Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.     

Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.     

A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-alpha were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.     

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.     

Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises

Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants ("7,5") appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.     

Whole-genome plasticity among Mycobacterium avium subspecies: insights from comparative genomic hybridizations

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is also implicated in cases of Crohn's disease in humans. Another closely related strain, M. avium subsp. avium, is a health problem for immunocompromised patients. To understand the molecular pathogenesis of M. avium subspecies, we analyzed the genome contents of isolates collected from humans and domesticated or wildlife animals. Comparative genomic hybridizations indicated distinct lineages for each subspecies where the closest genomic relatedness existed between M. avium subsp. paratuberculosis isolates collected from human and clinical cow samples. Genomic islands (n = 24) comprising 846 kb were present in the reference M. avium subsp. avium strain but absent from 95% of M. avium subsp. paratuberculosis isolates. Additional analysis identified a group of 18 M. avium subsp. paratuberculosis-associated islands comprising 240 kb that were absent from most of the M. avium subsp. avium isolates. Sequence analysis of DNA regions flanking the genomic islands identified three large inversions in addition to several small inversions that could play a role in regulation of gene expression. Analysis of genes encoded in the genomic islands reveals factors that are probably important for various mechanisms of virulence. Overall, M. avium subsp. avium isolates displayed a higher level of genomic diversity than M. avium subsp. paratuberculosis isolates. Among M. avium subsp. paratuberculosis isolates, those from wildlife animals displayed the highest level of genomic rearrangements that were not observed in other isolates. The presented findings will affect the future design of diagnostics and vaccines for Johne's and Crohn's diseases and provide a model for genomic analysis of closely related bacteria.     

Defining the stressome of Mycobacterium avium subsp. paratuberculosis in vitro and in naturally infected cows

Mycobacterium avium subsp. paratuberculosis causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. avium subsp. paratuberculosis exposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis of M. avium subsp. paratuberculosis. In this report, the stress response of M. avium subsp. paratuberculosis on a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups of M. avium subsp. paratuberculosis that were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n=597), suggesting the high sensitivity of M. avium subsp. paratuberculosis to acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar in M. avium subsp. paratuberculosis and Mycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g., sigH and sigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of six M. avium subsp. paratuberculosis mutants with inactivation of differentially regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could be applied to analyze other intracellular pathogens.     

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays

AIMS: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. METHODS AND RESULTS: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. CONCLUSIONS: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.     

Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is an important animal pathogen that has also been implicated in human disease. The major proteins expressed by M. paratuberculosis were analyzed by two-dimensional gel electrophoresis, and a superoxide dismutase (Sod) was identified from this protein profile. The M. paratuberculosis Sod has a molecular mass of 23 kDa and an isoelectric point of 6.1. Sequence analysis of the corresponding sodA gene from M. paratuberculosis indicates that this protein is a manganese-dependent enzyme. We show that the M. paratuberculosis Sod is actively secreted, suggesting that it may elicit a protective cellular immune response in the host during infection.     

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the central European Carpathians

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.     

Potential risk of Mycobacterium avium subspecies paratuberculosis spread by syrphid flies in infected cattle farms

The syrphid Eristalis tenax Linnaeus (Diptera: Syrphidae) may be found in and around dung storage pits at cattle farms at various developmental stages of their life cycle. The purpose of this study was to investigate the occurrence of Mycobacterium avium ssp. paratuberculosis in 1044 E. tenax samples at various developmental stages, as well as fresh and stored dung originating from nine cattle farms. Mycobacterium fortuitum was isolated from one (1.5%) larva from the vicinity of three paratuberculosis-free herds of cattle. Mycobacterium a. paratuberculosis was isolated from 111 (21.4%) of E. tenax larvae collected from two of seven farms known to be infected with the causal agent of paratuberculosis. Mycobacteria were not isolated from any of the 340 pupae, 41 adults of 78 samples of exoskeletal exuviae. Mycobacterium a. paratuberculosis isolates from E. tenax larvae were of the IS900 restriction fragment length polymorphism (RFLP) type B-C1, identical to that detected in faecal samples from cattle herds infected with paratuberculosis. Larvae artificially infected with mycobacteria of IS900 RFLP type B-C9 did not contain statistically more CFU of identical IS900 RFLP type B-C9 in the intestinal tract and internal organs than on the body surface. These results show that M. a. paratuberculosis can survive in the intestinal tract and internal organs of E. tenax.     

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.     

Diptera as vectors of mycobacterial infections in cattle and pigs

Mycobacteria were isolated from 14 (4.5%) of 314 samples, containing 7791 adult Diptera, which were collected in the Czech Republic and Slovakia in 1997-2000. These flies were collected from three cattle herds with paratuberculosis, two pig herds with mycobacterial infections and one farm that kept both cattle and pigs and that did not have problems of mycobacterial infections. Mycobacterium intracellulare was isolated from Eristalis tenax Linnaeus (Diptera: Syrphidae) captured from a pig herd. Mycobacterium avium ssp. avium (serotype 8) was isolated from flies of the genera Drosophila Fallen (Diptera: Drosophilidae) and Musca Linnaeus (Diptera: Muscidae) originating from a pig herd. Mycobacterium spp. were isolated from Musca spp. and Mycobacterium fortuitum was isolated from dung flies of the genus Scatophaga Meigen (Diptera: Scatophagidae), Musca spp. and Stomoxys calcitrans Linnaeus (Diptera: Muscidae) captured in the same herd. Mycobacterium scrofulaceum was isolated from S. calcitrans from the farm with both cattle and pigs. Mycobacterium avium ssp. paratuberculosis was isolated from Scatophaga spp. collected from pastures grazed by one of the cattle herds and from Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and Lucilia caesar Linnaeus (Diptera: Calliphoridae) captured in a slaughterhouse, where cattle infected with paratuberculosis were slaughtered. Mycobacterium phlei was isolated from flies of the genus Lucilia captured at a waste bin. These data indicate that mycobacteria may be spread by adult flies that have been in contact with material contaminated with these pathogens.     

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.