Ovine footrot is a contagious disease affecting the hooves of sheep. The principal causative agent is the anaerobic bacterium, 'Dichelobacter nodosus'. Different strains of 'D. nodosus' cause disease of differing severity, ranging from benign to virulent. The severity of the disease is also affected by environmental factors, such as temperature and rainfall, and by the breed of sheep. In the early stages of infection, it is difficult to distinguish between benign and virulent footrot by examination of the hooves of affected sheep. However, early diagnosis is essential for footrot management, as sheep affected by virulent, (but not benign), footrot are subject to quarantine. A variety of laboratory tests have been used for the diagnosis of virulent footrot. ... At the University of New England, located in the prime wool producing area of the northern tablelands of NSW, we have been using DNA analysis to investigate genes involved with virulence in 'D. nodosus'.

The Cicerone Project was formed in 1998 to address problems faced by wool producers. In the New England area, the issue of suspected false positive diagnoses of virulent footrot, which can be a significant cause of economic loss to individual producers, was investigated. In New South Wales, footrot diagnosis is primarily a field diagnosis supported by the gelatin gel laboratory test. The principal causative agent of footrot is 'Dichelobacter nodosus'. If the gelatin gel test finds strains of 'D. nodosus' to be thermostable (gel stable), a finding of virulent footrot is likely and quarantine of the affected property follows. However, livestock producers and inspectors reported that there were a considerable number of cases where laboratory tests found strains to be stable but these strains did not cause virulent footrot in the field. Preliminary results using DNA markers associated with virulent footrot showed that one of these markers, 'intA', was absent in gel stable, field benign strains but present in all strains tested which caused field virulent footrot.Atrial conducted at Uralla, New South Wales, demonstrated conclusively that there were strains of 'D. nodosus' which were stable in the gelatin gel test but did not cause virulent footrot in the field. All of these strains were negative in the 'intA' DNA test. These results were confirmed in a second field trial at Molong, New South Wales. These trials were instrumental in establishing that the gelatin gel test at times gave results inconsistent with the clinical expression of footrot, potentially leading to a false positive diagnosis of virulent footrot. Subsequent research led to confirmation of the 'intA' test, which is now available as an additional tool for footrot diagnosis.

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, 'xprG2' revertants were isolated and shown to carry mutations in genes which we have named 'sogA-C' (suppressors of 'xprG'). The translocation breakpoint in the 'sogA1' mutant was localized to a homolog of 'Saccharomyces cerevisiae VPS5' and mapping data indicated that 'sogB' was tightly linked to a 'VPS17' homolog. Complementation of the 'sogA1' and 'sogB1' mutations and identification of nonsense mutations in the 'sogA2' and 'sogB1' alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in 'sogA-C' permit secretion of intracellular proteases, as in 'S. cerevisiae vps5' and 'vps17' mutants. In contrast to 'S. cerevisiae', the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG-independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' is the principal causative agent of footrot in sheep. The 'intA', 'intB' and 'intC' elements are mobile genetic elements which integrate into two tRNA genes downstream from 'csrA' (formerly 'glpA') and 'pnpA' in the 'D. nodosus' chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of 'pnpA'. We have proposed a model in which virulence in 'D. nodosus' is controlled in part by the integration of genetic elements downstream from 'csrA' and 'pnpA', altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the 'intD' element, which is 32 kb in size and contains an integrase gene, 'intD', several genes related to genes on other integrated elements of 'D. nodosus', a type IV secretion system and a putative mobilisation region, suggesting that the 'intD' element has a role in the transfer of other genetic elements. Most of the 'D. nodosus' strains examined which contained the 'intD' gene were benign, with 'intD' integrated next to 'pnpA', supporting our previous observation that virulent strains of 'D. nodosus' have the 'intA' element next to 'pnpA'.

Based on morphological criteria, fungi are often separated into two groups: yeasts and filamentous fungi. Uncellular fungi are known as yeasts. The extraction of nucleic acids from yeasts cells is described in Chapter 9. Filamentous fungi form a mycelia consisting of multinucleate, tubular hyphae, which may be separated into compartments by septa. Some fungi are dimorphic, that is, they can exist in a unicellular or multinucleate form. The switch from filamentous to pathogenic yeast form is temperature dependent in the human dimorphic pathogens 'Coccidioides immitis', 'Blastomyces dermatitidis', 'Histoplasma capsulatum', and 'Paracoccidioides brasiliensis'. In the dimorphic plant pathogen, 'Ustilago maydis', the filamentous form, which is produced by mating, is infectious while the fungus grows in culture as a yeast. Some groups of fungi belonging to the phylum Chytridiomycota do not form a true mycelium. Unlike other fungi, chytrids produce motile spores possessing a flagellum.

Footrot is a mixed bacterial infection of the hooves of sheep. The Gram-negative anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of 'D. nodosus' from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for 'D. nodosus' strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.

In 'Aspergillus nidulans', production of extracellular proteases in response to carbon starvation and to a lesser extent nitrogen starvation is controlled by XprG, a putative transcriptional activator. In this study the role of genes involved in carbon catabolite repression and the role of protein as an inducer of extracellular protease gene expression were examined. The addition of exogenous protein to the growth medium did not increase extracellular protease activity whether or not additional carbon or nitrogen sources were present indicating that induction does not play a major role in the regulation of extracellular proteases. Northern blot analysis confirmed that protein is not an inducer of the major 'A. nidulans' protease, PrtA. Mutations in the 'creA', 'creB' and 'creC' genes increased extracellular protease levels in medium lacking a carbon source suggesting that they may have a role in the response to carbon starvation as well as carbon catabolite repression. Analysis of 'glkA4 frA2' and 'creAΔ4' mutants showed that the loss of glucose signalling or the DNA-binding protein which mediates carbon catabolite repression did not abolish glucose repression but did increase extracellular protease activity. This increase was XprG-dependent indicating that the effect of these genes may be through modulation of XprG activity.

The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' carries several genetic elements that integrate into the chromosome. These include the 'intA', 'intB', 'intC' and 'intD' elements, which integrate adjacent to 'csrA' and 'pnpA', two putative global regulators of virulence and the virulence-related locus, 'vrl', which integrates into 'ssrA'. Treatment of 'D. nodosus' strains with ultraviolet light resulted in the isolation of DinoHI, a member of the 'Siphoviridae' and the first bacteriophage to be identified in 'D. nodosus'. Part of the DinoHI genome containing the packaging site is found in all 'D. nodosus' strains tested and is located at the end of the 'vrl', suggesting a role for DinoHI in the transfer of the 'vrl' by transduction. Like the intB element, the DinoHI genome contains a copy of 'regA' which has similarity to the repressors of lambdoid bacteriophages, suggesting that the maintenance of DinoHI and the intB element may be co-ordinately controlled.

'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof 'D. nodosus' strains are classified as benign, intermediate or virulent depending upon the severity of disease they cause in sheep. Previous work has led to the identification of a virulence-associated DNA region in 'D. nodosus' virulent strain Al98, called the 'vap' element which may have a role in virulence. However, there is no transformation system for 'D. nodosus' which precludes direct testing of the role of these and other genes in virulence. In this work, a native 'D. nodosus' plasmid pDN1 was isolated, sequenced, characterised and modified to contain appropriate antibiotic resistance markers and a multiple cloning site. Derivatives of pDN1 were subsequently used in transformation experiments in an effort to develop a transformation system for 'D. nodosus'. In the absence of a transformation system, more indirect methods were utilised to determine whether the 'vap' genes of 'D. nodosus' have a role in virulence. Past investigations have concentrated on analysis of the 'vap' genes at the DNA level. In this work, northern blot experiments were undertaken to determine whether the 'vap' genes are expressed at the RNA level in the virulent and/or benign strains in which they are present. Results indicated that in general, the 'vap' genes are expressed in the virulent and benign strains in which they are present, and although differential expression of the 'vap' genes was observed, the differences observed were not related to the virulence of the isolates. In addition to the 'vap' element, part of an 'intB' element was previously identified. In an effort to further characterise the 'intB' element, chromosome walking 4.2 kb downstream of regions which were determined previously was undertaken. Results suggest that these downstream sequences, which include genes 'gepC-gepG' are not part of an integrated genetic element. In addition, Southern blot analysis was utilised in order to determine the prevalence, arrangement and the integrity of the 'intB' element in seventeen strains of 'D. nodosus'.

The Aspergillus nidulans xprG gene is involved in the regulation of extracellular proteases. A plasmid which complemented the xprG2 mutation was shown to carry the phoG gene, reported to encode an acid phosphatase. Two phoGDelta mutants were constructed and were identical in phenotype to an xprG2 mutant. Null mutants were unable to use protein as a carbon or nitrogen source, have lost a repressible acid phosphatase and have pale conidial color. XprG shows similarity to the Ndt80 transcriptional activator, which regulates the expression of genes during meiosis in Saccharomyces cerevisiae. The xprG1 gain-of-function mutant contains a missense mutation in the region encoding the putative DNA-binding domain. The response to carbon, nitrogen, sulfur, and phosphate limitation is altered in xprG(-) mutants suggesting that XprG is involved in a general response to starvation. Ndt80 may also be involved in sensing nutritional status and control of commitment to meiosis in S. cerevisiae.