Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.     

Molecular profiling and comparison of field transmissible spongiform encephalopathy cases diagnosed in Catalunya

Molecular profiling of the proteinase K resistant prion protein (PrP(res)) is a technique that has been applied to the characterisation of transmissible spongiform encephalopathy (TSE) strains. An interesting example of the application of this technique is the ability to differentiate, at the experimental level, between bovine spongiform encephalopathy (BSE) and scrapie infection in sheep, and to distinguish between classical and atypical BSE and scrapie cases. Twenty-six BSE cases and two scrapie cases from an active TSE surveillance program and diagnosed at the PRIOCAT, TSE Reference Laboratory (Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona, Catalunya, Spain) were examined by Western blotting. Molecular profiling was achieved by comparing the glycosylation profile, deglycosylated PrP molecular weight and 6H4/P4 monoclonal antibody binding ratio. The results obtained during the characterisation of these field cases indicated an absence of atypical BSE cases in Catalunya.     

PrP genetics in ruminant transmissible spongiform encephalopathies

Scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) are prion diseases in ruminants with considerable impact on animal health and welfare. They can also pose a risk to human health and control is therefore an important issue. Prion protein (PrP) genetics may be used to control and eventually eradicate animal prion diseases. The PrP gene in sheep and other representatives of the order Artiodactyles has many polymorphisms of which several are crucial determinants of susceptibility to prion diseases, also known as transmissible spongiform encephalopathies (TSE). This review will present the current understanding of PrP genetics in ruminants highlighting similarity and difference between the species in the context of TSE.     

朊蛋白在物种内、物种间的致病机理

朊蛋白(prion)是传染性海绵状脑病(transmissible spongiform encephalopathy,TSE)的唯一致病因子。在细胞内存在两种形式的朊蛋白,即正常形式PrP~c和致病形式PrP~(sc)(PrP~(res))。PrP~(sc)的出现是TSE发生的关键因素。本文阐述了朊蛋白的发现与意义及其在物种内、物种间的致病机理。     

Stability of bovine spongiform encephalopathy prions: absence of prion protein degradation by bovine gut microbiota

Bovine spongiform encephalopathy (BSE) is transmitted by the oral route. However, the impacts of anaerobic fermentation processes in cattle on the stability of BSE-associated prion protein (PrP(Sc)) are still unresolved. In this study, experiments were designed to assess the ability of complex ruminal and colonic contents of bovines to degrade BSE-derived PrP(Sc). No significant decrease in PrP(Sc) levels in BSE brain homogenates was detected by Western blotting after up to 66 h of co-incubation with intestinal fluids. These results indicate that BSE-associated PrP(Sc) survive gastrointestinal digestion processes in cattle and might be excreted via faeces.     

Detection of prions in the faeces of sheep naturally infected with classical scrapie

ABSTRACT: Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.     

Correlation between egg-shedding and uterus development in Fasciola hepatica human and animal isolates: applied implications

The emission of Fasciola hepatica eggs in faeces is usually subject to oscillations along time in animals as well as humans. Thus, looking for alternative biological markers reflecting eggs shed per gram of faeces (epg) with lower oscillations may be useful. This study analyzes the possible relationship between liver-fluke uterus area and epg. Uterus area (UA) development of adult F. hepatica obtained at different days post infection (dpi) in a Wistar rat model with isolates obtained from cattle, sheep, pigs and humans from the endemic human fascioliasis zone of the Northern Bolivian Altiplano was analyzed and compared with the number of eggs shed per gram of faeces as obtained through the Kato-Katz technique. The morphometric study of the UA of liver flukes was carried out using image analysis software. The multiple regression model shows that UA is dependent on dpi and isolate. The evolution of UA vs dpi followed a damped model. This work shows a positive relationship between liver-fluke UA and egg production. The complete absence of eggs in the uteri of some parasite individuals at 300 dpi was observed, which corresponds to the cessation of egg shedding in the advanced chronic stage. The results obtained suggest the necessity to characterize the isolates employed with regard to geographical as well as host origin in fascioliasis studies in which egg production is used as a biological tag.     

Alymphoplasia mice are resistant to prion infection via oral route

The major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff. There is evidence that the enteric nerve system (ENS) and gut-associated lymphoid tissues (GATL) are involved in the establishment of prion infection through alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alymphoplasia (aly) mice showing a deficiency in systemic lymph nodes and Peyer's patches. The aly/aly mice were susceptible to prion infection by intra-cranial inoculation and there were no differences in incubation periods between aly/aly mice and wild-type C57BL/6J mice. Incubation periods in aly/aly mice were about 20 days longer than those in C57BL/6J mice with the intra-peritoneal inoculation. The aly/aly mice were completely resistant to prion infection by per os administration, while C57BL/6J mice were sensitive as they entered the terminal stage of disease around 300 days post inoculation. PrP(Sc) were detected in the intestine and spleen of C57BL/6J mice inoculated with prion intraperitoneally or orally; however PrP(Sc) was not detected in the spleen and intestine of aly/aly mice. Prion infectivity was detected in the intestines and spleens of prion-inoculated C57BL/6J mice, even after the early stages of exposure, while no infectivity was detected in these tissues of prion-inoculated aly/aly mice. No apparent differences were observed in the organization of the enteric nerve system between wild-type and aly/aly mice. These results indicate that GALT rather than ENS acts as the primary entry port for prion after oral exposure.     

Excretion of BSE and scrapie prions in stools from murine models

Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10mug of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.     

Development of monoclonal antibodies against the abnormal prion protein isoform (PrPres) associated with chronic wasting disease (CWD)

Monoclonal antibodies (mAbs) specific for the abnormal prion protein isoform (PrP^res) are indispensable for diagnosing chronic wasting disease (CWD). In this study, eight mAbs were developed by immunizing PrP knockout mice with recombinant elk PrP and an immunogenic PrP peptide. The reactivity of the mAbs to recombinant PrP and the PrP peptide was measured, and their isotypes were subsequently determined. Among them, four mAbs (B85-05, B85-08, B85-12, and B77-75) were shown by Western blotting to recognize proteinase K-treated brain homogenate derived from an elk suffering from CWD.     

Primary infection protects pigs against re-infection with Lawsonia intracellularis in experimental challenge studies

In two separate trials pigs were experimentally infected with Lawsonia intracellularis at 5-6 weeks of age followed by antibiotic treatment and resolution of the primary infection and then re-inoculated at 12-13 weeks of age. A treatment-control group of pigs received the primary infection and antibiotic treatment only, and served as control for the antibiotic treatment of the primary infection. A challenge-control group of pigs received the second inoculation dose only at 12-13 weeks of age to control infectivity of the challenge-dose and susceptibility of pigs to L. intracellularis at this age. Pigs were monitored for shedding of L. intracellularis in faeces by PCR, and for the development of antibodies and responses of acute phase proteins in serum. The presence of L. intracellularis antigen in the intestinal mucosa was examined in post mortem samples by immunohistochemistry. In both trials primary infected pigs were protected from infection after challenge inoculation as evidenced by absence of faecal shedding of L. intracellularis, lack of changes in acute phase protein concentrations after challenge and with low levels of bacterial antigen in the intestinal mucosa of re-inoculated pigs comparable to that of the treatment-control pigs. In contrast, challenge-control pigs shed L. intracellularis in faeces, had L. intracellularis antigen extensively present within all layers of the intestinal mucosa and developed a significant acute phase protein response in serum after the experimental infection. The acute phase protein response to L. intracellularis infection was detected as an increased rise in the serum concentrations of C-reactive protein and haptoglobin from day-6 post infection, and increased serum concentrations of haptoglobin were generally seen 2-3 weeks after inoculation both at 5-6 and 12-13 weeks of age. In conclusion substantial protection against L. intracellularis infection was found in the re-inoculated pigs in contrast to the development of infection in age-matched control pigs. The acute phase protein responses reflected both the observed protection against L. intracellularis infection upon secondary challenge and that increased resistance to the infection develops with age.     

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.     

Recent developments in prion disease research: diagnostic tools and in vitro cell culture models

After prion infection, an abnormal isoform of prion protein (PrP(Sc)) converts the cellular isoform of prion protein (PrP(C)) into PrP(Sc). PrP(C)-to-PrP(Sc) conversion leads to PrP(Sc) accumulation and PrP(C) deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP(Sc) detection. Highly sensitive/accurate specific detection of PrP(Sc) in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP(Sc), namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP(C) functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology.     

A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep

A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.     

Propagation of scrapie in peripheral nerves after footpad infection in normal and neurotoxin exposed hamsters

As is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP(Sc)) in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP(Sc) for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component ;a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin beta,beta-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP(Sc) in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP(Sc). Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP(Sc) along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP(Sc) is not mediated by the slow component ;a' of the axonal transport mechanism.     

中国奶牛PrP基因型分析

根据已报道奶牛朊蛋白(PrP)基因序列设计引物.采用PCR法扩增了28头黑白花奶牛的PrP基因.序列测定及分析结果表明,所克隆的奶牛PrP基因片段为677 bp.编码225个氨基酸的前体蛋白.对其136、154、171位点氨基酸多态性分析结果发现,对TSE中度易感的ARQ型占检测奶牛的82.9%,对TSE抗性的ARR型占7.1%,对TSE高度易感的VRQ型占10%.这说明中国奶牛的基因型对TSE中度易感,为防制TsE的发生提供基础数据.     

中国奶牛PrP基因型分析

根据已报道奶牛朊蛋白(PrP)基因序列设计引物,采用PCR法扩增了28头黑白花奶牛的PrP基因。序列测定及分析结果表明,所克隆的奶牛PrP基因片段为677 bp,编码225个氨基酸的前体蛋白。对其136、154、171位点氨基酸多态性分析结果发现,对TSE中度易感的ARQ型占检测奶牛的82.9%,对TSE抗性的ARR型占7.1%,对TSE高度易感的VRQ型占10%。这说明中国奶牛的基因型对TSE中度易感,为防制TSE的发生提供基础数据。     

Infectivity of scrapie prion protein (PrPSc) following in vitro digestion with bovine gastrointestinal microbiota

The influence of a complex microflora residing in the gastrointestinal tract of cattle on the prion protein plays a crucial role with respect to early pathogenesis and the potential infectivity of faeces resulting in contamination of the environment. It is unknown whether infectious prion proteins, considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals during digestion. In our previous study it was shown that the scrapie-associated prion protein was degraded by ruminal and colonic microbiota of cattle, as indicated by a loss of anti-prion antibody 3F4 immunoreactivity in Western blot. Subsequently, in this study hamster bioassays with the pre-treated samples were performed. Although the PrP(Sc) signal was reduced up to immunochemically undetectable levels within 40 h of pre-treatment, significant residual prion infectivity was retained after degradation of infected hamster brain through the gastrointestinal microflora of cattle. The data presented here show that the loss of anti-prion antibody 3F4 immunoreactivity is obviously not correlated with a biological inactivation of PrP(Sc). These results highlight the deficiency of using Western blot in transmissible spongiform encephalopathies inactivation assessment studies and, additionally, point to the possibility of environmental contamination with faeces containing PrP(Sc) following an oral ingestion of prions.