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From: TSS ()
Subject: Survey for transmissible spongiform encephalopathies in Irish pigs fed meat and bone meal
Date: August 9, 2006 at 1:55 pm PST

##################### Bovine Spongiform Encephalopathy #####################

© 2006 British Veterinary Association

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Papers and Articles

Survey for transmissible spongiform encephalopathies in Irish pigs fed meat and bone meal

H. Jahns, MVM, VetSurgMun1, J. J. Callanan, PhD, DipECVP, CertVR, FRCPath, MVB, MRCVS2, D. J. Sammin, PhD, CertCHP, MVM, MVB, MRCVS1, M. C. McElroy, PhD, MVB1 and H. F. Bassett, PhD, MVB, MRCVS2
1 Central Veterinary Research Laboratory, Backweston Campus, Celbridge, County Kildare, Ireland
2 School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

Samples of brain and lymphoid tissues from 1107 meat and bone meal-fed, culled adult pigs from 24 Irish farms were examined for evidence of transmissible spongiform encephalopathy (TSE) by histopathological, immunohistochemical and Western blotting techniques. No evidence of deposits of abnormal prion protein suggesting the presence of TSE was found. Neuropil vacuolation was apparent in the rostral colliculus in 64 per cent of the brains examined and neuronal vacuolation was present in the dorsal vagal nucleus in 15·4 per cent of the brains. However, similar lesions have been described in pigs used as controls in a bovine spongiform encephalopathy challenge experiment. Age-related changes were also observed, including spheroids in the funicular nucleus of 24·5 per cent of the pigs, deposits of lipofuscin in the trigeminal neurons of 13·75 per cent, and mineral deposits in the walls of vessels in the dorsal vagal nucleus of 0·6 per cent. Low-grade non-suppurative inflammatory changes of uncertain origin were observed in 4 per cent of the animals.

http://veterinaryrecord.bvapublications.com/cgi/content/abstract/159/5/137

AS implied in the Inset 25 we must not _ASSUME_ that
transmission of BSE to other species will invariably
present pathology typical of a scrapie-like disease.

snip...

http://www.bseinquiry.gov.uk/files/yb/1991/01/04004001.pdf


Copyright © 2000 Harcourt Publishers Ltd. All rights reserved.
Regular Article
The Neuropathology of Experimental Bovine Spongiform Encephalopathy in the Pig

S. J. Ryder, S. A. C. Hawkins, M. Dawson and G. A. H. Wells

Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB, UK

Received 29 April 1999; accepted 13 September 1999. ; Available online 26 March 2002.


Abstract
In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1–2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals.


http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WHW-45FC860-1W&_coverDate=02%2F29%2F2000&_alid=432071172&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6861&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d8be1054aae304f337838df9991a2341


Neurobiology of Disease
Subclinical Bovine Spongiform Encephalopathy Infection in Transgenic Mice Expressing Porcine Prion Protein

Joaquín Castilla,1 Alfonso Gutiérrez-Adán,2 Alejandro Brun,1 Deirdre Doyle,3 Belén Pintado,2 Miguel A. Ramírez,2 Francisco J. Salguero,1 Beatriz Parra,1 Fayna Díaz San Segundo,1 José M. Sánchez-Vizcaíno,1 Mark Rogers,3 and Juan M. Torres1

1Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, 28130 Madrid, Spain, 2Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, 28040 Madrid, Spain, and 3Department of Zoology and Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

The bovine-porcine species barrier to bovine spongiform encephalopathy (BSE) infection was explored by generating transgenic mouse lines expressing the porcine prion protein (PrP) gene. All of the porcine transgenic (poTg) mice showed clinical signs of BSE after intracerebral inoculation with a high-titer BSE inoculum. The protease-resistant PrP (PrPres) was detected in 14% (3 of 22) of the BSE-infected poTg mice by immunohistochemical or immunoblot analysis. Despite being able to infect 42% (5 of 12) of control mice, a low-dose BSE inoculum failed to penetrate the species barrier in our poTg mouse model. The findings of these infectivity studies suggest that there is a strong species barrier between cows and pigs. However, after second-passage infection of poTg mice using brain homogenates of BSE-inoculated mice scoring negative for the incoming prion protein as inoculum, it was possible to detect the presence of the infectious agent. Thus, porcine-adapted BSE inocula were efficient at infecting poTg mice, giving rise to an incubation period substantially reduced from 300 to 177 d after inoculation and to the presence of PrPres in 100% (21 of 21) of the mice. We were therefore able to conclude that initial exposure to the bovine prion may lead to subclinical infection such that brain homogenates from poTg mice classified as uninfected on the basis of the absence of PrPres are infectious when used to reinoculate poTg mice. Collectively, our findings suggest that these poTg mice could be used as a sensitive bioassay model for prion detection in pigs.


Introduction

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative disorders that affect humans and animals, and are usually characterized by the presence of protease-resistant prion protein (PrPres), an abnormal, protease-resistant isoform of the normal cell protein denoted PrPC (Prusiner, 1991). Sheep scrapie, the best recognized TSE disease in animals, has been identified in sheep for over 200 years (Pattison and Jones, 1967), but since the 1960s, TSEs have also been identified in mink (Hartsough and Burger, 1965) and mule deer (Williams and Young, 1980). The most recent and extensive outbreak of an animal TSE disease was bovine spongiform encephalopathy (BSE) in cattle occurring in the United Kingdom in the 1980s. BSE has important implications for human health, and the consumption of BSE-contaminated meat or meat products has been linked to the advent of a new human TSE disease, variant Creutzfeldt-Jakob disease (Collinge and Rossor, 1996; Collinge et al., 1996; Will and Zeidler, 1996; Scott et al., 1999). The potential spread of BSE to other domestic animals such as sheep and pigs has implications for human health, and these populations of animals need to be monitored for signs of BSE infection.

It is well recognized that the transmission of a TSE disease within the same species occurs efficiently, but between certain pairs of species, there is a strong barrier to transmission (Pattison and Jones, 1968; Prusiner, 1990; Kocisko et al., 1995). This barrier has been defined in terms of the amino acid identity of the incoming PrPres and the resident PrPC; 100% identity usually indicates the lack of a species barrier (Prusiner, 1990). However, recent data suggest that conformational differences, independent of the amino acid sequence, can also be a key determining factor in the species barrier (Peretz et al., 2001).

Understanding the species barrier is crucial in predicting the spread of a TSE disease among species. In the past, the species barrier was considered sufficiently strong to prevent casual transmission of prion disease from one species to another, yet recent evidence shows that BSE has been transmitted to >15 species, including humans, with 134 diagnosed cases to date (see www.dh.gov.uk/Home/fs/en). However, rather than being an indication of a weak species barrier, the permissive spread of BSE to a wide variety of species may reflect the level of BSE contamination in meat and bone meal. Experimentally, it has been possible to infect mice, calves, sheep, goats, and mink via both the parenteral and oral routes, although pigs are only susceptible to the parenteral route of infection (Ryder et al., 2000), with disease failing to occur in pigs monitored for 7 years after oral exposure (Wells et al., 2003). Despite a susceptibility of pigs to infection with BSE, there is no evidence of BSE disease in the domestic pig population, at least using the conventional method of detecting clinical signs confirmed by the histopathological analysis of brain sections or the presence of PrPres. However, recent evidence suggests that these criteria may not be sufficiently strict, because prion infection can be transmitted by animals that show no clinical signs of disease (Race and Chesebro, 1998; Hill et al., 2000) and by mice that show clinical signs but have no detectable PrPres (Lasmezas et al., 1997). Because pigs are susceptible to BSE, it could be that subclinical infection, although undetected up until now, occurs in the domestic population. Given the resources and time scale (2-3 years before clinical signs appear in response to intracerebral BSE inoculation of pigs) (Ryder et al., 2000) required in studies on TSE diseases in large animals, so far much of the work on prions has been performed in mouse models (Dickinson et al., 1968; Scott et al., 1989; Collinge et al., 1995; Scott et al., 1997a). Transgenic mice expressing the PrPC of different species are generally easily infected with PrPres from the particular species being tested and are useful species-specific models. Although PrPres-PrPC interaction occurs in a mouse cell background, these models have greatly contributed to understanding the species barrier. The use of Prnp null mice to create these species-specific models generally leads to a shorter incubation period (Telling et al., 1995; Scott et al., 1997a).

In this study, we developed several lines of transgenic mice expressing different levels of porcine PrPC (poPrPC) in a Prnp null background to test for clinical or subclinical BSE infection in the pig population. Our ultimate goal was to gain additional insight into the species barrier between cattle and pigs, which appears to be stronger than barriers between the cow and mouse, cow and sheep, cow and goat, or cow and mink. We present evidence for a strong species barrier to the transmission of BSE from cattle to pigs. However, our most significant finding was the detection of subclinical infection in our transgenic mice expressing the porcine PrP gene, raising the possibility that pigs could harbor the BSE agent without showing the classic signs of its presence.

snip...

Susceptibility of poTg mice to BSE prions
PoTg001 and poTg027 mice inoculated with the BSE1 inoculum showed survival times similar to those seen for PBS-inoculated controls (Table 1). PrPres accumulation was nondetectable by Western blotting or IHC in any of the inoculated animals (see Figs. 2A, 4B1,4). However, all of the poTg001 and poTg027 mice inoculated with boTgBSE1 or BSE2 (Fig. 3) showed clinical signs of BSE, and some of these mice were found to have amounts of PrPres detectable by Western blotting and IHC. After inoculation with BSE2, both poTg027 and poTg001 mice showed an 100 d reduction in survival time compared with control inoculations. Moreover, PrPres was detectable by immunoblotting (Fig. 2) and IHC (Fig. 4C1,4) in one of five poTg027 mice and one of six poTg001 mice. Similarly, inoculation of poTg027 mice with boTgBSE1 gave rise to a 100 d reduction in survival time with respect to PBS-inoculated controls, and the PrPres protein could be detected by Western blotting (Fig. 2A) in one of the five infected mice. Bovine TgBSE1 inoculation of poTg001 mice led to an 60 d reduction in survival time compared with controls (Table 1), but it was not possible to detect PrPres in any of the six infected mice (data not shown).

To confirm the infectivity of the inocula used in these experiments, we also inoculated boTg mice (Castilla et al., 2003) and nontransgenic mice. All of the inocula (BSE1, boTgBSE1, and BSE2) were equally competent at transmitting BSE to boTg110 mice (Figs. 2A, 4C, Table 1). The BSE1 inoculum was able to infect 5 of 12 non-Tg mice, as determined by the presence of PrPres (data not shown and Table 1). These data confirm that all of the inocula were capable of efficiently transmitting BSE, including the low-dose BSE1 inoculum that was found to successfully infect both wild-type mice and transgenic mice expressing bovine PrP.

We then performed a series of second-passage experiments in which brain material from poTg027 mice showing detectable amounts of PrPres in response to boTgBSE1 inoculation was used to infect poTg001, poTg027, and boTg110 mice. The signal observed by Western blotting of porcine PrPres used as the inoculum (denoted poTgBSE1) was similar to that of the boTgBSE1 and BSE2 inocula (data not shown). All of the poTgBSE1-inoculated poTg mice proved positive for porcine PrPres by Western blotting and showed considerably reduced survival times compared with their counterparts infected with first-passage inocula (Table 1), indicating species barrier abrogation (Fig. 2B). Bovine Tg110 mice were also efficiently infected with the poTgBSE1 inoculum, with 100% of inoculated boTg110 mice showing bovine PrPres with Western blotting (Table 1). The PrPres patterns of the three different inocula used were identical according to Western blotting analysis. However, as expected, the PrPres pattern observed after the first passage in poTg mice clearly differed from the PrPres pattern of the inocula used, confirming the change of PrP species. The new pattern was maintained in subsequent passages of inoculation in poTg mice (data not shown). These data are consistent with other studies using ovine, murine, and bovine PrP transgenic mice (Scott et al., 1999; Vilotte et al., 2001; Castilla et al., 2003).

Given that poTg mice are susceptible to BSE infection, we reasoned that poTg mice inoculated with BSE1 could become subclinically infected. To test this hypothesis, we used the poTg001 mouse line to test poTg mice previously scoring negative for PrPres and clinical signs of BSE. To this end, the inocula poTgBSE1-N1 and poTgBSE1-N2 (BSE1- and boTgBSE1-inoculated poTg001 mice not producing PrPres, respectively) were used in second-passage experiments. None (0 of 9) of the poTgBSE1-N1-inoculated poTg001 mice scored positive for PrPres by Western blotting (Table 1), but survival times were reduced from 587 ± 17 to 406 ± 23 d, indicating the infectivity of the inoculum. In contrast, the poTgBSE1-N2-inoculated poTg001 mice showed neurological signs of disease and had a survival time that was shortened by 269 ± 23 d; in addition, two of six (33%) mice scored positive for PrPres by Western blotting (Table 1). Thus, clinically healthy PrPres-negative poTg mice were able to replicate porcine-adapted BSE and were also competent at transmitting the disease.

Histopathology and immunohistochemistry of BSE-inoculated mouse brains
In agreement with the biochemical data, histopathological and immunohistological studies showed no PrPres deposition in brain sections from BSE1-inoculated poTg001 mouse lines, which were indistinguishable from PBS-inoculated poTg001 brain sections (Fig. 4A,B). We observed no differences between BSE1-inoculated and PBS-inoculated animals killed at 520-730 d of age. In mice that were successfully infected with the BSE2 or boTgBSE1 inocula, granular neuronal cytoplasmic and perineuronal labeling (Fig. 4C4) and punctate neuropil labeling (Fig. 4C1) appeared as the most common histological patterns. Severe vacuolation was always accompanied by astrocytic gliosis. Astrocyte prolongations enveloped the vacuoles. Occasionally, the astrocyte cytoplasm was enlarged (Fig. 4C2,5).

In second-passage studies, histopathological signs and IHC findings were similar to those noted in the first-passage experiments using BSE2 (Fig. 4C,B). In comparative IHC studies of poTgBSE1-inoculated porcine PrP transgenic mice and BSE1-inoculated bovine PrP transgenic mice (Castilla et al., 2003), similar characteristics were observed (Fig. 5B,C). These histopathological findings essentially included vacuolation of the neuropil and an astrocyte response. These changes, along with the PrPres deposition detected by immunohistochemistry, resembled those reported in BSE (Wells and Wilesmith, 1995) and in pigs inoculated with BSE by intravenous, intraperitoneal, and intracerebral routes (Ryder et al., 2000).

snip...

Discussion

The transgenic mouse lines developed expressed the porcine PrP transgene at different levels. This is characteristic of random transgene integration in the mouse genome by the microinjection technique. Given that high expression levels promote reduced incubation times for heterologous and homologous prion propagation in mice (Scott et al., 1997b; Castilla et al., 2003), we selected two poTg lines, poTg001 and poTg027, expressing fourfold and 16-fold, respectively, the levels of PrP protein found in pig brain.

We observed that mice expressing higher levels of poPrP spontaneously developed clinical signs. A similar neurological syndrome was described previously by Westaway et al. (1994) in older Tg PrP mice expressing high levels of hamster, ovine, or murine PrP transgenes. This phenomenon may be related to the observed toxicity of overexpressed PrP in certain cell lines, which suggests that lack of physiological PrPC expression may render pathogenic in mice. However, the lifespan of poTg027 mice was much longer than the time needed by porcine prions to propagate in these animals, and the confirmation of infection could be tested using proteinase K (PK)-resistant studies. In none of the cases did the noninoculated animals presenting late clinical signs show PK-resistant protein.

We observed substantial evidence of subclinical BSE infection in our poTg mice. PoTg mice inoculated with BSE1 showed no clinical signs of BSE or detectable PrPres protein. However, subsequent passage of brain homogenates from these mice indicated the high level of infectivity of one of these animals. The presence of subclinical infection was particularly evident when we used the poTgBSE1-N2 inoculum (first-passage boTgBSE1 in poTg PrPres-negative mice), which led to a mean incubation time of 269 d and to PrPres that was detectable by Western blotting in two of six mice. The presence of subclinical infection has been reported in other species (Race and Chesebro, 1998; Hill et al., 2000). Although there is no evidence of clinical BSE disease in the domestic pig population, pigs are susceptible to BSE, and our observations raise the possibility of subclinical infection occurring in pigs. The poTg model could be used as an assay for subclinical infection in suspected cases of prion disease in pigs.

Three inocula (Fig. 3) were used to infect the poTg mice. These inocula are known to efficiently infect transgenic mice expressing the bovine PrP gene (boTg110 line) (Castilla et al., 2003). We used the same vector to express the porcine and bovine PrP genes under the mouse PrP promoter. In the boTg110 model, increasing the PrPres titer had no effect on the incubation time. When the low-dose BSE1 inoculum was tested in a normal mouse line, the animals showed neurological signs of disease, and 5 of 12 (42%) scored positive for PrPres. These data indicate that the BSE1 inoculum can cross the bovine-murine species barrier, although the expression level of the mouse PrPC is approximately half that shown by our transgenic lines.

However, the low-dose BSE1 inoculum provided evidence for a strong bovine-porcine species barrier, because it produced no signs of infection in the poTg001 or poTg027 mice. Survival times were unchanged compared with those observed in control PBS-inoculated poTg001 or poTg027 mice, and no PrPres was detected in any of the 39 inoculated mice (Table 1). In contrast, the higher titer BSE2 and boTgBSE1 inocula were able to breach the bovine-porcine species barrier, and PrPres was detected in 3 of 22 infected poTg mice (14%). Additional evidence for the bovine-porcine species barrier was obtained in second-passage transmission from BSE-infected poTg mice. The survival time dropped from 488 to 198 d postinoculation (dpi) for poTg001 and from 300 to 177 dpi for the poTg027 mice. The presence of a strong barrier may explain the resistance to infection shown by pigs during the BSE epidemic in the United Kingdom.

Contrary to the strong species barrier observed when poTg mice were inoculated with BSE, there was little evidence of a species barrier in the opposite direction (i.e., when we infected boTg110 mice with poTgBSE1). All of the boTg mice infected with this inoculum scored positive for PrPres, suggesting that the barrier has different difficulty levels depending on the direction of the infection. Western blotting analysis confirmed that the PrPres observed in the bo110Tg mice displayed the same pattern (band size, glycoform ratio) as the boTgBSE1 or BSE1 inocula but a pattern that is different from that of the newly generated porcine prion (po027Tg) (Fig. 2C). A characteristic feature of the BSE prion is that it retains its biological properties when transmitted to other species such as humans (Collinge and Rossor, 1996; Collinge et al., 1996; Will and Zeidler, 1996; Scott et al., 1999), sheep (Foster et al., 1993, 2001), or mice (Fraser et al., 1992; Lasmezas et al., 1997). Thus, the lack of a strong species barrier observed for transmission in the direction of pig to cow might be explained if the initial BSE inoculum infecting the pig confers BSE-like properties on the porcine prion, although the primary amino acid sequence of this prion is the porcine one. Alternatively, these results could be explained as follows: (1) the bovine PrP is a very permissive protein, more easily transformed by other heterologous prions or (2) the new porcine prion is highly infectious compared with others. This second possibility will be studied using other transgenic mice expressing ovine and human PrP.

The species barrier is related to amino acid sequence differences in the globular domain of the PrP protein, which undergoes a conformational change from -helix to -pleated sheet structures. The porcine PrP shows the most unique amino acid sequence (5) in this domain when compared with the mouse, cow, sheep, hamster, and human PrP sequences. Figure 6 compares the globular domains of porcine, bovine, and mouse PrP. It may be observed that four of the five unique amino acids occur in helix 3, and that there are two additional differences in this helix between the porcine and bovine sequence, I to V and R to K. The K residue is known to alter the length and quality of definition of helix 3 (Calzolai et al., 2000), and it is possible that this combination of amino acid variants alters the structure of helix 3 sufficiently to inhibit interactions between porcine PrPC and PrPres. Nuclear magnetic resonance analysis indicates that the global architecture of this region is similar for all species analyzed to date (Riek et al., 1998; Lopez Garcia et al., 2000; Zahn et al., 2000), but individual amino acid changes have been shown to affect local conformation or surface charge (Lopez Garcia et al., 2000). These subtle differences may be sufficient to strengthen or weaken a species barrier. ............

snip...full text ;

http://www.jneurosci.org/cgi/content/full/24/21/5063

Confindential
pigs & pharmaceuticals

http://www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf

http://www.bseinquiry.gov.uk/files/yb/1990/08/23002001.pdf

http://www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf

http://www.bseinquiry.gov.uk/files/yb/1990/08/29003001.pdf

TSS

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