Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.     

Uptake of orally administered polystyrene latex and poly(D,L-lactic/glycolic acid) microspheres into intestinal lymphoid tissues in chickens.

Fluorescein-labeled microspheres were orally administered to chickens and their distribution in intestinal lymphoid tissues was investigated. Polystyrene latex microspheres were observed in Peyer's patches, and also in the Meckel's diverticulum and the jejunum. Their density, however, seemed to be lower than that in Peyer's patches. Microspheres were rarely observed in the other intestinal tissues examined, including the bursa of Fabricius. Of note is that, although microspheres were present in the lumen, few, if any, were observed in the lamina propria of the caecal tonsil and caecum. Polystyrene latex microspheres of diameter 2.0 microm or 4.5 microm were also observed in Peyer's patches, but their density seemed to be lower as compared with the 0.75 microm microspheres. Poly(D,L-lactic/glycolic acid) (PLGA) microspheres were prepared using PLGAs of various molecular weights (MW) and their uptake into Peyer's patches was compared. Microspheres prepared with PLGA of average MW of 20000 were not taken up into Peyer's patches, but those prepared with PLGA of average MW of 61000 or 99 800 were taken up into Peyer's patches.     

Immuno-electromicroscopic approach for the study of neural stem cell niches

Lymphoproliferative response (LPR) was studied in 19 lambs orally infected (Group I) with Mycobacterium avium subsp. paratuberculosis (MAP) with in vitro lymphocyte stimulation test using MTT dye reduction assay. The non-specific LPR against Con A and specific LPR against sonicated antigen and johnin PPD (purified protein derivatives) were estimated on preinfection (0 day) and various days postinfection period (15 to 330 dpi) in the animals, which were classified according to histological and bacteriological evidence of paratuberculosis infection. Of the two antigens used, johnin PPD was found to be superior in terms of consistency and uniformity of response over an observation period of about a year. Significantly (P < 0.05) higher LPR were observed in the infected sheep during postinfection period, as compared with preinfection values and values from uninfected control sheep. It was evident from the present study that the LPR in histologically infected animals fluctuated during the long course of infection and had a definite relationship with the gut pathology and the mycobacterial load. The LPR were stronger but variable in sheep with grades 1, 2 and 3 lesions (paucibacillary) and increased progressively from 30 dpi onwards. The sheep with the advanced lesions (grade 4, multibacillary) showed progressive decline in LPR till 120 dpi after initial stronger response at 30 dpi. Most of the animals were detected by LPR before initiation of faecal shedding of MAP. The results suggested that repeated testing was required while screening an infected flock for detecting most of the positive animals.     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

Changes in distribution and numbers of CD4+ and CD8+ T-lymphocytes in lymphoid tissues and intestinal mucosa in the early phase of experimentally induced early onset mucosal disease in cattle

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune-mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post-inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post-inoculation was paralleled by a decrease of B-lymphocytes and an increase of CD4+ T-lymphocytes. An increased number of CD8+ T-lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T-lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T-lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T-lymphocytes in sites with lesions. This might indicate a cell-mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T-lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.     

Evidence of porcine circovirus infection in pigs with wasting disease syndrome from 1985 to 1999 in Hokkaido, Japan

An epizootiological survey with histopathological methods was conducted for porcine circovirus in 220 diseased pigs (1-200 days old) in 49 farms from 1985 to 1999. Histopathological lesions containing PCV antigen were detected mainly in the lymphoid tissues from 42 of 189 diseased pigs (22.2%) in 4 of 45 farms (8.9%) from 1990 to 1999. The rate of positive pigs gradually increased from 1997 onward and PCV infection was found in 50% of diseased pigs in 1999. Histopathologically, the lesions in the lymphoid tissues (including lymph nodes, Peyer's patches, tonsil and spleen) were highly correlated with the presence of numerous spherical basophilic intracytoplasmic inclusion bodies with PCV antigen, and consisted of lymphocellular depletion and infiltration of macrophages. Although most affected cells showed cytoplasmic reactivity for PCV, intranuclear antigen was also seen in the lymphocytes, macrophages and ileal epithelial cells. Ultrastructurally, macrophages and giant cells contained electron-dense, round to ovoid lysosomal bodies, in which there were concentric circle or paracrystalline arrays of small nonenveloped icosahedral viral particles, approximately 15-17 nm in diameter. Other consistent infectious agents were present in 90.5% of cases, and porcine reproductive and respiratory syndrome virus infection was in 52.4% of the cases with PCV. The histopathological findings suggested that PCV induced systemic immunosuppression in the infected pigs and made them more susceptible to infection of the organisms. Because of the presence of PCV antigens in the intestinal epithelium, feces may play a significant role in dissemination of PCV.     

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.     

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development

Unlike the Peyer's patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyer's patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyer's patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyer's patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyer's patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyer's patches with respect to their lymphocyte population and the ileal Peyer's patch with respect to their follicle-associated epithelium.     

Lymphocyte migration studies

For maintenance of immunity and tolerance, the organs and tissues of the organism are connected by migrating lymphoid cells. Understanding lymphocyte migration is essential for many disorders and diseases-- especially in the mucosa-lined organs. Detailed analyses of migrating lymphocytes have been performed in many species, especially in laboratory animals. However, important experiments in lymphocyte migration have been carried out in large animals, for example sheep, cattle and pigs. These species allow experimental procedures like in situ-organ labelling, lymphocyte retransfusion studies or lymph vessel cannulations. Such studies have made an important contribution to the understanding of the overall principles of lymphocyte migration especially in the mucosal immune system. Major results on the specific migration of na?ve and memory T cells through lymphoid organs, the re-distribution of gamma/delta T cells in the intestinal immune system and the emigration of newly produced B cells from the ileal Peyer's patches have been obtained in large animals. Since there are growing numbers of markers for large animals, and molecular biology methods are available in these species, experiments in large animals will be an essential tool for the understanding of lymphocyte migration especially in mucosal organs.     

Necrotizing enteritis in a calf infected with adenovirus

A three week old Holstein calf that died after a period of anorexia and diarrhea had a severe necrotizing ileitis. The lesions which were most severe above Peyer's patches contained numerous filamentous bacteria. Prominent intranuclear inclusion bodies in vascular endothelial cells in both the affected intestine and the mesenteric lymph nodes were accompanied by vasculitis and thrombosis. Adenovirus-like particles were demonstrated ultrastructurally in the inclusions.

This case demonstrates a preferential association of the intestinal necrosis with the gut associated lymphoid tissue. Also, the significance of viral infection of vascular endothelium versus intestinal epithelium in determining the type and severity of injury, is deduced from this and other previously reported cases.

     

Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge

Sixty Romney sheep of three prion protein genotypes were dosed orally at six months of age with an inoculum prepared from the brains of cattle clinically affected with BSE, and 15 sheep were left undosed as controls. They were randomly assigned within genotype to groups and were sequentially euthanased and examined postmortem at intervals of six or 12 months, depending on their predicted susceptibility. Tissue pools prepared from the three, four or five dosed animals in each group were inoculated into groups of 20 RIII mice as a bioassay for infectivity. Separate inocula were prepared from the matched control sheep killed at each time. In the ARQ/ARQ sheep killed four months after inoculation, infectivity was detected in the Peyer's patch tissue pool, and at 10 months it was detected in the spleen pool; from 16 months, infectivity was detected in a range of nervous and lymphoreticular tissues, including the spinal cord pool, distal ileum excluding Peyer's patches, liver, Peyer's patches, mesenteric and prescapular lymph nodes, spleen, tonsil and cervical thymus. No infectivity was detected in the tissue pools from the ARQ/ARR and ARR/ARR sheep killed 10 months or 22 months after infection.     

Pathological and immunohistochemical study of experimental peste des petits ruminants virus infection in goats

Peste des petits ruminants (PPR) is an emerging, economically important viral disease of goats and sheep in the Indian subcontinent. In the present investigation, 15 hill goats were experimentally infected with 2 ml of 10% splenic suspension of a virulent isolate of PPR virus (PPR/Izatnagar/94) that had caused heavy mortality (>75%) in goats during 1994 outbreaks in northern India. More than 86% (13 of 15) animals died between 9 and 13 days post inoculation at the height of temperature or when temperatures were declining. Necropsy findings included congestion of gastrointestinal tract (GIT), nasal sinuses, consolidation of antero-ventral lobes of lungs, engorged spleen, and occasionally oedematous lymph nodes. Histopathological examination of major organs of GIT revealed degeneration and necrosis of labial mucosa, severe mucosal and submucosal congestion, degeneration and necrosis of intestinal epithelium and lymphoid cell depletion from Peyer's patches along with presence of syncytia at times. Lungs showed broncho-interstitial changes and presence of intracytoplasmic and intranuclear eosinophilic inclusions in alveolar macrophages and syncytial cells. These changes in lungs were frequently complicated with serofibrinous pneumonia (57%, eight of 14). Lymphocytolysis and occasional syncytia formation were evident in the lymphoid tissues. Immunohistochemical (IHC) findings included presence of PPR virus antigen in the labial, intestinal, and bronchiolar epithelial cells, pneumocytes, macrophages and syncytial cells in lungs, and lymphoid (intact and necrotic) and reticular cells in lymphoid organs. The findings of the study indicated the highly virulent nature of the PPR virus isolate (PPR/Izatnagar/94), causing 100% mortality and characteristic pathological changes in the target organs such as lungs, intestines and lymphoid tissues. The results of the IHC study suggested that indirect immunoperoxidase could be an alternative method in the absence of more sophisticated methods of laboratory diagnosis of PPR virus infection in goats.     

Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues

We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT.     

White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation

ABSTRACT: Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to the agent of scrapie after intracerebral inoculation and to compare clinical signs and lesions to those reported for chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 mL of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. A non-inoculated deer was maintained as a negative control. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of scrapie were noted. One animal died 7 months post inoculation (pi) due to intercurrent disease. Examinations of brain tissue for the presence of the disease-associated abnormal prion protein (PrPSc) by western blot (WB) and immunohistochemistry (IHC) were negative whereas IHC of lymphoid tissues was positive. Deer necropsied at 15-22 months pi were positive for scrapie by IHC and WB. Deer necropsied after 20 months pi had clinical signs of depression and progressive weight loss. Tissues with PrPSc immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum, and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord, and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, Peyer's patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.     

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.     

Differential antibody responses to Corynebacterium pseudotuberculosis in sheep with naturally acquired caseous lymphadenitis

Enzyme-linked immunosorbent assays (ELISA) with Corynebacterium pseudotuberculosis cell wall and bacteria-free supernatant with exotoxin preparations as antigens, and hemolysis inhibition tests were used to detect antibodies in the sera of adult range sheep with naturally acquired caseous lymphadenitis (CL). The extent and severity of lesions were quantitated on the basis of a lesion score, derived from an examination of the carcass (peripheral lymphoid tissue) and viscera (including internal lymphoid tissue) at the time of slaughter. The overall prevalence of C pseudotuberculosis-positive CL lesions in 104 sheep was 31.7%. The cell wall ELISA detected antibodies in 96.9% (32/33) of sheep with C pseudotuberculosis-positive CL lesions. The exotoxin ELISA detected antibodies in 84.8% (28/33) of positive sheep in the same group. Both ELISA resulted in a high number of apparent false-positives, with 64.7% and 49.2%, respectively, positive optical density (OD) values in sheep with no gross CL lesions and no apparent C pseudotuberculosis infection. There was no significant relationship between the extent of lesion development (lesion score) and OD values in both cell wall (r = 0.472) and exotoxin (r = 0.464) ELISA. Similarly, there was no significant relationship between the titer of antitoxin antibodies, as measured by the hemolysis inhibition test, and the extent of disease. These investigations indicate that those ELISA that use crude C pseudotuberculosis antigens are of questionable utility in the field, where C pseudotuberculosis infection is endemic in many sheep populations. Furthermore, these studies suggest that antibodies that are reactive with components of C pseudotuberculosis and that develop in response to infection may have little impact on the recovery of the host.     

Association of severity of enteric granulomatous inflammation with disseminated Mycobacterium avium subspecies paratuberculosis infection and antemortem test results for paratuberculosis in dairy cows

Disseminated infection (DI) of Mycobacterium avium subspecies paratuberculosis (MAP) in cattle may impair cow health, potentiate spread of disease, and is a potential food-safety risk. The objectives of this study were to determine the association between severity of histologic enteric lesions and the occurrence of DI, clinical signs, and positive fecal culture and serum ELISA results. Bacteriologic fecal culture and serum ELISA were performed on 40 dairy cows from MAP-infected herds. Cows were classified as having DI if MAP was isolated from any of 11 extra-intestinal tissues collected postmortem. A grade of 0-3, corresponding to the severity of histologically evident granulomatous inflammation was determined for sections of ileum, jejunum, mesenteric lymph node, and ileocolic lymph node. An overall intestinal inflammation (OII) grade of 0-3 was assigned to each cow. The proportion of cows with DI increased with tissue-specific lesion grade and OII grade. All cows with grade 3 inflammation in any single tissue had DI, however, some cows with DI had grade 1 inflammation or no lesions. In general, there was a positive association between OII grade and clinical signs, gross enteric lesions, and positive ELISA and fecal culture results. However, 12% of OII grade 0 cows had clinical signs (explained by other conditions recognized with necropsy), and the proportion of positive ELISA results was lower for OII grade 3 cows relative to grade 2 cows. Although MAP dissemination may occur early in the disease process, histopathology of intestinal tissues may be used to detect a substantial proportion of DI cows.     

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.