M.J. Stack

Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update

Further preliminary observations are reported of an experiment to examine the spread of infectivity and the occurrence of pathological changes in cattle exposed orally to infection with bovine spongiform encephalopathy. Calves were dosed at four months of age and clinically monitored groups were killed sequentially from two to 40 months after inoculation. Tissues were collected for bioassay, for histopathological examinations and for the detection of PrP. Previous reported observations have included the presence of infectivity in the distal ileum of cattle killed after six to 18 months, the earliest onset of clinical signs in an exposed animal after 35 months, and diagnostic histopathological changes in the brain, in association with clinical disease, after 36, 38 and 40 months. In spite of the relative inefficiency of the bioassay of scrapie-like agents across a species barrier the new observations confirm that the onset of clinical signs and pathological changes in the central nervous system (CNS) occur at approximately the same time. The earliest pathological change, the presence of abnormal PrP 32 months after inoculation, coincided with the earliest detected infectivity in the CNS and occurred shortly before there was evidence of typical spongiform changes in the brain 36 months after inoculation. Infectivity has now been demonstrated in the peripheral nervous system, in the cervical and thoracic dorsal root ganglia 32 to 40 months after inoculation and in the trigeminal ganglion 36 and 38 months after inoculation. At the time of writing evidence of infectivity in other tissues is confined to the distal ileum, not only after six to 18 months but also after 38 and 40 months, but these findings may be supplemented by the results of further mouse assays. Nevertheless, they are in general agreement with current knowledge of the pathogenesis of scrapie.

Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies

Abstract. A panel of ruminant brain tissues were subjected to a Western immunoblotting technique using two monoclonal antibodies (mAbs). The resultant prion protein (PrP) glycoforms showed that three distinctions can be made between natural ovine scrapie cases and sheep experimentally inoculated with bovine spongiform encephalopathy (BSE). Differentiation between BSE-infected cattle and natural cases of sheep scrapie was also possible using these two antibodies. There were subtle differences in the molecular weight positions of the di-glycosylated, mono-glycosylated and unglycosylated forms of the abnormal PrP (PrPSc) associated with these ruminant transmissible spongiform encephalopathies. In particular, a distinct difference for the unglycosylated protein band was observed. For ovine scrapie samples, this band was noticeably of a higher molecular weight than that found for brain samples from the Romney and Cheviot breed sheep infected with BSE and, to a lesser degree, higher than that observed for bovine BSE samples. Using the comparison of glycoform ratios, the technique provided a distinction between the sheep experimentally infected with BSE and natural cases of sheep scrapie but did not provide a distinction between natural cases of bovine BSE and ovine scrapie. The sheep-passaged CH1641 scrapie strain gave molecular weights similar to, but not identical to BSE, and a glycoform ratio similar to ovine scrapie cases. The SSBP1 experimental scrapie strain gave molecular weights that were akin to natural scrapie cases but the glycoform ratio was different to that found for all the other samples. When mAb P4 was substituted for mAb 6H4 in the technique, only the natural scrapie samples and SSBP1 gave strong signals. BSE in sheep and the CH1641 strain gave weak reactions and PrPSc from BSE-infected cattle could not be detected at all. The results suggest that this combination of molecular weight and glycoform ratio analyses, and differentiation with two specific antibodies could be used to provide a possible screening test for BSE in the UK sheep flock, if confirmed as accurate by bioassay and lesion profile analysis in mice inoculated with brain tissue from suspect field cases.

Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE)

Abstract In this report we document the results of several independent studies testing the sensitivity, specificity and reliability of the Prionics Western blotting (PWB) procedure to detect bovine and ovine disease-specific, protease-resistant prion protein (PrPSc). Validation of the technique was obtained by blind analysis of samples from cattle affected with bovine spongiform encephalopathy (BSE), clinically normal animals or cattle with neurological diseases unrelated to BSE. Overall, very high sensitivity, specificity and reliability was observed. It became clear that sampling of the correct brain region and the method used for protein extraction are important factors for correct diagnosis. Furthermore, we tested the usefulness of the PWB technique as an instrument for surveillance purposes. We analyzed animals from a culling scheme as well as older animals from abattoirs to determine the number of subclinical BSE cases detectable by histopathological examination, immunohistochemistry for PrPSc and PWB. In both studies, BSE-affected animals with no overt clinical symptoms were detected. These results demonstrate the usefulness of the PWB procedure in surveillance systems serving as a rapid diagnostic tool to identify animals subclinically infected with BSE.

Diagnosis of transmissible spongiform encephalopathies in animals: a review

Transmissible spongiform encephalopathies (TSEs) in animals include, among others, bovine spongiform encephalopathy (BSE), scrapie, chronic wasting disease, and atypical forms of prion diseases. Diagnosis of TSEs is based on identification of characteristic lesions or on detection of the abnormal prion proteins in tissues, often by use of their partial proteinase K resistance property. Correctly sampling of target tissues is of utmost importance as this has a considerable effect on test sensitivity. Most of the rapid or screening tests are based on ELISA or Western immunoblot (WB) analysis, and many are officially approved. Confirmatory testing is normally performed by use of histologic examination, immunohistochemical analysis, certain WB protocols, or detection of prion fibrils by use of electron microscopy (scrapie-associated fibril). The discriminatory methods for diagnostic use are mostly based on WB technology and provide initial identification of the prion strain, particularly for differentiation of BSE from scrapie in small ruminants. Definitive prion strain characterization is performed by use of bioassays, usually in mice. A burgeoning number of transgenic mice have been developed for TSE studies. Development of new tests with higher sensitivity and of more reliable diagnostic applications for live animals tested for food safety reasons is a rapidly developing field. Ultimately, the choice of a test for TSE diagnosis depends on the rationale for the testing.

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa

Samples of tissue from the central nervous system (CNS), the lymphoreticular system (LRS) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrPd). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97·1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrPd was detected in 86·4 to 91·5 per cent of the other LRS tissues of the healthy sheep examined and in 77·7 per cent of their CNS tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrPd were observed in the rectal mucosa and other LRS tissues of VRQ/ARR sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.

Poxviral disease in red squirrels Sciurus vulgaris in the UK: spatial and temporal trends of an emerging threat.

The squirrel poxvirus (SQPV) is the probable mediator of apparent competition between the introduced invading gray squirrel (Sciurus carolinensis) and the red squirrel (Sciurus vulgaris) in the UK, and modeling studies have shown that this viral disease has had a significant impact on the decline of the red squirrel in the UK. However, given our limited understanding of the epidemiology of the disease, and more generally the effects of invasive species on parasite ecology, there is a need to investigate the transmission dynamics and the relative pathogenicity of the virus between species. We aimed to increase our knowledge of these processes through an empirical study in which we: (i) used pathological signs and transmission electron microscopy (TEM) to diagnose SQPV disease in red squirrels found dead during scanning surveillance between 1993 and 2005; (ii) detected antibody to SQPV using an enzyme-linked immunosorbent assay (ELISA) in the same animals; and (iii) mapped cases of the disease, and the gray squirrel distribution, using a geographical information system. We analyzed the distribution of cases of SQPV disease according to woodland type, a measure of squirrel density. SQPV disease occurred only in areas of England also inhabited by seropositive gray squirrels, and as the geographical range of gray squirrels expanded, SQPV disease occurred in these new gray squirrel habitats, supporting a role for the gray squirrel as a reservoir host of the virus. There was a delay between the establishment of invading gray squirrels and cases of the disease in red squirrels which implies gray squirrels must reach a threshold number or density before the virus is transmitted to red squirrels. The spatial and temporal trend in SQPV disease outbreaks suggested that SQPV disease will have a significant effect on Scottish populations of red squirrels within 25 years. The even spread of cases of disease across months suggested a direct rather than vector-borne transmission route is more likely. Eight juvenile and sub-adult free-living red squirrels apparently survived exposure to SQPV by mounting an immune response, the first evidence of immunity to SQPV in free-living red squirrels, which possibly suggests a changing host-parasite relationship and that the use of a vaccine may be an effective management tool to protect remnant red squirrel populations.

Natural transmission of BSE between sheep within an experimental flock.

SIR, – The recognition of bovine spongiform encephalopathy (BSE) in a French goat ([Eloit and others 2005][1]) has heightened the debate in Europe as to whether BSE has been maintained in small ruminants following historical exposure via feed. Key to the debate and associated risk assessments,

Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE)

Semen from 13 bulls, eight with clinical bovine spongiform encephalopathy (BsE), was used to artificially inseminate (AI) 167 cows with clinical BSE, and their resultant embryos were collected non-surgically seven days after Al. The viable and non-viable embryos with intact zonae pellucidae were washed 10 times (as recommended by the International Embryo Transfer Society) then frozen. Later, 587 of the viable embryos were transferred singly into 347 recipient heifers imported from New Zealand, and 266 live offspring were born of which 54.1 per cent had a BSE-positive sire and a BSE-positive dam. The recipients were monitored for clinical signs of BSE for seven years after the transfer, and the offspring were monitored for seven years after birth. Twenty-seven of the recipients and 20 offspring died while being monitored but none showed signs of BSE. Their brains, and the brains of the recipients and offspring killed after seven years, were examined for BSE by histopathology, PrP immunohistochemistry, and by electron microscopy for scrapieassociated fibrils. They were all negative. In addition, 1020 non-viable embryos were sonicated and injected intracerebrally into susceptible mice (20 embryos per mouse) which were monitored for up to 700 days, after which their brains were examined for spongiform lesions. They were all negative. It is concluded that embryos are unlikely to carry BSE infectivity even if they have been collected at the end-stage of the disease, when the risk of maternal transmission is believed to be highest.

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

BackgroundGiven the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic.Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.ResultsDisease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.ConclusionThe study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.

Experimental H-type and L-type bovine spongiform encephalopathy in cattle: observation of two clinical syndromes and diagnostic challenges

BackgroundThe 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.ResultsTwo 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.ConclusionsThe 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.