J. Michael Sharp

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johnes disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohns disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpsons index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Molecular Characterization of Pigmented and Nonpigmented Isolates of Mycobacterium avium subsp. paratuberculosis

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

Jaagsiekte Sheep Retrovirus Proviral Clone JSRV JS7 , Derived from the JS7 Lung Tumor Cell Line, Induces Ovine Pulmonary Carcinoma and Is Integrated into the Surfactant Protein A Gene

ABSTRACT Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRVJS7) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRVJS7 is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

Do non-ruminant wildlife pose a risk of paratuberculosis to domestic livestock and vice versa in Scotland?

Paratuberculosis (Johnes disease) was long considered only a disease of ruminants. Recently non-ruminant wildlife species have been shown to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis. We review the known non-ruminant wildlife host range of M. avium subsp. paratuberculosis and consider their role in the epidemiology of paratuberculosis in domestic ruminant livestock. Mycobacterium avium subsp. paratuberculosis has been isolated from lagomorph, canid, mustelid, corvid, and murid species. In agricultural environments domestic ruminants may contact wildlife and/or their excreta when grazing or feeding on farm-stored feed contaminated with wildlife feces, opening up the possibility of inter-species transmission. Of the wildlife species known to harbor M. avium subsp. paratuberculosis in Scotland, the rabbit is likely to pose the greatest risk to grazing livestock. Paratuberculosis in domestic ruminants is a notoriously difficult disease to control; the participation of non-ruminant wildlife in the epidemiology of the disease may partially account for this difficulty.

Sheep pulmonary adenomatosis: A unique model of retrovirus associated lung cancer

Sheep pulmonary adenomatosis (SPA) is a contagious bronchiolo-alveolar carcinoma of sheep associated with an exogenous type D/B retrovirus known as jaagsiekte sheep retrovirus (JSRV). SPA represents a unique model for lung cancer, and studies on its aetiopathogenesis can provide further insight into the mechanisms of epithelial neoplasms.

Analysis of Integration Sites of Jaagsiekte Sheep Retrovirus in Ovine Pulmonary Adenocarcinoma

ABSTRACT Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Complete sequence of the gene encoding the bacterioferritin subunit of Mycobacterium avium subspecies silvaticum

A gene encoding the bacterioferritin subunit (Bfr) of Mycobacterium avium (Ma) subspecies silvaticum has been cloned, sequenced and expressed. The 477-bp open reading frame codes for 159 amino acids, which were shown to share up to 92% identity with the Bfr of five bacterial genera. The recombinant Bfr exhibits serological cross-reactivity with Ma paratuberculosis antigen D, a protein of approx. 20 kDa in cell lysates of Ma paratuberculosis and Ma silvaticum and a protein of 20-22 kDa in sonicates of M. leprae.

Characterisation of IS901 integration sites in the Mycobacterium avium genome.

Data are presented on the identification and characterisation of 17 chromosomal integration loci of the insertion element IS901 in the Mycobacterium avium (cervine strain JD88/118) genome. Thirteen of these integration loci have been mapped to their corresponding positions on the M. avium strain 104 (an IS901(-) strain) genome (The Institute for Genome Research (TIGR) unfinished genome-sequencing project). Sequence data for both upstream and downstream sequence flanking regions were obtained for 12 insertion loci, while upstream sequence was obtained for five others. A consensus IS901 insertion target sequence compiled from all 17 integration sites was in broad agreement with earlier reports that were based on only two such loci. Analysis of IS901 integration site flanking sequences revealed that, like IS900 in M. avium subspecies paratuberculosis, IS901 inserts preferentially between a putative ribosome-binding sequence (RBS) and the translational start codon of an open reading frame (ORF). In BLAST X and BLAST P searches of the GenBank database, these ORFs were shown to share significant homologies with a number of other prokaryotic genes.

Assessing virulence of vaccine strains of Mycobacterium avium subspecies paratuberculosis in a calf model

The purpose of this investigation was to characterise the virulence of two Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis) vaccine strains and compare them with a recent virulent isolate in new born calves over a time course of 8 months post-inoculation. Paratuberculosis-free new born calves were inoculated orally with either a vaccine strain (2e or 316F) or a wild type strain (F13) of M.a. paratuberculosis. Blood and faecal samples were collected throughout the experiment to analyse immune responses to infection and assess faecal shedding of M.a. paratuberculosis. Tissue samples were taken at post-mortem for histological examination and bacteriological culture. Cell-mediated immune responses were measured using a Bovigam (CSL) interferon-gamma assay. At 20 weeks post-inoculation there was a significant increase in the cell-mediated immune responses in calves infected with the wild type strain relative to the two vaccine strains. Acid fast bacteria were detected in the faeces of calves in all three groups between 4 and 8 weeks post-inoculation. Histopathology was unrewarding in all three groups. M.a. paratuberculosis was recovered only from tissues of calves inoculated with the wild type strain. Therefore, it appeared that the vaccine strains used in this study had reduced virulence. Identifying the genes responsible for pathogenesis observed in the wild type isolate and reduced or inactive in these vaccine isolates may offer a valuable resource for improving our knowledge of pathogenesis and permit the development of improved diagnostic reagents and vaccines for the control of M.a. paratuberculosis in livestock.

Unique expression of a highly conserved mycobacterial gene in IS901(+) Mycobacterium avium.

Expression of a gene encoding a novel protein antigen of 40 kDa (p40) was detected in IS901(+) strains of Mycobacterium avium, but not in any other species or subspecies of Mycobacterium tested, including IS901(-) M. avium and the other members of the M. avium complex. Although Southern hybridization revealed that the p40 gene is widely distributed within the genus, expression of the antigen could not be detected on Western blots of mycobacterial cell lysates. Nucleotide sequence analysis of the cloned p40 gene, and a database search, revealed high levels of sequence identity with a homologous gene in IS901(-) M. avium, M. avium subsp. paratuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium smegmatis and Mycobacterium tuberculosis. Further analysis of upstream sequences identified a putative promoter region. The p40 gene is the first example of a gene that is widely distributed within the genus Mycobacterium but expressed only in association with the presence of a genomic insertion element, in this case IS901, in strains of M. avium isolated from birds and domestic livestock.