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.

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.

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.

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.

Hens were vaccinated during the rearing phase with infectious bronchitis virus (IBV) vaccines commercially available in Australia (Vic S and A3) or left unvaccinated and then challenged with the N1/88 strain of IBV at 30 wk of age. Oviduct and fecal samples were collected at regular intervals after N1/88 challenge. A locked nucleic acid probe-based reverse transcription real-time PCR test was designed and used to detect the IBV strain N1/88 from the oviduct and feces of unvaccinated and vaccinated laying hens. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 100 copies and independent assay runs showed reproducible threshold cycle values. Viral RNA was detected in the oviduct of 12 unvaccinated then challenged hens and viral RNA increased sharply on d 10 and 12 postinfection (p.i.). By contrast, among the hens in the vaccinated group, N1/88 was detectable only in the oviduct of 2 hens at 8 and 12 d p.i. N1/88 challenge. Viral RNA was detected in feces of 2 unvaccinated hens up to 4 wk p.i. and in 1 vaccinated hen up to 3 wk p.i. This shows that rearing phase vaccination lowers the total viral RNA of the strain N1/88, even though this strain shows considerable antigenic and genetic variation from the vaccine strain. This new test will be useful for the rapid identification of the N1/88 strain of IBV from oviduct and fecal samples.

The anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof. Although the bacterium only survives for a few days in soil, this period is crucial for transmission of the disease as sheep are infected by walking through soil or pasture contaminated with infectious bacteria. The 'D. nodosus' genome is only 1.3Mb in size and has a dearth of genes encoding regulatory proteins. A series of genetic elements which integrate into the genome has been identified and we have proposed that these integrated genetic elements control the expression of adjacent genes encoding global regulators of virulence. The intA, intB, intC and intD elements integrate next to csrA or pnpA while the vrl integrates next to ssrA. CsrA, PnpA and the ssrA gene product, a 10SaRNA, have been shown to act as global virulence regulators in other bacteria. We have also identified a bacteriophage, DinoHI, which is integrated into the genome of some 'D. nodosus' strains. Sequence analyses suggest that there are many possible interactions between these integrated genetic elements. The vrl contains a copy of the DinoHI packaging site, indicating that the vrl may be transferred between strains by the bacteriophage. DinoHI and the intB element have a common repressor gene, suggesting that maintenance of the integrated state of these two genetic elements is co-ordinately controlled. Similarly, a DNA segment resembling the bacteriophage P4 immunity region is present on the intA, intC and intD elements and may be responsible for maintaining these three genetic elements in the integrated state. The features of the intD element suggest that it is self-transmissible and also capable of mobilising the intA element. Exchange of sequences between these genetic elements may also occur. We discuss here evidence for a network of interactions between these genetic elements with implications for the control of virulence in 'D. nodosus'.

The Gram-negative anaerobe 'Dichelobacter nodosus' is the causative agent of footrot in sheep. Different strains of 'D. nodosus' cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of 'D. nodosus' and found that PNPase activity is lower in virulent strains. We disrupted the 'pnpA' gene in three benign 'D. nodosus' strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the 'pnpA' gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of 'D. nodosus'.

Two real-time PCR assays were developed which enable quantification and differentiation between pathogenic Australian isolates of 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.

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with 'meq' gene polymorphism. Unvaccinated and herpes virus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94%)A, of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDVI vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The 'meq' gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the 'meq' sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four pro lines (PPPP repeats) in the 'meq' gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.