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.     

Exposure of workers to dust and bioaerosol on a poultry farm

Poultry houses are known for generating excessive dust, which originates from bedding materials, fiberglass insulations, feed, dried fecal materials, and feather particles. Dust may contain microorganisms, including endotoxins, fungi, and bacteria, that may affect living things when inhaled. Dust that contains living organisms is referred to as bioaerosol, and its particle size may range from 0.5 to 100 µm. Respirable dust, which has an aerodynamic diameter of less than or equal to 4 µm, can travel to and be deposited in the gas-exchange region of the human respiratory system. This is of particular concern because of the greater health hazard that it poses. The concentrations of respirable dust and bioaerosol measured with samplers attached to the workers (worker-exposure concentrations) were more than 3 (0.82 vs. 0.26 mg/m³) and one-and-a-half times (58.46 vs. 33.79 cfu/m³) higher, respectively, than the concentrations measured with stationary samplers indoors. The respirable dust is still below the permissible exposure limit (5 mg/m³) set by the Occupational Safety and Health Administration, but beyond the limit for animal buildings suggested by other researchers.     

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.     

Effects of stable dust on farm animals--a review

The relationship between air quality--its dust content--and respiratory diseases of animals in the food industry and otherwise is examined. The correlation between dust content and incidence of disease in farm animals is also discussed. Lesions caused either directly or indirectly by dust-transported microorganisms are reviewed. Effects of dust on animal performance are included. This review is an attempt to compile available information on the natural occurrence of stable dust and the possibilities for its measurement and control.     

Effects of stable dust on farm animals-a review

The relationship between air quality-its dust content-and respiratory diseases of animals in the food industry and otherwise in examined. The correlation between dust content and incidence of disease in farm animals is also discussed. Lesions caused either directly or indirectly by dusttransported microorganisms are reviewd. Effects of dust on animal performance are included.This review is an attempt to compile available information on the natural occurrence of stable dust and the possibilities for its measurement and control.     

Spiroplasma found in the eyes of scrapie affected sheep

Objective Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. Procedure The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). Results The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie‐affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. Conclusion These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.     

Faecal shedding, alimentary clearance and intestinal spread of prions in hamsters fed with scrapie

Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrP(TSE)) during the course of infection. The intestinal fate of ingested PrP(TSE) was further investigated by monitoring the deposition of the protein in components of the gut wall using immunohistochemistry and paraffin-embedded tissue (PET) blotting. Western blotting of faecal extracts showed shedding of PrP(TSE) in the excrement at 24-72 h post infection (hpi), but not at 0-24 hpi or at later preclinical or clinical time points. About 5% of the ingested PrP(TSE) were excreted via the faeces. However, the bulk of PrP(TSE) was cleared from the alimentary canal, most probably by degradation, while an indiscernible proportion of the inoculum triggered intestinal infection. Components of the gut-associated lymphoid tissue (GALT) and the enteric nervous system (ENS) showed progressing accumulation of PrP(TSE) from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrP(TSE) were found in the GALT in close vicinity to the intestinal lumen. Despite an apparent possibility of shedding from Peyer's patches that may involve the follicle-associated epithelium (FAE), only small amounts of PrP(TSE) were detected in faeces from clinically infected animals by serial protein misfolding cyclic amplification (sPMCA). Although excrement may thus provide a vehicle for the release of endogenously formed PrP(TSE), intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.     

Derivation of a scrapie-free sheep flock from the progeny of a flock affected by scrapie

The Cheviot flock at the Institute for Animal Health's Neuropathogenesis Unit (npu) has endemic scrapie, which affects primarily vrq/vrq sheep and at high frequency. A new flock with a full range of PrP genotypes, including the highly susceptible vrq/vrq, has been produced on a separate site, from animals in the npu breeding flock, and it remains scrapie-free after eight years. In contrast, in a parallel flock at the npu farm, scrapie has reappeared after five years, although the animals were kept in separate accommodation from the scrapie-affected sheep. During this time the npu breeding flock continued to have scrapie cases. Although it is known that highly susceptible sheep can remain free of infection in a clean environment, this is the first report of the infection being removed successfully from the bloodlines of scrapie-affected sheep. The results confirm that scrapie is not a genetic disease dependent only on the PrP gene sequence, but requires both genetic susceptibility and an infectious agent.     

Evidence for maternal transmission of scrapie in naturally affected flocks

It has been known for many years that the offspring of scrapie affected ewes are at increased risk of developing scrapie but whether this is simply the result of an increased genetic susceptibility or transmission of infection has always been unclear. To contribute to clarify this we analysed the data collected in a detailed study of scrapie occurrence in a number of naturally affected commercial sheep flocks in Great Britain (GB) to investigate the association between PrP genotype and parental scrapie status and the incidence of scrapie. Our analyses confirmed the strong association between PrP genotype and the incidence of scrapie found in previous studies and a low incidence of scrapie in animals carrying the ARR allele and a high risk in homozygous VRQ animals. However, we also demonstrate an increased incidence of scrapie in the offspring of scrapie affected ewes controlling for the confounding effect of PrP genotype, but no increased scrapie incidence in the offspring of scrapie affected sires. Our results suggest that some of the increased incidence of scrapie in the offspring of scrapie affected ewes is the result of transmission of infection from mother to offspring. Our results confirm that a breeding policy aimed at decreasing the genetic susceptibility of the population should decrease the incidence of scrapie and that removing the offspring of scrapie affected animals from affected flocks could contribute to the control of this disease.     

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP^Sc) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP^Sc is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.     

Variable nature of border disease on a single farm: the infection status of affected sheep

Study of the virological and serological status of abnormal sheep derived from a single outbreak of Border disease indicated a relationship between infection status and diverse pathological lesions. Persistent infection was associated with typical Border disease whereas an active, even exaggerated, serological response correlated with severe intracranial malformations which probably resulted from fetal infections occurring during the early stages of acquisition of immune responsiveness.     

Variable nature of border disease on a single farm: clinical and pathological description of affected sheep

The clinical, pathological and virological findings in cases of Border disease occurring on a farm were studied. Fourteen abnormal lambs approximately three months old were obtained and were necropsied within 18 months at death or slaughter. Another lamb born during the study to a persistently infected ewe was also examined. The findings of these 15 animals are presented in three groupings. The animals of group A were persistently viraemic and hypomyelinogenesis was present in two lambs at one and 14 weeks old. Systemic, chronic, multifocal inflammation including nephritis, myocarditis and pneumonitis was found in older sheep. Group B lambs were non-viraemic and the predominant necropsy finding was cystic cerebral cavitation of variable severity. Histological examination showed cerebellar dysplasia and in two animals the retinas also showed multifocal photoreceptor cell atrophy. Lambs in group C were also non-viraemic and although these failed to thrive, no consistent pathological changes were found. It is concluded that the dams of animals in group A were infected in early pregnancy while those of animals in groups B and C were infected at mid-gestation or later.     

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks

ABSTRACT: Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.     

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.     

CNA42 monoclonal antibody identifies FDC as PrPsc accumulating cells in the spleen of scrapie affected sheep

Natural scrapie, new variant Creutzfeldt-Jakob disease and murine experimental transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders. The agent responsible for these diseases is closely related to PrPsc, an abnormal isoform of the cellular prion protein. Before reaching the brain, it invades and replicates in lymphoid organs such as spleen, tonsils and lymph nodes. Follicular dendritic cells (FDC) may support the prion replication in lymphoid tissues of sheep as shown in murine models infected with scrapie. In sheep, specific antibodies recognising FDC are lacking. The CNA42 mAb, directed against human FDC was used to identify these cells in sheep spleen. As well as showing that the pre-treatments needed for immunohistochemical detection of PrPsc did not prevent labelling by the CNA42 mAb, accumulation of PrPsc in FDC of spleens of scrapie affected sheep was demonstrated using a double immunolabelling strategy. Thus, the CNA42 antibody represents a suitable tool to identify FDC and investigate their role in natural sheep scrapie.     

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrP(Sc) in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrP(Sc) depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrP(Sc) was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrP(Sc) exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.