriakov J, Carriere C, Yu S, Vaamonde C, McAdam R A, Bloom B R, Hatfull G F, Jacobs W R., Jr Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 1997;94:10961–10966. [PMC free article] [PubMed]
12. Bashyam M D, Kaushai D, Dasgupta S K, Tyagi A K. A study of the mycobacterial transcriptional apparatus: identification of novel features in promoter elements. J Bacteriol. 1996;178:4847–4853. [PMC free article] [PubMed]
13. Bashyam M D, Tyagi A K. Identification and analysis of “extended −10” promoters from mycobacteria. J Bacteriol. 1998;180:2568–2573. [PMC free article] [PubMed]
14. Bassey E O E, Collins M T. Study of T-lymphocyte subsets of healthy and Mycobacterium avium subsp. paratuberculosis-infected cattle. Infect Immun. 1997;65:4869–4872. [PMC free article] [PubMed]
15. Bauerfeind R, Benazzi S, Weiss R, Schliesser T, Willems H, Balger G. Molecular characterization of Mycobacterium paratuberculosis isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900. J Clin Microbiol. 1996;34:1617–1621. [PMC free article] [PubMed]
16. Bech-Nielsen S, Jorgensen J B, Ahrens P, Feld N C. Diagnostic accuracy of a Mycobacterium phlei-absorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows. J Clin Microbiol. 1992;30:613–618. [PMC free article] [PubMed]
17. Bech-Nielsen S, Shulaw W P, Frandsen P L, Berg-Jorgensen J, Ahrens P, Feld N C. Use of a dot enzyme-linked immunosorbent assay on absorbed sera for the diagnosis of bovine paratuberculosis. Rev Sci Technol. 1992;12:617–627. [PubMed]
18. Bech-Nielsen S, Shulaw W P, Frandsen P L, Jorgensen J B, Feld N C, Ahrens P. Comparison of subjective and objective test evaluations for use of Mycobacterium phlei-absorbed serum in a dot-enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in cattle. Am J Vet Res. 1993;53:1386–1391. [PubMed]
19. Bercovier H, Kafri O, Sela S. Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. Biochem Biophys Res Commun. 1986;136:1136–41. [PubMed]
20. Berg-Jorgensen J. Survival of Mycobacterium paratuberculosis in slurry. Nord Vet Med. 1977;29:267–270. [PubMed]
21. Bermudez L E, Kolonoski P, Young L S. Natural killer cell activity and macrophage-dependent inhibition of growth or killing of Mycobacterium avium complex in a mouse model. J Leukoc Biol. 1990;47:135–141. [PubMed]
22. Bermudez L E, Petrofsky M, Kolonski P, Young L S. An animal model of Mycobacterium avium complex disseminated infection after colonization of the intestinal tract. J Infect Dis. 1992;165:75–79. [PubMed]
23. Billman-Jacobe H, Carrigan M, Cockram F, Corner L A, Gill I J, Hill J F, Jessep T, Milner A R, Wood P R. A comparison of the interferon gamma assay with the absorbed ELISA for the diagnosis of Johne's disease in cattle. Aust Vet J. 1992;69:25–28. [PubMed]
24. Blackwell J M, Searle S. Genetic regulation of macrophage activation: understanding the function of Nramp1 (= Ity/Lsh/Bcg) Immunol Lett. 1999;65:73–80. [PubMed]
25. Boermans H J, Percy D H, Stirtzinger T, Croy B A. Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues. Vet Immunol Immunopathol. 1992;34:273–289. [PubMed]
26. Brown E J, Goodwin J L. Fibronectin receptors of phagocytosis: characterization of the Arg-Gly-Asp binding proteins of human monocytes and polymorphonuclear leukocytes. J Exp Med. 1988;167:777–793. [PMC free article] [PubMed]
27. Buergelt C D, Hall C, McEntee K, Duncan J R. Pathological evaluation of paratuberculosis in naturally infected cattle. Vet Pathol. 1978;15:196–207. [PubMed]
28. Buergelt C D, Layton A W, ginn P E, Taylor M, King J M, Habecker P L, Mauldin E, Whitlock R, Rossiter C, Collins M T. The pathology of spontaneous paratuberculosis in the North American bison (Bison bison) Vet Pathol. 2000;37:428–438. [PubMed]
29. Bull T J, Hermon-Taylor J, Pavlik I, El-Zaatari F, Tizard M. Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing. Microbiology. 2000;146:2185–2197. [PubMed]
30. Burnside D M, Rowley B O. Evaluation of an enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in goats. Am J Vet Res. 1994;55:465–466. [PubMed]
31. Burrells C, Clarke C J, Colston A, Kay J M, Porter J, Little D, Sharp J M. A study of immunological responses of sheep clinically affected with paratuberculosis (Johne's disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology. Vet Immunol Immunopathol. 1998;66:343–358. [PubMed]
32. Byrd T F, Horwitz M A. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J Clin Investig. 1989;83:1457–1465. [PMC free article] [PubMed]
33. Cameron R M, Stevenson K, Inglis N F, Kalusen J, Sharp J M. Identification and characterization of a putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis. Microbiology. 1994;140:1977–1982. [PubMed]
34. Canonne-Hergaux F, Gruenheid S, Govoni G, Gros P. The Nramp1 protein and its role in resistance to infection and macrophage function. Proc Assoc Am Physicians. 1999;111:283–289. [PubMed]
35. Cavaignac S M, White S J, de Lisle G W, Collins D M. Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries. Arch Microbiol. 2000;173:229–231. [PubMed]
36. Cellier C, De Beenhouwer H, Berger A, Penna C, Carbonnel F, Parc R, Cugnenc P H, Le Quintrec Y, Gendre J P, Barbier J P, Portaels F. Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum DNA cannot be detected by PCR in Crohn's disease tissue. Gastroenterol Clin Biol. 1998;22:675–678. [PubMed]
37. Cetinkaya B, Erdogan H M, Morgan K L. Relationships between the presence of Johne's disease and farm and management factors in dairy cattle in England Prev. Vet Med. 1997;32:253–266. [PubMed]
38. Chan J, Xing Y, Magliozzo R S, Bloom B R. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med. 1992;175:1111–1122. [PMC free article] [PubMed]
39. Chandler R L. Infection of laboratory animals with Mycobacterium johnei. IV. Comparative susceptibility to infection of C.57, C.B.A. and Swiss white mice. J Comp Pathol. 1961;71:233–242. [PubMed]
40. Chandler R L. Infection of laboratory animals with Mycobacterium johnei. V. Further studies on the comparative susceptibility of C57 black mice. J Comp Pathol. 1962;72:198–213. [PubMed]
41. Chen L, Xie Q W, Nathan C. Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates. Mol Cell. 1998;1:795–805. [PubMed]
42. Chenuse S W, Warmingon K S, Berger A E, Tracey D E. Immunohistochemical demonstration of interleukin-1 receptor antagonist protein and interleukin-1 in human lymphoid tissue and granulomas. Am J Pathol. 1992;140:269–275. [PMC free article] [PubMed]
43. Chiba M, Fukushima T, Horie Y, Iizuka M, Masamune O. No Mycobacterium paratuberculosis detected in intestinal tissue, including Peyer's patches and lymph follicles, of Crohn's disease. J Gastroenterol. 1998;33:482–487. [PubMed]
44. Chiodini R J, Van Kruiningen H J. Eastern white-tailed deer as a reservoir of ruminant paratuberculosis. J Am Vet Med Assoc. 1983;182:168–169. [PubMed]
45. Chiodini R J, Van Kruiningen H J, Merkal R S, Thayer W R, Coutu J A. Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. J Clin Microbiol. 1984;20:966–971. [PMC free article] [PubMed]
46. Chiodini R J, Van Kruiningen H J, Merkal R S. Ruminant paratuberculosis Johne's disease: the current status and future prospects. Cornell Vet. 1984;74:218–262. [PubMed]
47. Chiodini R J. The genetic relationship between Mycobacterium paratuberculosis and the M. avium complex. Acta Leprol. 1989;7:249–251. [PubMed]
48. Chiodini R J. Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region. J Clin Microbiol. 1990;28:489–494. [PMC free article] [PubMed]
49. Chiodini R J. Bactericidal activities of various antimicrobial agents against human and animal isolates of Mycobacterium paratuberculosis. Antimicrob Agents Chemother. 1990;34:366–367. [PMC free article] [PubMed]
50. Chiodini R J, Buergelt C D. Susceptibility of Balb/c C57/B6 and C57/B10 mice to infection with Mycobacterium paratuberculosis. J Comp Pathol. 1993;109:309–319. [PubMed]
51. Chiodini R J, Davis W C. The cellular immunology of bovine paratuberculosis: immunity may be regulated by CD4+ helper and CD8+ immunoregulatory T lymphocytes which down-regulate γ/δ+ T-cell cytotoxicity. Microb Pathog. 1993;14:355–347. [PubMed]
52. Chiodini R J, Kreeger J M, Thayer W R. Use of rifabutin in treatment of systemic Mycobacterium paratuberculosis infection in mice. Antimicrob Agents Chemother. 1993;37:1645–1648. [PMC free article] [PubMed]
53. Chiodini R J. M. paratuberculosis in foods and the public health implications. In: Chiodini R J, Hines M E Jr, Collins M T, editors. Fifth Colloquium on Paratuberculosis. Rehoboth, Mass: International Association for Paratuberculosis, Inc.; 1997. pp. 353–365.
54. Choy E, Whittington R J, Marsh I, Marshall J, Campbell M T. A method for purification and characterisation of Mycobacterium avium subsp. paratuberculosis from the intestinal mucosa of sheep with Johne's disease. Vet Microbiol. 1998;64:51–60. [PubMed]
55. Cirillo J D, Stover C K, Bloom B R, Jacobs W R, Jr, Barletta R G. Bacterial vaccine vectors and bacillus Calmette-Guerin. Clin Infect Dis. 1995;20:1001–1009. [PubMed]
56. Clarke C J. Host responses to Mycobacterium paratuberculosis/M. avium infection. In: Chiodini R J, Collins M T, Bassey E O, editors. Proceedings of the 4th International Colloquium on Paratuberculosis. East Providence, R.I: International Association for Paratuberculosis, Inc.; 1994. pp. 345–354.
57. Clarke C J, Patterson I A P, Armstrong K E, Low J C. Comparison of the absorbed ELISA and agar gel immunodiffusion test with clinicopathological findings in ovine clinical paratuberculosis. Vet Rec. 1996;139:618–621. [PubMed]
58. Clarkston W K, Presti M E, Petersen P F, Zachary P E, Jr, Fan W X, Leonardi C L, Vernava III A M, Longo W E, Kreeger J M. Role of Mycobacterium paratuberculosis in Crohn's disease: a prospective, controlled study using polymerase chain reaction. Dis Colon Rectum. 1998;41:195–199. [PubMed]
59. Clemens D L, Horwitz M A. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med. 1995;181:257–270. [PMC free article] [PubMed]
60. Cobb A J, Frothingham R. The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis. Vet Microbiol. 1999;67:31–35. [PubMed]
61. Cocito C, Gilot P, Coene M, de Kesel M, Poupart P, Vannuffel P. Paratuberculosis. Clin Microbiol Rev. 1994;7:328–345. [PMC free article] [PubMed]
62. Coetsier C, Havaux X, Mattelard F, Sadatte S, Cormont F, Buergelt K, Limbourg B, Latinne D, Bazin H, Denef J F, Cocito C. Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures. Clin Diagn Lab Immunol. 1998;5:446–451. [PMC free article] [PubMed]
63. Coetsier C, Vannuffel P, Blondeel N, Denef J F, Cocito C, Gala J L. Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues from cattle. J Clin Microbiol. 2000;38:3048–3054. [PMC free article] [PubMed]
64. Coffin J W, Condon C, Compston C A, Potter K N, Lamontagne L R, Shafiq J, Kunimoto D Y. Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes. J Clin Microbiol. 1992;30:1829–1836. [PMC free article] [PubMed]
65. Cole S T, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon S V, Eiglmeier K, Gas S, Barry III C E, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Barrell B G, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998;393:537–544. [PubMed]
66. Collins D M, de Lisle G W. DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG. J Gen Microbiol. 1984;130:1019–1021. [PubMed]
67. Collins D M, de Lisle G W. Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle. Am J Vet Res. 1986;47:2226–2229. [PubMed]
68. Collins D M, Gabric D M, de Lisle G W. Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis. FEMS Microbiol Lett. 1989;60:175–178. [PubMed]
69. Collins D M, Gabric D M, de Lisle G W. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J Clin Microbiol. 1990;28:1591–1596. [PMC free article] [PubMed]
70. Collins D M, Stephens D M. Identification of an insertion sequence, IS1081, in Mycobacterium bovis. FEMS Microbiol Lett. 1991;67:11–15. [PubMed]
71. Collins D M, Hilbink F, West D M, Hosie B D, Cooke M M, de Lisle G W. Investigation of Mycobacterium paratuberculosis in sheep by faecal culture, DNA characterisation and polymerase chain reaction. Vet Rec. 1993;133:599–600. [PubMed]
72. Collins D M, Stephens D M, de Lisle G W. Comparison of polymerase chain reaction tests and faecal culture for detecting Mycobacterium paratuberculosis in bovine faeces. Vet Microbiol. 1993;36:289–299. [PubMed]
73. Collins D M, Kawakami R P, de Lisle G W, Pascopella L, Bloom B R, Jacobs W R., Jr Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA. 1995;92:8036–8040. [PMC free article] [PubMed]
74. Collins D M, Cavignac S, de Lisle G W. Use of four DNA insertion sequences to characterize strains of the Mycobacterium avium complex isolated from animals. Mol Cell Probes. 1997;11:373–380. [PubMed]
75. Collins M T, Sockett D C. Accuracy and economics of the USDA-licensed enzyme-linked immunosorbent assay for bovine paratuberculosis. J Am Vet Med Assoc. 1993;203:1456–1463. [PubMed]
76. Collins M T. Clinical approach to control of bovine paratuberculosis. J Am Vet Med Assoc. 1994;204:208–210. [PubMed]
77. Collins M T, Sockett D C, Googdger W J, Conrad T A, Thomas C B, Carr D J. Herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in Wisconsin. J Am Vet Med Assoc. 1994;204:636–641. [PubMed]
78. Collins M T. Diagnosis of paratuberculosis. Vet Clin North Am Food Anim Pract. 1996;12:357–371. [PubMed]
79. Cook W E, Cornish T E, Shideler S, Lasley B, Collins M T. Radiometric culture of Mycobacterium avium paratuberculosis from the feces of tule elk. J Wildp Dis. 1997;33:635–637. [PubMed]
80. Cousins D V, Whittington R, Marsh I, Masters A, Evans R J, Kluver P. Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable by IS900 polymerase chain reaction: implications for diagnosis. Mol Cell Probes. 1999;13:431–442. [PubMed]
81. Cox J C, Drane D P, Jones S L, Ridge S, Milner A R. Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne's disease in cattle. Aust Vet J. 1991;68:157–160. [PubMed]
82. Cranwell M P. Control of Johne's disease in a flock of sheep by vaccination. Vet Rec. 1993;133:219–220. [PubMed]
83. Croy B A, Chapeau C. Evaluation of the pregnancy immunotrophism hypothesis by assessment of the reproductive performance of young adult mice of genotype scid/scid.bg/bg. J Reprod Fertil. 1990;88:231–239. [PubMed]
84. Darcel C. Diagnosis of infection with Mycobacterium paratuberculosis. Can Vet J. 1995;36:199–200. [PMC free article] [PubMed]
85. Darcel C, Logan-Handsaeme B. ELISA testing for antibody to Mycobacterium paratuberculosis. Can Vet J. 1998;39:335–336. [PMC free article] [PubMed]
86. Davis E O, Jenner P J, Brooks P C, Colston M J, Sedgwick S G. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell. 1992;71:201–210. [PubMed]
87. De Kesel M, Gilot P, Coene M, Cocito C. Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis. Scand J Immunol. 1992;36:201–212. [PubMed]
88. De Kesel M, Gilot P, Misonne M C, Coene M, Cocito C. Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease. J Clin Microbiol. 1993;31:947–954. [PMC free article] [PubMed]
89. de Lisle G W, Collins D M, Huchzermeyer H F A K. Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization. Onderstepoort J Vet Res. 1992;59:163–165. [PubMed]
90. de Lisle G W, Yates G F, Collins D M. Paratuberculosis in farmed deer case reports and DNA characterization of isolates of Mycobacterium paratuberculosis. J Vet Diagn Investig. 1993;5:567–571. [PubMed]
91. Dellagostin O A, Wall S, Norman E, O'Shaughnessy T, Dale J W, McFadden J J. Construction and use of integrative vectors to express foreign genes in mycobacteria. Mol Microbiol. 1993;10:983–993. [PubMed]
92. Denny S L, Brackett N Y, Mann F A, Kreeger J M, Estes D M. Antigen-specific humoral immune responses to a hapten-carrier conjugate in SCID mice engrafted with bovine fetal hematopoietic tissues. Lab Anim Sci. 1996;46:48–55. [PubMed]
93. De Voss J J, Rutter K, Schroeder B G, Su H, Zhu Y, Barry C E., III The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc Natl Acad Sci USA. 2000;97:1252–1257. [PMC free article] [PubMed]
94. Doran T J, Davies J K, Radford A J, Hodgson A L M. Putative functional domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902. Immunol Cell Biol. 1994;72:427–434. [PubMed]
95. Doran T, Tizard M, Millar D, Ford J, Sumar N, Loughlin M, Hermon-Taylor J. IS900 targets translation initiation signals in Mycobacterium avium subsp. paratuberculosis to facilitate expression of its hed gene. Microbiology. 1997;143:547–4552. [PubMed]
96. Dubash K, Shulaw W P, Bech-Nielsen S, Stills H F, Jr, Slemons R D. Evaluation of an enzyme-linked immunosorbent assay licensed by the USDA for use in cattle for diagnosis of ovine paratuberculosis. J Vet Diagn Investig. 1995;7:347–351. [PubMed]
97. Dubash K, Shulaw W P, Bech-Nielsen S, Stills H F, Jr, Slemons R D. Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep. J Am Vet Med Assoc. 1996;208:401–403. [PubMed]
98. Ellingson J L E, Bolin C A, Stabel J R. Identification of a gene unique to Mycobacterium avium subspecies paratuberculosis and application to diagnosis of paratuberculosis. Mol Cell Probes. 1998;12:133–142. [PubMed]
99. Ellingson J L E, Stabel J R, Bishai W R, Frothingham R, Miller J M. Evaluation of the accuracy and reproducibility of a practical PCR panel assay for rapid detection and differentiation of Mycobacterium avium species. Mol Cell Probes. 2000;14:153–161. [PubMed]
100. El-Zaatari F A K, Naser S A, Engstrand L, Hachem C Y, Graham D Y. Identification and characterization of Mycobacterium paratuberculosis recombinant proteins expressed in E. coli. Curr Microbiol. 1994;29:177–184. [PubMed]
101. El-Zaatari F A K, Naser S A, Engstrand L, Burch P E, Hachem C Y, Whipple D L, Graham D Y. Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis. Clin Diagn Lab Immunol. 1995;2:657–664. [PMC free article] [PubMed]
102. El-Zaatari F A K, Naser S A, Graham D Y. Characterization of a specific Mycobacterium paratuberculosis recombinant clone expressing 35,000-molecular-weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease. J Clin Microbiol. 1997;35:1794–1799. [PMC free article] [PubMed]
103. England P M, Wall S, McFadden J. IS900-promoted stable integration of a foreign gene into mycobacteria. Mol Microbiol. 1991;5:2047–2052. [PubMed]
104. Englund S, Ballagi-Pordány A, Bölske G, Johansson K E. Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagn Microbiol Infect Dis. 1999;33:163–171. [PubMed]
105. Eriks I S, Munck K T, Besser T E, Cantor G H, Kapur V. Rapid differentiation of Mycobacterium avium and M. paratuberculosis by PCR and restriction enzyme analysis. J Clin Microbiol. 1996;34:734–737. [PMC free article] [PubMed]
106. Falkingham J O, Crawford J T. Plasmids. In: Bloom B R, editor. Tuberculosis. Washington, D.C.: ASM Press; 1994. pp. 188–190.
107. Fang Z, Doig C, Morrison N, Watt B, Forbes K J. Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110. J Bacteriol. 1999;181:1021–1024. [PMC free article] [PubMed]
108. Fawcett A R, Goddard P J, McKelvey W A C, Buxton D, Reid H W, Greig A, Macdonald A J. Johne's disease in a herd of farmed red deer. Vet Rec. 1995;136:165–169. [PubMed]
109. Feola R P, Collins M T, Czuprynski C J. Hormonal modulation of phagocytosis and intracellular growth of Mycobacterium avium ss. paratuberculosis in bovine peripheral blood monocytes. Microb Pathog. 1999;26:1–11. [PubMed]
110. Fidler H M, Thurrell W, Johnson N M, Rook G A, McFadden J J. Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn's disease. Gut. 1994;35:506–510. [PMC free article] [PubMed]
111. Fine P E M, Sterne A A C, Ponnighaus J M, Rees R J W. Delayed-type hypersensitivity, mycobacterial vaccines and protective immunity. Lancet. 1994;344:1245–1249. [PubMed]
112. Foley-Thomas E M, Whipple D L, Bermudez L E, Barletta R G. Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology. 1995;141:1173–1181. [PubMed]
113. Follett D M, Czuprynski C J. Cyclophosphamide and prednisolone exacerbate the severity of intestinal paratuberculosis in Mycobacterium paratuberculosis monoassociated mice. Microb Pathog. 1990;9:407–415. [PubMed]
114. Francis J. Infection of laboratory animals with Mycobacterium johnei. J Comp Pathol. 1943;53:140–150.
115. François B, Krishnamoorthy R, Elion J. Comparative study of Mycobacterium paratuberculosis strains isolated from Crohn's disease and Johne's disease using restriction fragment length polymorphism and arbitrarily primed polymerase chain reaction. Epidemiol Infect. 1997;118:227–233. [PMC free article] [PubMed]
116. Frelier P F, Templetion J W, Estes M, Whitford H W, Kienle R D. Genetic regulation of Mycobacterium paratuberculosis infection in recombinant inbred mice. Vet Pathol. 1990;27:362–364. [PubMed]
117. Freytag L C, Clements J D. Bacterial toxins as mucosal adjuvants. Curr Top Microbiol Immunol. 1999;236:215–236. [PubMed]
118. Frischkorn K, Sander P, Scholz M, Teschner K, Prammananan T, Bottger E C. Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination. Mol Microbiol. 1998;29:1203–1214. [PubMed]
119. Frost A J, Bland A P, Wallis T S. The early dynamic response of the calf ileal epithelium to Salmonella typhimurium. Vet Pathol. 1997;34:369–386. [PubMed]
120. Frothingham R. Evolutionary bottlenecks in the agents of tuberculosis, leprosy, and paratuberculosis. Med Hypotheses. 1999;52:95–99. [PubMed]
121. Fujimura Y, Owen R L. M cells as portals of infection: clinical and pathophysiological aspects. Infect Agents Dis. 1996;5:144–156. [PubMed]
122. Gangadharam P R J. Beige mouse model for Mycobacterium avium complex disease. Antimicrob Agents Chemother. 1995;39:1647–1654. [PMC free article] [PubMed]
123. Georgopoulos C, Ang D, Liberek K, Zylicz M. Stress proteins in biology and medicine. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1990.
124. Gezon H M, Bither H D, Gibbs H C, Acker E J, Hanson L A, Thompson J K, Jorgenson R D. Identification and control of paratuberculosis in a large goat herd. Am J Vet Res. 1988;49:1817–1823. [PubMed]
125. Gilmour N J L, Angus K W. Effect of revaccination on Mycobacterium johnei infections in sheep. J Comp Path. 1973;83:437–445. [PubMed]
126. Gilmour N J L, Angus K W. Absence of immunogenicity of an oral vaccine against Mycobacterium johnei in sheep. Res Vet Sci. 1974;16:269–270. [PubMed]
127. Gilot P, De Kesel M, Machtelinckx L, Coene M, Cocito C. Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis. J Bacteriol. 1993;175:4930–4935. [PMC free article] [PubMed]
128. Godfroid J, Boelaert F, Heier A, Clavareau C, Wellemans V, Desmecht M, Roels S, Walravens K. First evidence of Johne's disease in farmed red deer (Cervus elaphus) in Belgium. Vet Microbiol. 2000;77:283–290. [PubMed]
129. Gomes M S, Appelberg R. Evidence for a link between iron metabolism and Nramp1 gene function in innate resistance against Mycobacterium avium. Immunology. 1998;95:165–168. [PMC free article] [PubMed]
130. Gomez J E, Chen J M, Bishai W R. Sigma factors of Mycobacterium tuberculosis. Tubercle Lung Dis. 1997;78:175–183. [PubMed]
131. Gonzalez-y-Merchand J A, Colston M J, Cox R A. Effects of growth conditions on expression of mycobacterial murA and tyrS genes and contributions of their transcripts to precursor rRNA synthesis. J Bacteriol. 1999;181:4617–4627. [PMC free article] [PubMed]
132. Goodger W J, Collins M T, Nordlund K V, Eisele C, Pelletier J, Thomas C B, Sockett D C. Epidemiologic study of on-farm management practices associated with prevalence of Mycobacterium paratuberculosis infections in dairy cattle. J Am Vet Med Assoc. 1996;208:1877–1881. [PubMed]
133. Gormley E, Sandall L, Hong C, Lawton D, Murray A. Identification and differentiation of mycobacteria using the PAN promoter sequence from Mycobacterium paratuberculosis as a DNA probe. FEMS Microbiol Lett. 1997;147:63–68. [PubMed]
134. Grange J M, Gibson J A. Strain to strain variation in the immunogenicity of BCG. Dev Biol Stand. 1986;58:37–41. [PubMed]
135. Grant I R, Ball H J, Rowe M T. Effect of high-temperature, short-time HTST pasteurization on milk containing low numbers of Mycobacterium paratuberculosis. Lett Appl Microbiol. 1998;26:166–170. [PubMed]
136. Grant I R, Ball H J, Rowe M T. Effect of higher pasteurization temperatures, and longer holding times at 72 degrees C, on the inactivation of Mycobacterium paratuberculosis in milk. Lett Appl Microbiol. 1999;28:461–465. [PubMed]
137. Green E P, Tizard M L, Moss M T, Thompson J, Winterbourne D J, McFadden J J, Hermon-Taylor J. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 1989;17:9063–9073. [PMC free article] [PubMed]
138. Greenwood J D, Croy B A. A study on the engraftment and trafficking of bovine peripheral blood leukocytes in severe combined immunodeficient mice. Vet Immunol Immunopathol. 1993;38:21–44. [PubMed]
139. Greenwood J D, Croy B A, Trout D R, Wilcock B P. Xenogenic (bovine) peripheral blood leukocytes engrafted into severe combined immunodeficient mice retain primary immune function. Vet Immunol Immunopathol. 1997;59:93–112. [PubMed]
140. Greig A, Stevenson K, Perez V, Pirie A A, Grant J M, Sharp J M. Paratuberculosis in wild rabbits (Oryctolagus cuniculus) Vet Rec. 1997;140:141–143. [PubMed]
141. Greig A, Stevenson K, Henderson D, Perez V, Hughes V, Pavlik I, Hines II M E, McKendrick I, Sharp J M. Epidemiological study of paratuberculosis in wild rabbits in Scotland. J Clin Microbiol. 1999;37:1746–1751. [PMC free article] [PubMed]
142. Gresham H D, Goodwin J L, Anderson D C, Brown E J. A novel member of the integrin receptor family mediates Arg-Gly-Asp-stimulated neutrophil phagocytosis. J Cell Biol. 1989;108:1935–1943. [PMC free article] [PubMed]
143. Guerrero C, Bernasconi C, Burki D, Bodmer T, Telenti A. A novel insertion element from Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness. J Clin Microbiol. 1995;33:304–307. [PMC free article] [PubMed]
144. Guilhot C, Gicquel B, Davies J, Martin C. Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis. Mol Microbiol. 1992;6:107–113. [PubMed]
145. Gwozdz J M, Thompson K G, Manktelow B W, Murray A, West D M. Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis. Aust Vet J. 2000;78:560–566. [PubMed]
146. Hamilton H L, Follett D M, Siegfried L M, Czprynski C J. Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice. Infect Immun. 1989;57:225–230. [PMC free article] [PubMed]
147. Hamilton H L, Cooley A J, Adams J L, Czuprynski C J. Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model. Vet Pathol. 1991;28:146–155. [PubMed]
148. Harding H P. The histopathology of Mycobacterium johnei infection in small lab animals. J Pathol Bacteriol. 1959;78:157–169. [PubMed]
149. Harris N B, Feng Z, Liu X, Cirillo C L, Cirillo J D, Barletta R G. Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett. 1999;175:21–26. [PubMed]
150. Hein W R, Mackay C R. Prominence of γ/δ T cells in the ruminant immune system. Immunol Today. 1991;32:30–34. [PubMed]
151. Hermon-Taylor J, Bull T J, Sheridan J M, Cheng J, Stellakis M L, Sumar N. Causation of Crohn's disease by Mycobacterium avium subsp. paratuberculosis. Can J Gastroenterol. 2000;14:521–539. [PubMed]
152. Hernandez-Perez M, Fomukong N G, Hellyer T, Brown I N, Dale J W. Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium. Mol Microbiol. 1994;12:717–724. [PubMed]
153. Hilbink F, West D M, deLisle G W, Kittelberger R, Hosie B D, Hutton J, Cooke M M, Penrose M. Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep. Vet Microbiol. 1994;41:107–116. [PubMed]
154. Hinds J, Mahenthiralingam E, Kempsell K E, Duncan K, Stokes R W, Parish T, Stoker N G. Enhanced gene replacement in mycobacteria. Microbiology. 1999;145:519–527. [PubMed]
155. Hines S A, Buergelt C D, Wilson J H, Bliss E R. Disseminated Mycobacterium paratuberculosis infection in a cow. J Am Vet Med Assoc. 1987;190:681–683. [PubMed]
156. Hirch A. Infection of hamsters and rabbits with Mycobacterium johnei. J Comp Pathol. 1956;66:260–269. [PubMed]
157. Hoffner S E, Svenson S B, Kallenius G. Synergistic effects of antimycobacterial drug combinations on Mycobacterium avium complex determined radiometrically in liquid medium. Eur J Clin Microbiol. 1987;6:530–535. [PubMed]
158. Horwitz M A, Lee B W, Dillon B J, Harth G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 1995;92:1530–1534. [PMC free article] [PubMed]
159. Hurley S S, Splitter G A, Welch R A. Deoxyribonucleic acid relatedness of Mycobacterium paratuberculosis to other members of the family Mycobacteriaceae. Int J Syst Bacteriol. 1988;38:143–146.
160. Jark U, Ringena I, Franz B, Gerald F G, Beyerbach M, Franz B. Development of an ELISA technique for serodiagnosis of bovine paratuberculosis. Vet Microbiol. 1997;51:189–198. [PubMed]
161. Jessup D A, Abbas B, Behymer D. Paratuberculosis in tule elk in California. J Am Vet Med Assoc. 1981;179:1252–1253. [PubMed]
162. Johnson-Ifearulundu Y J, Kaneene J B. Relationship between soil type and Mycobacterium paratuberculosis. J Am Vet Med Assoc. 1997;210:1735–1740. [PubMed]
163. Johnson-Ifearulundu Y, Kaneene J B. Management-related risk factors for M. paratuberculosis infection in Michigan, USA, dairy herds. Prev Vet Med. 1998;37:41–54. [PubMed]
164. Johnson-Ifearulundu Y, Kaneene J B. Distribution and environmental risk factors for paratuberculosis in dairy cattle herds in Michigan. Am J Vet Res. 1999;5:589–596. [PubMed]
165. Johnson-Ifearulundu Y, Kaneene J B, Lloyd J W. Herd-level economic analysis of the impact of paratuberculosis on dairy herds. J Am Vet Med Assoc. 1999;214:822–825. [PubMed]
166. Juste R A, Garcia Marin J F, Peris B, Saez de Ocariz C, Badiola J J. Experimental infection of vaccinated and non-vaccinated lambs with Mycobacterium paratuberculosis. J Comp Pathol. 1994;110:185–194. [PubMed]
167. Kanazawa K, Haga Y, Funakoshi O, Nakajima H, Munakata A, Yoshida Y. Absence of Mycobacterium paratuberculosis DNA in intestinal tissues from Crohn's disease by nested polymerase chain reaction. J Gastroenterol. 1999;34:200–206. [PubMed]
168. Kaufmann S H E. Immunity to intracellular bacteria. Annu Rev Immunol. 1993;11:129–163. [PubMed]
169. Kenefick K B, Adams J L, Steinberg H, Czuprynski C J. In vivo administration of a monoclonal antibody against the type I IL-1 receptor inhibits the ability of mice to eliminate Mycobacterium paratuberculosis. J Leukoc Biol. 1994;55:719–722. [PubMed]
170. Kindler V A, Sappino P, Grau G E, Piguet P F, Vassalli P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell. 1989;56:731–740. [PubMed]
171. Kishima M, Yokomizo Y, Nonomura I, Goto N. Suppressive effect of nonviable Mycobacterium paratuberculosis on the delayed-type hpersensitivity reaction to sheep erythrocytes in mice. Lab Anim. 1991;25:310–318. [PubMed]
172. Kontoghiorghes G J, Weinberg E D. Iron: mammalian defense systems, mechanisms of disease, and chelation therapy approaches. Blood Rev. 1995;9:33–45. [PubMed]
173. Kopecky K E, Larsen A B, Merkal R S. Uterine infection in bovine paratuberculosis. Am J Vet Res. 1967;28:1043–1045. [PubMed]
174. Kormendy B. Paratuberculosis vaccine in a large dairy herd. Acta Vet Hung. 1992;40:171–184. [PubMed]
175. Kormendy B. The effect of vaccination on the prevalence of paratuberculosis in large dairy herds. Vet Microbiol. 1994;41:117–125. [PubMed]
176. Kreeger J M. Ruminant paratuberculosis—a century of progress and frustration. J Vet Diagn Investig. 1991;3:373–382. [PubMed]
177. Kunze A M, Wall S, Appelberg R, Silva M T, Portaels F, McFadden J J. IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium. Mol Microbiol. 1991;5:2265–2272. [PubMed]
178. Labidi A, Dauguet C, Goh J S, David H L. Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol. 1984;11:235–240.
179. Larsen A B, Merkal R S, Vardaman T H. Survival time of Mycobacterium paratuberculosis. Am J Vet Res. 1956;17:549–551. [PubMed]
180. Larsen A B, Kopecky K E. Mycobacterium paratuberculosis in reproductive organs and semen of bulls. Am J Vet Res. 1970;31:255–258. [PubMed]
181. Larsen A B, Moon H W. Experimental Mycobacterium paratuberculosis infection in chickens. Am J Vet Res. 1972;33:1231–1235. [PubMed]
182. Larsen A B, Merkal R S, Moon H W. Evaluation of a paratuberculosis vaccine given to calves before infection. Am J Vet Res. 1974;35:367–369. [PubMed]
183. Lawrence W E. Congenital infection with Mycobacterium johnei in cattle. Vet Rec. 1956;68:312.
184. Le J, Vilcek J. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Investig. 1987;56:234–248. [PubMed]
185. Liesack W, Pitulle C, Sela S, Stackebrandt E. Nucleotide sequence of the 16S rRNA from Mycobacterium leprae. Nucleic Acids Res. 1990;18:5558. [PMC free article] [PubMed]
186. Liesack W, Sela S, Bercovier H, Pitulle C, Stackebrandt E. Complete nucleotide sequence of the Mycobacterium leprae 23 S and 5 S rRNA genes plus flanking regions and their potential in designing diagnostic oligonucleotide probes. FEBS Lett. 1991;281:114–118. [PubMed]
187. Lominski L, Cameron J, Roberts G B S. Experimental Johne's disease in mice. J Pathol Bacteriol. 1956;71:211–222. [PubMed]
188. Lovell R, Levi M, Francis J. Studies on the survival of Johne's bacilli. J Comp Pathol. 1944;54:120–128.
189. Lugton I W. Mucosa-associated lymphoid tissues as sites for uptake, carriage and excretion of tubercle bacilli and other pathogenic mycobacteria. Immunol Cell Biol. 1999;77:364–372. [PubMed]
190. MacDougall J R, Croy B A, Chapeau C, Clark D A. Demonstration of a splenic cytotoxic effector cell in mice of genotype SCID/SCID.BG/BG. Cell Immunol. 1990;130:106–117. [PubMed]
191. Mahillon J, Chandler M. Insertion sequences. Microbiol Mol Biol Rev. 1998;62:725–774. [PMC free article] [PubMed]
192. Manca C, Paul S, Barry III C E, Freedman V H, Kaplan G. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect Immun. 1999;67:74–79. [PMC free article] [PubMed]
193. Manganelli R, Dubnau E, Tyagi S, Kramer F R, Smith I. Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol Microbiol. 1999;31:715–724. [PubMed]
194. Manning E J, Steinberg H, Rossow K, Ruth G R, Collins M T. Epizootic of paratuberculosis in farmed elk. J Am Vet Med Assoc. 1998;213:1320–1322. [PubMed]
195. Marklund B I, Speert D P, Stokes R W. Gene replacement through homologous recombination in Mycobacterium intracellulare. J Bacteriol. 1995;177:6100–6105. [PMC free article] [PubMed]
196. Marsh L, Whittington R, Cousins D. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Mol Cell Probes. 1999;13:115–126. [PubMed]
197. McDonald W L, Ridge S E, Hope A F, Condron R J. Evaluation of diagnostic tests for Johne's disease in young cattle. Aust Vet J. 1999;77:113–119. [PubMed]
198. McFadden J, Collins J, Beamen B, Arthur M, Gitnick G. Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue. J Clin Microbiol. 1992;30:3070–3073. [PMC free article] [PubMed]
199. McNab W B, Meek A H, Duncan J R, Brooks B W, Van Dreumel A A, Martin S W, Nielsen K H, Sugden E A, Turcotte C. An evaluation of selected screening tests for bovine paratuberculosis. Can J Vet Res. 1991;55:252–259. [PMC free article] [PubMed]
200. McNab W B, Meek A H, Martin S W, Duncan J R. Associations between dairy production indices and lipoarabinomannan enzyme-immunoassay results for paratuberculosis. Can J Vet Res. 1991;55:356–361. [PMC free article] [PubMed]
201. Mdluli K, Swanson J, Fischer E, Lee R E, Barry C E., III Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium. Mol Microbiol. 1998;27:1223–1233. [PubMed]
202. Millar D S, Withey S J, Tizard M L V, Ford J G, Hermon-Taylor J. Solid-phase hybridization capture of low-abundance target DNA sequences: application to the polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum. Anal Biochem. 1995;226:325–330. [PubMed]
203. Miller J, Jenny A, Rhyan J, Saari D, Suarez D. Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms. J Vet Diagn Investig. 1997;9:244–249. [PubMed]
204. Mishina D, Katsel P, Brown S T, Gilberts E C, Greenstein R J. On the etiology of Crohn disease. Proc Natl Acad Sci USA. 1996;93:9816–9820. [PMC free article] [PubMed]
205. Mokresh A H, Czuprynski C J, Butler D G. A rabbit model for study of Mycobacterium paratuberculosis infections. Infect Immun. 1989;57:3798–3807. [PMC free article] [PubMed]
206. Mokresh A H, Butler D G. Granulomatous enteritis following oral inoculation of newborn rabbits with Mycobacterium paratuberculosis of bovine origin. Can J Vet Res. 1990;54:313–319. [PMC free article] [PubMed]
207. Molina J M, Anguiano A, Ferrer O. Study on immune response of goats vaccinated with a live strain of Mycobacterium paratuberculosis. Comp Immunol Microbiol Infect Dis. 1996;19:9–15. [PubMed]
208. Momotani E, Whipple D L, Thiermann A B, Cheville N F. Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves. Vet Pathol. 1988;25:131–137. [PubMed]
209. Moss M T, Green E P, Tizard M L, Malik Z P, Hermon-Taylor J. Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900. Gut. 1991;32:395–398. [PMC free article] [PubMed]
210. Moss M T, Malik Z P, Tizard M L V, Green E P, Sanderson J D, Hermon-Taylor J. IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum. J Gen Microbiol. 1992;138:129–145. [PubMed]
211. Mowat A M, Hutton A K, Garside P, Steel M. A role for interleukin-1 in immunologically mediated intestinal pathology. Immunology. 1993;80:110–115. [PMC free article] [PubMed]
212. Mulder M A, Zappe H, Steyn L M. Mycobacterial promoters. Tubercle Lung Dis. 1997;78:211–223. [PubMed]
213. Murray A, Winter N, Lagranderie M, Hill D F, Rauzier J, Timm J, Leclerc C, Moriarty K M, Gheoghiu M, Gicquel B. Expression of Escherichia coli β-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses. Mol Microbiol. 1992;6:3331–3342. [PubMed]
214. Mutharia L M, Moreno W, Raymond M. Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies. Infect Immun. 1997;65:387–394. [PMC free article] [PubMed]
215. Mutwiri G K, Butler D G, Rosendal S, Yager J. Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin. Infect Immun. 1992;60:4074–4079. [PMC free article] [PubMed]
216. Mutwiri G K, Butler D G, Rosendal S, Woodward B. The role of restricted food intake in the pathogenesis of cachexia in severe combined immunodeficient beige mice infected with Mycobacterium paratuberculosis. Can J Vet Res. 1995;59:40–45. [PMC free article] [PubMed]
217. Naser S A, Gillespie R F, Naser N A, El-Zaatari F A K. Effect of IS900 gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis. Curr Microbiol. 1998;37:373–379. [PubMed]
218. Naser S A, Felix J, Liping H, Romero C, Naser N, Walsh A, Safranek W. Occurrence of the IS900 gene in Mycobacterium avium complex derived from HIV patients. Mol Cell Probes. 1999;13:367–372. [PubMed]
219. National Animal Health Monitoring System. Johne's disease on U.S. dairy operations. Report N245.1087. USDA:APHIS:VS, CEAH. Fort Collins, Colo: National Animal Health Monitoring System; 1997.
220. Nebbia P, Robino P, Ferroglio E, Rossi L, Meneguz G, Rosati S. Paratuberculosis in red deer (Cervus elaphus hippelaphus) in the Western Alps. Vet Res Commun. 2000;24:435–443. [PubMed]
221. Oh Y K, Straubinger R M. Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction. Infect Immun. 1996;64:319–25. [PMC free article] [PubMed]
222. Olsen I, Reitan L J, Holstad G, Wiker H G. Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp. paratuberculosis. Infect Immun. 2000;68:801–808. [PMC free article] [PubMed]
223. Ott S L, Wells S J, Wagner B A. Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med. 1999;40:179–192. [PubMed]
224. Parish T, Gordhan B G, McAdam R A, Duncan K, Mizrahi V, Stoiker N G. Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology. 1999;145:3497–3503. [PubMed]
225. Parish T, Stoker N G. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology. 2000;146:1969–1975. [PubMed]
226. Patterson C J, La Venture M, Hurley S S, Davis J P. Accidental self-inoculation with Mycobacterium paratuberculosis bacterin (Johne's bacterin) by veterinarians in Wisconsin. J Am Vet Med Assoc. 1988;192:1197–1199. [PubMed]
227. Pavelka M S, Jr, Jacobs W R., Jr Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol. 1999;181:4780–4789. [PMC free article] [PubMed]
228. Pavlik I, Bejková L L, Pavlas M, Rozxypalová Z, Kosková S. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet Microbiol. 1995;45:311–3118. [PubMed]
229. Pavlik I, Horvathova A, Dvorska L, Bartl J, Svastova P, de Maine R, Rychlik I. Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. J Microbiol Methods. 1999;38:155–167. [PubMed]
230. Pearson J K L, McClelland T G. Uterine infection and congenital Johne's disease in cattle. Vet Rec. 1955;67:615–616.
231. Pelicic V, Reyrat J M, Gicquel B. Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol Microbiol. 1996;20:919–925. [PubMed]
232. Pelicic V, Reyrat J M, Gicquel B. Positive selection of allelic exchange mutants in Mycobacterium bovis BCG. FEMS Microbiol Lett. 1996;144:161–166. [PubMed]
233. Pelicic V, Jackson M, Reyrat J M, Jacobs W R, Jr, Gicquel B, Guilhot C. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 1997;94:10955–10960. [PMC free article] [PubMed]
234. Perez V, Chavez G, Gutierrez M, Tellechea J, Badiola J J, Garcia-Marin J F. Evaluation of the AGID and γ-interferon tests in lambs infected with Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. paratuberculosis. In: Chiodini R J, Collins M T, Bassey E O, editors. Proceedings of the 4th International Colloquium on Paratuberculosis. East Providence, R.I: International Association for Paratuberculosis Inc.; 1994. pp. 91–96.
235. Perez V, Tellechea J, Badiola J J, Gutierrez M, Garcia-Marin J F. Relation between serologic response and pathologic findings in sheep with naturally acquired paratuberculosis. Am J Vet Res. 1997;58:799–803. [PubMed]
236. Perez V, Tellechea J, Corpa J M, Gutierrez M, Garcia-Marin J F. Relation between pathologic findings and cellular immune responses in sheep with naturally acquired paratuberculosis. Am J Vet Res. 1999;60:123–127. [PubMed]
237. Pernodet J L, Boccard F, Alegre M T, Gagnat J, Guerineau M. Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens. Gene. 1989;79:33–46. [PubMed]
238. Picardeau M, Varnerot A, Rauzier J, Gicquel B, Vincent V. Mycobacterium xenopi IS1395, a novel insertion sequence expanding the IS256 family. Microbiology. 1996;142:2453–2461. [PubMed]
239. Picardeau M, Bull T J, Prodhom G, Pozniak A L, Shanson D C, Vincent V. Comparison of a new insertion element, IS1407, with established molecular markers for the characterization of Mycobacterium celatum. Int J Syst Bacteriol. 1997;47:640–644. [PubMed]
240. Pierschbacher M D, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984;309:30–33. [PubMed]
241. Poupart P, Coene M, van Heuverswyn H, Cocito C. Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease. J Clin Microbiol. 1993;31:1601–1605. [PMC free article] [PubMed]
242. Power S B, Haagsma J, Smyth D P. Paratuberculosis in farmed red deer (Cervus elaphus) in Ireland: Vet Rec. 1993;132:213–216. [PubMed]
243. Prescott J F, Baggot J D. Miscellaneous antibiotics: ionophores, nitrofurans, nitroimidazoles, rifampin, and others. In: Prescott J F, Baggot J D, editors. Antimicrobial therapy in veterinary medicine. Ames: Iowa State University Press; 1993. pp. 288–289.
244. Puyang X, Lee K, Pawlichuk C, Kunimoto D Y. IS1626, a new IS900-related Mycobacterium avium insertion sequence. Microbiology. 1999;145:3163–3168. [PubMed]
245. Rankin J D. The experimental production of Johne's disease in laboratory rabbits. J Pathol Bacteriol. 1958;75:363–366. [PubMed]
246. Rastogi N, Goh K S, Labrousse V. Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol. Antimicrob Agents Chemother. 1992;36:2843–2846. [PMC free article] [PubMed]
247. Rauzier J, Gormley E, Gutierrez M C, Kassa-Kelembho E, Sandall L J, Dupont C, Gicquel B, Murray A. A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex. Microbiology. 1999;145:1695–1701. [PubMed]
248. Reviriego F J, Moreno M A, Dominguez L. Soil type as a putative risk factor of ovine and caprine paratuberculosis seropositivity in Spain. Prev Vet Med. 2000;43:43–51. [PubMed]
249. Ridge S E, Harkin J T, Badman R T, Mellor A M, Larsen J W A. Johne's disease in alpacas (Lama pacos) in Australia. Aust Vet J. 1995;72:150–153. [PubMed]
250. Ridley D S. The histopathological spectrum of the mycobacterioses. In: Ratledge C, Stanford J, editors. The biology of the mycobacteria. Vol. 2. New York, N.Y: Academic Press, Inc.; 1983. pp. 129–172.
251. Rogall T, Wolters J, Florh T, Böttger E C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol. 1990;40:323–330. [PubMed]
252. Rohonczy E B, Balachandran A V, Dukes T W, Payeur J B, Rhyan J C, Saari D A, Whiting T L, Wilson S H, Jarnagin J L. A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus) Can J Vet Res. 1996;60:108–114. [PMC free article] [PubMed]
253. Roiz M P, Palenque E, Guerrero C, Garcia M J. Use of restriction fragment length polymorphism as a genetic marker for typing Mycobacterium avium strains. J Clin Microbiol. 1995;33:1389–1391. [PMC free article] [PubMed]
254. Rossiter C A, Burhans W S. Farm-specific approach to paratuberculosis Johne's disease control. Vet Clin North Am Food Anim Pract. 1996;12:383–413. [PubMed]
255. Rothel J S, Jones S L, Corner L A, Cox J C, Wood P R. A sandwich enzyme immunoassay for bovine interferon-γ and its use for the detection of tuberculosis in cattle. Aust Vet J. 1990;67:134–137. [PubMed]
256. Saito H, Tomioka H, Sato K, Tasaka H, Tsukamura M, Kuze F, Asano K. Identification and partial characterization of Mycobacterium avium and Mycobacterium intracellulare by using DNA probes. J Clin Microbiol. 1989;27:994–997. [PMC free article] [PubMed]
257. Sanderson J D, Moss M T, Tizard M L V, Hermon-Taylor J. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut. 1992;33:890–896. [PMC free article] [PubMed]
258. Saxegaard F, Fodstad F H. Control of paratuberculosis (Johne's disease) in goats by vaccination. Vet Rec. 1985;116:439–441. [PubMed]
259. Saxegaard F, Baess I, Jantzen E. Characterization of clinical isolates of Mycobacterium paratuberculosis by DNA-DNA hybridization and cellular fatty acid analysis. Acta Pathol Microbiol Immunol Scand. 1988;96:497–502. [PubMed]
260. Shinnick T M. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol. 1987;169:1080–1088. [PMC free article] [PubMed]
261. Shulaw W P, Gordon J C, Bech-Nielsen S, Pretzman C I, Hoffsis G F. Evidence of paratuberculosis in Ohio's white-tailed deer, as determined by an enzyme-linked immunosorbent assay. Am J Vet Res. 1986;47:2539–2542. [PubMed]
262. Shulaw W P, Bech-Nielsen S, Rings D M, Getzy D M, Woodruff T S. Serodiagnosis of paratuberculosis in sheep by use of agar gel immunodiffusion. Am J Vet Res. 1993;54:13–19. [PubMed]
263. Sigurdardottir O G, Press C M, Saxegaard F, Evensen O. Bacterial isolation, immunological response, and histopathological lesions during the early subclinical phase of experimental infection of goat kids with Mycobacterium avium subsp. paratuberculosis. Vet Pathol. 1999;36:542–550. [PubMed]
264. Silbaq F S, Cho S N, Cole S T, Brennan P J. Characterization of a 34-kilodalton protein of Mycobacterium leprae that is isologous to the immunodominant 34-kilodalton antigen of Mycobacterium paratuberculosis. Infect Immun. 1998;66:5576–5579. [PMC free article] [PubMed]
265. Skamene E, Philippe G, Forget A, Kongshavn P A L, St. Charles C, Taylor B A. Genetic regulation of resistance to intracellular pathogens. Nature. 1982;297:506–509. [PubMed]
266. Skamene E. Genetic control of susceptibility to mycobacterial infections. Rev Infect Dis. 1989;11:S394–S399. [PubMed]
267. Smith I, Dussurget O, Rodriguez G M, Timm J, Gomez M, Dubnau J, Gold B, Manganelli R. Extra and intracellular expression of Mycobacterium tuberculosis genes. Tubercle Lung Dis. 1998;79:91–97. [PubMed]
268. Smith R A, Kreeger J M, Alvarez A J, Goin J C, Davis W C, Whipple D L, Estes D M. Role of CD8+ and WC-1 γ/δ T cells in resistance to Mycobacterium bovis infection I the SCID-bo mouse. J Leukoc Biol. 1999;65:28–34. [PubMed]
269. Snow G A. Mycobactins: iron-chelating growth factors from mycobacteria. Bacteriol Rev. 1970;34:99–125. [PMC free article] [PubMed]
270. Sockett D C, Conrad T A, Thomas C B, Collins M T. Evaluation of four serological tests for bovine paratuberculosis. J Clin Microbiol. 1992;30:1134–1139. [PMC free article] [PubMed]
271. Spangler E, Heider J E, Bech-Nielsen S, Dorn C R. Serologic enzyme-linked immunosorbent assay responses of calves vaccinated with a killed Mycobacterium paratuberculosis vaccine. Am J Vet Res. 1991;52:1197–1200. [PubMed]
272. Stabel J R. Temporal effects of tumor necrosis factor-α on intracellular survival of Mycobacterium paratuberculosis. Vet Immunol Immunopathol. 1995;45:321–332. [PubMed]
273. Stabel J R, Stabel T J. Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA. Vet Immunol Immunopathol. 1995;45:211–220. [PubMed]
274. Stabel J R. Production of γ-interferon by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis. J Vet Diagn Investig. 1996;8:345–350. [PubMed]
275. Stabel J R, Goff J P. Influence of vitamin D3 infusion and dietary calcium on secretion of interleukin 1, interleukin 6, and tumor necrosis factor in mice infected with Mycobacterium paratuberculosis. Am J Vet Res. 1996;57:825–829. [PubMed]
276. Stabel J R, Goff J P, Whipple D L, Ackermann M R, Reinhardt T A. Low calcium diet and 1,25-dihydroxyvitamin D3 infusion modulate immune responses during Mycobacterium paratuberculosis infection in beige mice. Vet Immunol Immunopathol. 1996;50:127–143. [PubMed]
277. Stabel J R, Steadham E M, Bolin C A. Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective? Appl Environ Microbiol. 1997;63:4975–4977. [PMC free article] [PubMed]
278. Stabel J R, Goff J P, Ackermann M R. Dietary calcium modulates Mycobacterium paratuberculosis infection in beige mice. Vet Immunol Immunopathol. 1998;66:377–390. [PubMed]
279. Stabel J R. Johne's disease: a hidden threat. J Dairy Sci. 1998;81:283–288. [PubMed]
280. Stahl D A, Urbance J W. The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J Bacteriol. 1990;172:116–124. [PMC free article] [PubMed]
281. St.-Jean G, Jernigan A D. Treatment of Mycobacterium paratuberculosis infection in ruminants. Vet Clin North Am Food Anim Pract. 1991;7:793–804. [PubMed]
282. Stuart P. Vaccination against Johne's disease in cattle exposed to experimental infection. Brit Vet J. 1965;121:289–318. [PubMed]
283. Sturgill-Koszycki S, Schlesinger P H, Chakraborty P, Haddix P L, Collins H L, Fok A K, Allen R D, Gluck S L, Heuser J, Russell D G. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science. 1994;263:678–681. [PubMed]
284. Sugden E A, Stilwell K, Michaelides A. A comparison of lipoarabinomannan with other antigens used in absorbed enzyme immunoassays for the serological detection of cattle infected with mycobacterium paratuberculosis. J Vet Diagn Investig. 1997;9:413–417. [PubMed]
285. Sung N, Collins M T. Thermal tolerance of Mycobacterium paratuberculosis. Appl Environ Microbiol. 1998;64:999–1005. [PMC free article] [PubMed]
286. Suzuki Y, Yoshinaga K, Ono Y, Nagata A, Yamada T. Organization of rRNA genes in Mycobacterium bovis BCG. J Bacteriol. 1987;169:839–843. [PMC free article] [PubMed]
287. Suzuki Y, Ono Y, Nagata A, Yamada T. Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J Bacteriol. 1988;170:1631–1636. [PMC free article] [PubMed]
288. Sweeney R W, Whitlock R H, Buckley C L, Spencer P, Rosenberger A E, Hutchinson L J. Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk. Am J Vet Res. 1994;55:905–909. [PubMed]
289. Sweeney R W, Whitlock R H, Buckley C L, Spencer P A. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J Vet Diagn Investig. 1995;7:488–493. [PubMed]
290. Sweeney R W. Transmission of paratuberculosis. Vet Clin North Am Food Anim Pract. 1996;12:305–312. [PubMed]
291. Sweeney R W, Jones D E, Habecker P, Scott P. Interferon-γ and interleukin 4 gene expression in cows infected with Mycobacterium paratuberculosis. Am J Vet Res. 1998;59:842–847. [PubMed]
292. Takewaki S I, Okuzumi K, Manabe I, Tanimura M, Miyamura K, Nakahara K I, Yazaki Y, Ohkubo A, Nagai R. Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species. Int J Syst Bacteriol. 1994;44:159–166. [PubMed]
293. Tanaka S, Sato M, Taniguchi T, Yokomizo Y. Histopathological and morphometrical comparison of granulomatous lesions in BALB/c and C3H/HeJ mice inoculated with Mycobacterium paratuberculosis. J Comp Pathol. 1994;110:381–388. [PubMed]
294. Temple R M S, Muscoplat C C, Thoen C O, Hines E M, Johnson D W. Observations on diagnostic tests for paratuberculosis in a deer herd. J Am Vet Assoc. 1979;175:914–915. [PubMed]
295. Thoen C O, Jarnagin J L, Richards W D. Isolation and identification of mycobacteria from porcine tissues: a three-year study. Am J Vet Res. 1975;36:1383–1386. [PubMed]
296. Thole J E, Keulen W J, De Bruyn J, Kolk A H, Groothuis D G, Berwald L G, Tiesjema R H, van Embden J D. Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in Escherichia coli K-12. Infect Immun. 1987;55:1466–1475. [PMC free article] [PubMed]
297. Thole J E, van Schooten W C, Keulen W J, Hermans P W, Janson A A, de Vries R R, Kolk A H, van Embden J D. Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG. Infect Immun. 1988;56:1633–1640. [PMC free article] [PubMed]
298. Thorel M F, Drichevsky M, Lévy-Frébault V V. Numerical taxonomy of mycobactin-dependent Mycobacteria, amended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov. Int J Syst Bacteriol. 1990;40:254–260. [PubMed]
299. Thoresen O V, Olsaker I. Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of Mycobacterium paratuberculosis. Vet Microbiol. 1994;40:293–303. [PubMed]
300. Tizard M L V, Moss M T, Sanderson J D, Austen B M, Hermon-Taylor J. P43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis. J Gen Microbiol. 1992;138:1729–1736. [PubMed]
301. Tizard M, Bull T, Millar D, Doran T, Martin H, Sumar N, Ford J, Hermon-Taylor J. A low G C content genetic island in Mycobacterium avium subsp. paratuberculosis and M. avium subsp. silvaticum with homologous genes in Mycobacterium tuberculosis. Microbiology. 1998;144:3413–1423. [PubMed]
302. Valente C, Cuteri V, Quondam Giandomenico R, Gialletti L, Franciosini M P. Use of an experimental chicks model for paratuberculosis enteritis (Johne's disease) Vet Res. 1997;28:239–246. [PubMed]
303. van der Giessen J W B, Haring R M, van der Zeijst B A M. Comparison of the 23S ribosomal RNA genes and the spacer region between the 16S and 23S rRNA genes of the closely related Mycobacterium avium and Mycobacterium paratuberculosis and the fast-growing Mycobacterium phlei. Microbiology. 1994;140:1103–1108. [PubMed]
304. van Schaik G, Kalis C H J, Benedictus G, Dijkhuizen A A, Huirne R B M. Cost-benefit analysis of vaccination against paratuberculosis in dairy cattle. Vet Rec. 1996;139:624–627. [PubMed]
305. Vary P H, Andersen P R, Green E, Hermon-Taylor J, McFadden J J. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. J Clin Microbiol. 1990;28:933–937. [PMC free article] [PubMed]
306. Veazey R S, Horohov D W, Krahenbuhl J L, Taylor H W, Oliver III J L, Snider T G., III Comparison of the resistance of C5BL/6 and C3H/He mice to infection with Mycobacterium paratuberculosis. Vet Microbiol. 1995;47:79–87. [PubMed]
307. Veazey R S, Taylor H W, Horohov D W, Krahenbuhl J L, Oliver III J L, Snider T G., III Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. J Comp Pathol. 1995;113:75–80. [PubMed]
308. Velaz-Faircloth M, Cobb A J, Horstman A L, Henry S C, Frothingham R. Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein. Infect Immun. 1999;67:4243–4250. [PMC free article] [PubMed]
309. Wards B J, Collins D M, de Lisle G W. Restriction endonuclease analysis of members of the Mycobacterium avium-M. intracellulare- M. scrofulaceum serocomplex. J Clin Microbiol. 1987;25:2309–2313. [PMC free article] [PubMed]
310. Waters W R, Stabel J R, Sacco R E, Harp J A, Pesch B A, Wannemuehler M J. Antigen-specific B-cell unresponsiveness induced by chronic Mycobacterium avium subsp. paratuberculosis infection of cattle. Infect Immun. 1999;67:1593–1598. [PMC free article] [PubMed]
311. Wayne L G, Kubica G P. The mycobacteria. In: Sneath P H A, Mair N S, Sharpe M E, Holt J G, editors. Bergey's manual of systematic bacteriology. Vol. 2. Baltimore, Md: The Williams & Wilkins Co.; 1986. pp. 1435–1457.
312. Weinberg E D. Iron and susceptibility to infectious disease. Science. 1974;184:952–956. [PubMed]
313. Wentink G H, Bongers J H, Vos J H, Zeeuwen A A P A. Relationship between negative skin test with Johnin after vaccination and post mortem findings. Vet Rec. 1993;132:38–39. [PubMed]
314. Wentink G H, Bongers J H, Zeeuwen A A P A, Jaartsveld F H J. Incidence of paratuberculosis after vaccination against M. paratuberculosis in two infected dairy herds. J Vet Med. 1994;41:517–522. [PubMed]
315. Whipple D L, Le Febvre R B, Andrews R E, Jr, Thiermann A B. Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from Mycobacterium paratuberculosis and other Mycobacterium species. J Clin Microbiol. 1987;25:1511–1515. [PMC free article] [PubMed]
316. Whipple D L, Kapke P A, Andrews R E., Jr Analysis of restriction endonuclease fragment patterns of DNA from Mycobacterium paratuberculosis. Vet Microbiol. 1989;19:189–194. [PubMed]
317. Whipple D L, Kapke P, Vary C. Identification of restriction fragment length polymorphisms in DNA from Mycobacterium paratuberculosis. J Clin Microbiol. 1990;28:2561–2564. [PMC free article] [PubMed]
318. Whipple D L, Klemens S P, Cynamon M H. Proceedings of the 3rd international colloquium on paratuberculosis. East Providence, R.I: International Association of Paratuberuclosis; 1991. Development of the beige mouse as an animal model for M. paratuberculosis infection; pp. 551–552.
319. Whipple D L, Kapke P A, Andersen P R. Comparison of a commercial DNA probe test and three cultivation procedures for detection of Mycobacterium paratuberculosis in bovine feces. J Vet Diagn Investig. 1992;4:23–27. [PubMed]
320. White W B, Whipple D L, Stabel J R, Bolin C A. Comparison of cellular and extracellular proteins expressed by various isolates of Mycobacterium paratuberculosis and other mycobacterial species. Am J Vet Res. 1994;10:1399–1405. [PubMed]
321. Whittington R, Marsh I, Choy E, Cousins D. Polymorphisms in IS1311, an insertion sequence common to Mycobacterium avium and M. avium subsp. paratuberculosis, can be used to distinguish between and within these species. Mol Cell Probes. 1998;12:349–358. [PubMed]
322. Williams E S, Snyder S P, Martin K L. Pathology of spontaneous and experimental infection of North American wild ruminants with Mycobacterium paratuberculosis. Vet Pathol. 1983a;20:274–291. [PubMed]
323. Williams E S, Snyder S P, Martin K L. Experimental infection of some North American wild ruminants and domestic sheep with Mycobacterium paratuberculosis: clinical and bacteriological findings. J Wildl Dis. 1983b;19:185–191. [PubMed]
324. Williams S L, Harris N B, Barletta R G. Development of a firefly luciferase-based assay for determining antimicrobial susceptibility of Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol. 1999;37:304–309. [PMC free article] [PubMed]
325. Wilson D J, Rossiter C, Han H R, Sears P M. Association of Mycobacterium paratuberculosis infection with reduced mastitis, but with decreased milk production and increased cull rate in clinically normal dairy cows. Am J Vet Res. 1993;54:1851–1857. [PubMed]
326. Wood P R, Corner L A, Plackett P. Development of a simple, rapid in vitro cellular assay for bovine tuberculosis based on the production of γ interferon. Res Vet Sci. 1990;49:46–49. [PubMed]
327. Yokomizo Y, Yugi H, Merkal R S. A method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay ELISA for the diagnosis of bovine paratuberculosis. Nippon Juigaku Zasshi. 1985;47:111–119. [PubMed]
328. Yoshimura H H, Graham D Y. Nucleic acid hybridization studies of mycobactin-dependent mycobacteria. J Clin Microbiol. 1988;26:1309–1312. [PMC free article] [PubMed]
329. Young D, Lathigra R, Hendrix R, Sweeter D, Young R A. Stress proteins are immune targets in leprosy and tuberculosis. Proc Natl Acad Sci USA. 1988;85:4267–4270. [PMC free article] [PubMed]
330. Zhao B, Collins M T, Czuprynski C J. Effects of γ-interferon and nitric oxide on the interaction of Mycobacterium avium subsp. paratuberculosis with bovine monocytes. Infect Immun. 1997;65:1761–1766. [PMC free article] [PubMed]
331. Zhao B Y, Czuprynski C J, Collins M T. Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method. Can J Vet Res. 1999;63:56–61. [PMC free article] [PubMed]
332. Zimmer K, Drager K G, Klawonn W, Hess R G. Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and commercially available DNA-ProbeR test in detecting Mycobacterium paratuberculosis in faeces from cattle. J Vet Med. 1999;46:137–140. [PubMed]
333. Zurbrick B G, Follett D M, Czuprynski C J. Cytokine regulation of the intracellular growth of Mycobacterium paratuberculosis in bovine monocytes. Infect Immun. 1988;56:1692–1697. [PMC free article] [PubMed]

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