Development and initial validation of quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's Disease Virus (MDV) are described. Also described is the development of an internal control qPCR assay that detects the chicken α2(VI) collagen gene. To reduce costs, a duplex assay for MDV1 and the internal control was also developed. The MDV qPCR assays were specific to their target gene and more sensitive than standard PCR when compared using Australian field and vaccine strains of MDV. All assays were found to have acceptable reproducibility. Relative abundance (RA) of MDVI and HVT viruses was quantified using the relative standard curve method in twenty experimentally infected chickens over a period of 7-35 days post infection and was shown to vary during the course of the infection. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a variety of applications.

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'.

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.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.

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'.

In Australia, currently, all pullets reared for egg production are vaccinated with live attenuated strains of infectious bronchitis virus. Various vaccines and protocols to control this viral disease have been developed, although the severity of the disease varies from place to place and flock to flock. In the present trial, the effects of vaccine strains A3 and Vic S on the oviduct of laying hens were assessed by histopathology, electron microscopy, serology and also by determining the presence and persistence of viral RNA in the oviduct by real time PCR following the experimental infection. Birds were either unvaccinated or vaccinated with both A3 and Vic S and then mock-infected, challenged with the same attenuated strains, either A3 or Vic S. Some respiratory signs were observed, but were mild and short-lived. There was no drop in egg production in any of the groups. However, there was visual loss of shell colour in the unvaccinated hens challenged with the Vic S strain. Mild histopathology was recorded only in terms of lymphocyte infiltration and occasional submucosal oedema in the infundibulum and very mild gland dilatation in the magnum. Microscopic lesions were not recorded in the isthmus, tubular shell gland or shell gland pouch. Cilia loss was not observed in any region of the oviduct using scanning electron microscopy. Both the A3 and Vic S vaccine strains were detected in the oviduct of vaccinated and unvaccinated hens, mainly on the 12th day p.i. These results indicate that the A3 and Vic S strains replicate at a low level in the oviduct without causing significant damage and hence are safe for the oviduct.

Methods for taqman real-time PCR assays to detect the three serotypes of MDV are available (Islam et al., 2004), and an absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Thus, it is now possible to perform qPCR assays for all three serotypes ofMDV on a sample. Absolute quantification of MDV2 in dust samples from poultry farms across Australia in a preliminary study, revealed the presence of MDV2 in 13 of 30 samples tested.

Quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's disease virus (MDV) have been developed. An internal control qPCR assay that detects chicken α2 (VI) collagen gene was also developed to allow quantitation of MDV. To reduce costs and time, the assays for MDV1 and the internal control were combined into a duplex assay. The sensitivity, specificity, precision, and reproducibility of each assay are reported. The MDV qPCR assays were specific to their target gene when compared using Australian field and vaccine strains of MDV and 10–100-fold more sensitive than standard PCR. Using DNA from infected spleen tissue, the lower limit of detection of total DNA (viral and host combined) was 0.025 ng for the MDV1 and collagen assays, and 0.25 ng for the HVT and MDV2 assays. All assays were found to be highly reproducible for Ct values, but less so for calculated concentrations. MDV1 and HVT were quantitated in spleen tissue of twenty experimentally infected chickens 7–35 days after infection. The relative abundance of MDV1 exhibited a clear peak at day 14 post-infection, whereas HVT displayed an increasing trend over the 35 days post-infection. The duplex assay was optimized such that it was able to accurately quantitate MDV1 in samples of very high, medium, and very low relative abundance of MDV1. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a range of research and industry applications.

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.