Prions are proteins that can exist in two (or more) folding states, a normal or cellular form and a series of infectious or prion forms, which are prone to aggregate. The prion form can induce conversion of the cellular form and so transmit phenotypic effects of this structural rearrangement within and between cells and organisms. The conversion of PrPC, the mammalian prion glycoprotein, to its prion form, PrPSc, in the brain is a precursor to progressive neurological degeneration, and the various folded forms of PrPSc (defined by the size and glycosylation of protease-resistant core peptides of the PrP aggregates, PrPres) are characteristic of a particular neurodegenerative phenotype or prion disease. Here, quantitative multiplex mass spectrometry was used for N-terminal amino acid profiling (N-TAAP) of PrPres from sheep affected by scrapie, the prion disease of small ruminants, to rapidly assess the diversity of prions within particular flocks. In 29 cases, PrPres concentrations varied from below the limit of detection (350fmol/g) to 15pmol/g wet brain. Although most had a single N-TAAP profile, two novel variants were identified: one common to the ARH/ARQ animals in this study and one in an animal of the wild-type sheep PrP genotype (ARQ/ARQ).

The dose-response of cattle exposed to the bovine spongiform encephalopathy (BSE) agent is an important component of modelling exposure risks for animals and humans and thereby, the modulation of surveillance and control strategies for BSE. In two experiments calves were dosed orally with a range of amounts of a pool of brainstems from BSE-affected cattle. Infectivity in the pool was determined by end-point titration in mice. Recipient cattle were monitored for clinical disease and, from the incidence of pathologically confirmed cases and their incubation periods (IPs), the attack rate and IP distribution according to dose were estimated. The dose at which 50 % of cattle would be clinically affected was estimated at 0.20 g brain material used in the experiment, with 95 % confidence intervals of 0.04-1.00 g. The IP was highly variable across all dose groups and followed a log-normal distribution, with decreasing mean as dose increased. There was no evidence of a threshold dose at which the probability of infection became vanishingly small, with 1/15 (7 %) of animals affected at the lowest dose (1 mg).

BACKGROUND:New and emerging diseases of livestock may impact animal welfare, trade and public health. Early detection of outbreaks can reduce the impact of these diseases by triggering control measures that limit the number of cases that occur. The aim of this study was to investigate whether prospective spatiotemporal methods could be used to identify outbreaks of new and emerging diseases in scanning surveillance data. SaTScan was used to identify clusters of unusually high levels of submissions where a diagnosis could not be reached (DNR) using different probability models and baselines. The clusters detected were subjected to a further selection process to reduce the number of false positives and a more detailed epidemiological analysis to ascertain whether they were likely to represent real outbreaks.RESULTS:187,925 submissions of clinical material from cattle were made to the Regional Laboratory of the Veterinary Laboratories Agency (VLA) between 2002 and 2007, and the results were stored on the VLA FarmFile database. 16,925 of these were classified as DNRs and included in the analyses. Variation in the number and proportion of DNRs was found between syndromes and regions, so a spatiotemporal analysis for each DNR syndrome was done. Six clusters were identified using the Bernoulli model after applying selection criteria (e.g. size of cluster). The further epidemiological analysis revealed that one of the systemic clusters could plausibly have been due to Johne's disease. The remainder were either due to misclassification or not consistent with a single diagnosis.CONCLUSIONS:Our analyses have demonstrated that spatiotemporal methods can be used to detect clusters of new or emerging diseases, identify clusters of known diseases that may not have been diagnosed and identify misclassification in the data, and highlighted the impact of data quality on the ability to detect outbreaks. Spatiotemporal methods should be used alongside current temporal methods for analysis of scanning surveillance data. These statistical analyses should be followed by further investigation of possible outbreaks to determine whether cases have common features suggesting that these are likely to represent real outbreaks, or whether issues with the collection or processing of information have resulted in false positives.

BACKGROUND:Various clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrPd) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations.RESULTS:Based on the display of selected behavioural, sensory and locomotor changes, 20 (67%) orally dosed and 17 (77%) intracerebrally inoculated pathologically confirmed BSE cases and 21 (13%) orally dosed and 18 (6%) intracerebrally inoculated but unconfirmed cases were considered clinical BSE suspects. None of 103 controls showed significant signs and were all negative on diagnostic postmortem examinations. Signs indicative of BSE suspects, particularly over-reactivity and ataxia, were more frequently displayed in confirmed cases with vacuolar changes in the brain. The display of several BSE-associated signs over time, including repeated startle responses and nervousness, was significantly more frequent in confirmed BSE cases compared to controls, but these two signs were also significantly more frequent in orally dosed cattle unconfirmed by postmortem examinations.CONCLUSIONS:The findings confirm that in experimentally infected cattle clinical abnormalities indicative of BSE are accompanied by vacuolar changes and PrPd accumulation in the brainstem. The presence of more frequently expressed signs in cases with vacuolar changes is consistent with this pathology representing a more advanced stage of disease. That BSE-like signs or sign combinations occur in inoculated animals that were not confirmed as BSE cases by postmortem examinations requires further study to investigate the potential causal relationship with prion disease.

New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease-resistant prion protein (PrPSc) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease-resistant products (PrPres) with partially variable N-termini. The conformation(s) of PrPSc and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrPres gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrPres N-terminal tryptic peptides by MS and thus to define the N-terminal amino acid profiles (N-TAAPs) of PrPres characteristic for various TSEs in sheep. The fragmentation behaviour of the N-terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrPres preparation methods were evaluated and the most effective approach applied to differentiate the N-TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N-TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE-strain specific diagnosis.

In parts of Great Britain and Ireland, Eurasian badgers (Meles meles) constitute a reservoir of Mycobacterium bovis infection and a potential source of infection for cattle. In vitro diagnostic tests for live badgers are an important component of strategies to control TB in this species. Immunological tests have been developed for badgers, although little is known about the influence of the age of the animal on test performance. To address this, we evaluated the performance of three immunological tests for badgers with respect to the age of the animal: the Brock Test and BrockTB STAT-PAK® serological tests and the recently developed interferon-gamma enzyme immunoassay (IFNγ EIA). Data published elsewhere suggested that seropositivity was associated with more progressive forms of TB in the badger. To gain further evidence for this, we used longitudinal data from a well-studied population of badgers to test for an association between the sensitivity of the Brock Test and the duration of TB infection.

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.

BACKGROUND:Although the epidemiology of scrapie has been broadly understood for many years, attempts to introduce voluntary or compulsory controls to eradicate the disease have frequently failed. Lack of precision in defining the risk factors on farm has been one of the challenges to designing control strategies. This study attempted to define which parts of the annual flock management cycle represented the greatest risk of infection to naive lambs exposed to the farm environment at different times.RESULTS:In VRQ/VRQ lambs exposed to infected sheep at pasture or during lambing, and exposed to the buildings in which lambing took place, the attack rate was high and survival times were short. Where exposure was to pasture alone the number of sheep affected in each experimental group was reduced, and survival times were longer and related to length of exposure.CONCLUSION:At the flock level, eradication and control strategies for scrapie must take into account the need to decontaminate buildings used for lambing, and to reduce (or prevent) the exposure of lambs to infected sheep, especially in the later stages of incubation, and at lambing. The potential for environmental contamination from pasture should also be considered. Genotype selection may still prove to be the only viable tool to prevent infection from contaminated pasture, reduce environmental contamination and limit direct transmission from sheep to sheep.

Milk specimens were collected from lactating cows that had previously been challenged with bovine spongiform encephalopathy (BSE)-infected brain at 4-6 months of age. One group of 10 animals received a single oral dose of 100 g, a second group received 1 g and the third was made up of unexposed controls. The cows were inseminated artificially, and calved at approximately 2 years of age and annually thereafter. Milking was done within the first week following calving and at 10-weekly intervals during the lactation period. Specimens were centrifuged to obtain a fraction enriched for somatic cells and these fractions were analysed for disease-associated, abnormal prion protein (PrP(BSE)) by using a modified commercial BSE ELISA and a different confirmatory assay. No abnormal prion protein has so far been identified in the cell fraction of milk from cattle incubating BSE by using these methods at their limits of detection.

We applied capture–recapture methodology (CRC) to data from three surveillance sources (statutory notification, abattoir survey (AS) and fallen stock (FS) survey) to estimate the number of holdings infected with scrapie in Great Britain and to assess the sensitivity of the surveillance network. Between January 1, 2002 and March 31, 2003, 144 holdings were identified by the three sources. Using CRC modelling techniques, we estimated a minimum lower bound for the total number of holdings infected as 642. A biologically plausible positive dependence between the statutory reporting and the fallen stock survey was found statistically significant. The sensitivity of the three sources combined was very low. The integration of the three overlapping sources provided a better understanding of the interactions within the surveillance network. However, the scarcity of the data and reduced overlapping among sources only allowed for very cautious inferences to be drawn about the true proportion of scrapie affected holdings in the national population. Future surveys and surveillance activities should be planned such that the resulting data can be used more effectively as part of CRC modelling approaches.

Background/aims: A number of subjective methods have been used to quantify the extent of the cutaneous delayed-type hypersensitivity (DTH) reaction. However, because of their subjective nature, significant differences in measurements may be seen between individual observers or laboratories unless thorough training is given to each observer.Methods: Objective measurement of the DTH reaction using a hand-held spectrophotometer is described. Guinea pigs were primed using inoculation with Mycobacterium bovis Bacille Calmette-Guerin and challenged five weeks later in the shaved flank with three doses of bovine purified protein derivative. The extent of the ensuing DTH reaction was measured 24 and 48 h later. Spectrophotometric measurement of the reaction site was compared with a control region of skin on each animal and expressed as the change within a standard colour space. Data obtained with the spectrophotometer was compared with the subjective measurement of the area of the DTH reaction by an experienced operator.Results: The measurements obtained with the spectrophotometer correlated very closely with conventional measurement of the reaction area by a trained operator. The reaction size in square mm and changes along the red/green colour axis was correlated most strongly.Conclusion: Spectrophotometric measurement of the DTH reaction had advantages over conventional measuring techniques in terms of speed, reproducibility and reduced operator to operator variation. We conclude that the cutaneous DTH reaction may be simply and objectively quantified with the use of a hand-held spectrophotometer.