Detection and quantification of Marek's disease viruses using real-time polymerase chain reaction in separate and duplex assays
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.
Mutations in Genes Encoding Sorting Nexins Alter Production of Intracellular and Extracellular Proteases in 'Aspergillus nidulans'
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.
Influence of egg shell translucency on egg shell penetration by bacteria
Egg shell translucency, which can be due to changes in the mammillary cores and mamillary layer during the early phases of eggshell formation, has the potential to increase the incidence of microcracks in egg shells, and hence, may facilitate bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by 'Salmonella' Infantis. 'Salmonella' Infantis was able to penetrate translucent egg shells even at very low doses. The penetration, however, appeared to be hindered in both translucent and non-translucent eggs at 4°C, as compared with room temperature which highlights the importance of storage of eggs at refrigeration temperatures.
Effects of infectious bronchitis virus vaccine on the oviduct of hens
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.
Improved Diagnosis of Footrot Using DNA Technology
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'.
Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR
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.
Recovery of 'Salmonella' and 'Escherichia coli' from commercial egg shells and effect of translucency on bacterial penetration in eggs
This experiment was conducted to study the prevalence of 'Salmonella' and 'Escherichia coli' ('E. coli'). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. 'Salmonella' spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five 'E. coli' isolates were isolated from the egg shell surface. Ten 'E. coli' strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five 'E. coli' isolates using primers for heat stable enterotoxin genes A and B (STa and STb) and also for colicin V gene (cvaC). STa gene was detected in four 'E. coli' isolates isolated from egg shell surfaces. All the 'E. coli' isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent 'E. coli' and 'Salmonella' spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of 'Salmonella' outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by 'Salmonella' Infantis and 'E coli'. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4°C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.
Producer-initiated field research leads to a new diagnostic test for footrot
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.
Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis
Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the 'meq' gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n = 20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.
The 'intD' mobile genetic element from 'Dichelobacter nodosus', the causative agent of ovine footrot, is associated with the benign phenotype
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'.