ABSTRACT Current European Commission (EC) surveillance regulations require discriminatory testing of all transmissible spongiform encephalopathy (TSE)-positive small ruminant (SR) samples in order to classify them as bovine spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests, including characterization by bioassay in mouse models. Since 2005, naturally occurring BSE has been identified in two goats. It has also been demonstrated that more than one distinct TSE strain can coinfect a single animal in natural field situations. This study assesses the ability of the statutory methods as listed in the regulation to identify BSE in a blinded series of brain samples, in which ovine BSE and distinct isolates of scrapie are mixed at various ratios ranging from 99% to 1%. Additionally, these current statutory tests were compared with a new in vitro discriminatory method, which uses serial protein misfolding cyclic amplification (sPMCA). Western blotting consistently detected 50% BSE within a mixture, but at higher dilutions it had variable success. The enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only when it was present as 99% of the mixture, with variable success at higher dilutions. Bioassay and sPMCA reported BSE in all samples where it was present, down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at 0.1%. While bioassay is the only validated method that allows comprehensive phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears to offer a fast and cost-effective alternative for the screening of unknown isolates when the purpose of the investigation was solely to determine the presence or absence of BSE.

Background The infectious agent responsible for the bovine spongiform encephalopathy (BSE) epidemic in Great Britain is a transmissible spongiform encephalopathy (TSE) strain with uniform properties but the origin of this strain remains unknown. Based on the hypothesis that classical BSE may have been caused by a TSE strain present in sheep, cattle were inoculated intracerebrally with two different pools of brains from scrapie-affected sheep sourced prior to and during the BSE epidemic to investigate resulting disease phenotypes and characterise their causal agents by transmission to rodents. Results As reported in 2006, intracerebral inoculation of cattle with pre-1975 and post-1990 scrapie brain pools produced two distinct disease phenotypes, which were unlike classical BSE. Subsequent to that report none of the remaining cattle, culled at 10 years post inoculation, developed a TSE. Retrospective Western immunoblot examination of the brains from TSE cases inoculated with the pre-1975 scrapie pool revealed a molecular profile similar to L-type BSE. The inoculation of transgenic mice expressing the bovine, ovine, porcine, murine or human prion protein gene and bank voles with brains from scrapie-affected cattle did not detect classical or atypical BSE strains but identified two previously characterised scrapie strains of sheep. Conclusions Characterisation of the causal agents of disease resulting from exposure of cattle to naturally occurring scrapie agents sourced in Great Britain did not reveal evidence of classical or atypical BSE, but did identify two distinct previously recognised strains of scrapie. Although scrapie was still recognizable upon cattle passage there were irreconcilable discrepancies between the results of biological strain typing approaches and molecular profiling methods, suggesting that the latter may not be appropriate for the identification and differentiation of atypical, particularly L-type, BSE agents from cattle experimentally infected with a potential mixture of classical scrapie strains from sheep sources.

BACKGROUND:Atypical bovine spongiform encephalopathies (BSEs), classified as H-type and L-type BSE based on the Western immunoblot profiles, are naturally occurring diseases in cattle, which are phenotypically different to classical BSE. Transmission studies in cattle using the intracerebral route resulted in disease where the phenotypes were maintained irrespective of BSE type but clinically affected cattle with a shorter survival time displayed a nervous form whereas cattle with a longer survival time displayed a dull form. A second transmission study is reported here where four cattle were intracerebrally inoculated with brain tissue from experimentally infected cattle presenting with either the nervous or dull form of H- or L-type BSE to determine whether the phenotype is maintained.RESULTS:The four inoculated cattle were culled at 16.5-19.5 months post inoculation after presenting with difficulty getting up, a positive scratch response (all) and dullness (three cattle), which was not observed in two non-inoculated control cattle, each housed with either group of inoculated cattle. Only the inoculated cattle had detectable prion protein in the brain based on immunohistochemical examination, and the Western immunoblot profile was consistent with the H-type or L-type BSE of the respective donor cattle.CONCLUSIONS:Second passage of H-type and L-type BSE in cattle produced a TSE where the majority of cattle displayed the dull form regardless of clinical disease form of the donor cattle. The pathological and molecular phenotypes of H- and L-type BSE were maintained.

Development of transgenic mouse models expressing heterologous prion protein (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimaeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the ovine AHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrP(AHQ))EM16 (EM16) mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154–178 days post-inoculation (p.i.), 100% disease penetrance and early Western blot detection of protease-resistant fragments (PrP(res)) of the disease-associated isoform, PrP(Sc), in EM16 brain from 110 days p.i. onwards. EM16 mice were also highly susceptible to classical scrapie and bovine spongiform encephalopathy (BSE), with mean IPs 320 and 246 days faster, respectively, than WT mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrP(res) profiles associated with disease in the natural host were faithfully maintained in EM16 mice, and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrP(Sc) deposition in brain sections from terminal phase transmissible spongiform encephalopathy-challenged EM16 mice. The findings indicate that EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle which was first observed in Great Britain (GB) in 1986. Throughout the subsequent BSE epidemic, cases identified by passive surveillance have shown consistent histopathological, immunohistochemical, biochemical and biological properties. However, since the start of active surveillance in 2001, across Europe and elsewhere, approximately 67 cases with different biochemical characteristics have been identified by Western blotting (WB). These cases fall into two categories; 'H-type' (H-BSE) or 'L-type' (L-BSE), based on the relatively heavy (H-BSE) or light (L-BSE) mass of the unglycosylated band of the prion protein, as compared with WB against that obtained from classical BSE (C-BSE) cases. Here we report the detection and confirmation of the first four L-BSE cases by active surveillance in GB, two of which were born after the reinforced feed ban of 1996 (BARB cases). These four L-BSE cases were found in relatively old cattle (age range; 11-21 years old) and the carcases did not enter the human food chain or animal feed chains.

Oral necrobacillosis (lumpy jaw) is known as one of the most common and significant diseases in captive macropods (Vogelnest and Portas 2008). The most important pathogen is the anaerobic bacteria, Fusobacterium necrophorum (Samuel 1983). Many studies have shown dental calculus as one of the risk factors for oral necrobacillosis, oral injury, feed contaminated with faeces, adverse environmental conditions, and molar exfoliation (Butler 1981, Oliphant and others 1984, Vogelnest and Portas 2008). In the present study, we conducted a retrospective study to reveal recurrence and evaluate the risk factors in swamp wallabies that presented with oral necrobacillosis. We showed that oral necrobacillosis recurred in all cases after apparent complete recovery, and that advanced age and cold weather were the contributing risk factors. Seventy-five swamp wallabies were housed at the Nogeyama Zoological Gardens from 1987 to 2011. For this retrospective study, we selected 54 individuals for which clinical records had been maintained from birth to death. The average age at the onset of the first lesion was examined. Further, the number of diseased individuals and the rate of diseased swamp wallabies were counted and calculated, respectively, at each age. The location(s) and number of lesion(s) were counted and analysed in each month, which also included recurrence cases. The average temperature of …

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).

In sheep infected experimentally with the bovine spongiform encephalopathy (BSE) agent, amplification of infectivity in peripheral organs during early preclinical stages is thought to contribute to high titres of the agent being detected in blood, with subsequent haematogenous neuroinvasion through the circumventricular organs (CVOs). In contrast, little disease-associated prion protein (PrP(d)) or infectivity is detected in the peripheral tissues of cattle during the preclinical and clinical stages of BSE. The aim of this study was to investigate immunohistochemically the role of haematogenous neuroinvasion in cattle with spontaneously arising and experimentally induced BSE. There was almost complete absence of PrP(d) in the peripheral organs of BSE infected cattle. Additionally, there was minimal involvement of the CVOs during preclinical disease and there was progressive caudorostral accumulation of PrP(d) in the brain. These findings do not support haematogenous neuroinvasion in the bovine disease.

BACKGROUND:The majority of atypical bovine spongiform encephalopathy (BSE) cases so far identified worldwide have been detected by active surveillance. Consequently the volume and quality of material available for detailed characterisation is very limiting. Here we report on a small transmission study of both atypical forms, H- and L-type BSE, in cattle to provide tissue for test evaluation and research, and to generate clinical, molecular and pathological data in a standardised way to enable more robust comparison of the two variants with particular reference to those aspects most relevant to case ascertainment and confirmatory diagnosis within existing regulated surveillance programmes.RESULTS:Two groups of four cattle, intracerebrally inoculated with L-type or H-type BSE, all presented with a nervous disease form with some similarities to classical BSE, which progressed to a more dull form in one animal from each group. Difficulty rising was a consistent feature of both disease forms and not seen in two BSE-free, non-inoculated cattle that served as controls. The pathology and molecular characteristics were distinct from classical BSE, and broadly consistent with published data, but with some variation in the pathological characteristics. Both atypical BSE types were readily detectable as BSE by current confirmatory methods using the medulla brain region at the obex, but making a clear diagnostic distinction between the forms was not consistently straightforward in this brain region. Cerebellum proved a more reliable sample for discrimination when using immunohistochemistry.CONCLUSIONS:The prominent feature of difficulty rising in atypical BSE cases may explain the detection of naturally occurring cases in emergency slaughter cattle and fallen stock. Current confirmatory diagnostic methods are effective for the detection of such atypical cases, but consistently and correctly identifying the variant forms may require modifications to the sampling regimes and methods that are currently in use.

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrPSc) in the duodenum, jejunum and ileum of cattle orally exposed to a 1g or 100g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrPSc was detected in the duodenum or jejunum of animals exposed to a 1g dose or in the duodenum of animals receiving a 100g dose. PrPSc was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100g dose. The frequency of PrPSc- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrPSc in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrPSc detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.

Prions are largely contained within the nervous and lymphoid tissue of transmissible spongiform encephalopathy (TSE) infected animals. However, following advances in diagnostic sensitivity, PrP(Sc), a marker for prion disease, can now be located in a wide range of viscera and body fluids including muscle, saliva, blood, urine and milk, raising concerns that exposure to these materials could contribute to the spread of disease in humans and animals. Previously we demonstrated low levels of infectivity in the liver of sheep experimentally challenged with bovine spongiform encephalopathy. In this study we show that PrP(Sc) accumulated in the liver of 89% of sheep naturally infected with scrapie and 100% of sheep challenged with BSE, at both clinical and preclinical stages of the disease. PrP(Sc) was demonstrated in the absence of obvious inflammatory foci and was restricted to isolated resident cells, most likely Kupffer cells.

Background Transmission of the prion disease bovine spongiform encephalopathy (BSE) occurred accidentally to cattle and several other mammalian species via feed supplemented with meat and bone meal contaminated with infected bovine tissue. Prior to United Kingdom controls in 1996 on the feeding of mammalian meat and bone meal to farmed animals, the domestic chicken was potentially exposed to feed contaminated with the causal agent of BSE. Although confirmed prion diseases are unrecorded in avian species a study was undertaken to transmit BSE to the domestic chicken by parenteral and oral inoculations. Transmissibility was assessed by clinical monitoring, histopathological examinations, detection of a putative disease form of an avian prion protein (PrP) in recipient tissues and by mouse bioassay of tissues. Occurrence of a progressive neurological syndrome in the primary transmission study was investigated by sub-passage experiments. Results No clinical, pathological or bioassay evidence of transmission of BSE to the chicken was obtained in the primary or sub-passage experiments. Survival data showed no significant differences between control and treatment groups. Neurological signs observed, not previously described in the domestic chicken, were not associated with significant pathology. The diagnostic techniques applied failed to detect a disease associated form of PrP. Conclusion Important from a risk assessment perspective, the present study has established that the domestic chicken does not develop a prion disease after large parenteral exposures to the BSE agent or after oral exposures equivalent to previous exposures via commercial diets. Future investigations into the potential susceptibility of avian species to mammalian prion diseases require species-specific immunochemical techniques and more refined experimental models.

Bovine spongiform encephalopathy (BSE) is a prion disease of domesticated cattle, first identified in Great Britain (GB) in 1986. The disease has been characterized by histopathological, immunohistochemical, biochemical and biological properties, which have shown a consistent disease phenotype among cases obtained by passive surveillance. With the advent of active surveillance in 2001, immunological tests for detection of the prion protein revealed some cases with different biochemical characteristics and, in certain instances, differences in pathology that have indicated variant phenotypes and the possibility of agent strain variation. This study examines a case set of 523 bovine brains derived from archived material identified through passive surveillance in GB. All cases conformed to the phenotype of classical BSE (BSE-C) by histopathological, immunohistochemical and biochemical approaches. The analyses consolidated an understanding of BSE-C and, by western blotting, confirmed differentiation from the known atypical BSE cases which exhibit higher or lower molecular masses than BSE-C (BSE-H and BSE-L respectively).

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.

Primary isolation of bovine spongiform encephalopathy (BSE) in RIII mice generates a lesion profile believed to be reproducible and distinct from that produced by classical scrapie. This profile, which is characterized by peaks at gray matter areas 1, 4 and 7 (dorsal medulla, hypothalamus and septal nuclei), is used to diagnose BSE on primary isolation. The aim of this study was to investigate whether the BSE agent could be present in sheep diagnosed with classical scrapie, using lesion profiles in RIII mice as a discriminatory method. Sixty-two positive scrapie field cases were collected from individual farms between 1996 and 1999 and bioassayed in RIII mice. Fifty-five of these isolates transmitted successfully to at least one mouse. Of the 31 that produced adequate data to allow lesion profile analysis, 10 showed a consistent profile with peaks at brain areas 1, 4 and 7. All inocula for this subgroup were derived from sheep of genotype ARQ/ARQ. While the 1-4-7-scrapie profile exhibited similarities to BSE in RIII mice at primary isolation, it was distinguishable based on histopathology, immunohistochemistry and cluster analysis. We conclude that caution should be taken to distinguish this profile from BSE and that additional parameters should be considered to reach a final diagnosis.

To investigate the relative involvement of the olfactory region in classical bovine spongiform encephalopathy (BSE), immunohistochemical labeling of prion protein scrapie (PrP(Sc)) was scored in the brainstem, frontal cerebral cortex, and olfactory bulb of cattle with natural and experimental clinical cases of BSE in Great Britain. The intensity of immunolabeling was greatest in the brainstem, but PrP(Sc) was also detected in the olfactory bulb and the cerebral cortex. A diffuse, nonparticulate labeling, possibly due to abundance of cellular PrP, was consistently observed in the olfactory glomeruli of the cases and negative controls. Involvement of the olfactory bulb in BSE and other naturally occurring TSEs of animals raises speculation as to an olfactory portal of infection or a route of excretion of the prion agent.