Mike said:
TimH: According to some, less than 1 gram of "infective" beef can trigger vCJD in humans.
I've not seen the research to support this statement.
In fact, just the opposite is true. From what I've read, the "Cross Species" (human) barrier makes it approximately 100 to 1000 times less likely that a human will be "infected" from eating the same amount of infectivity that cattle do.
The big worry is that cosmetics, the blood supply, and surgical procedures (among others) will transmit PrPsc.
Also, some scientists believe some people might/do have certain genetic immunities to PrPsc that were derived from eating meat back in the Cro-Magnon and Neanderthal days.
It's not the number of actual cases that worry me as much as the "Media Induced Hysteria" that goes along with it.
Just wait until the first case of "Home Grown" nCJD hits North America!
There will be second guessing all to hell and back if the proper precautions are not taken beforehand.
NO studies on humans have every been done to document load infectivity of BSE or any TSE to humans. ITS against the law to do those studies i suppose. i think instead of primates a good study would be with human volunteers on death row. boy that's another can of worms, but until then, we must look at these studies with great concern.
IT seems to take very little of BSE to infect primates in the only few studies they have every done. my opinion, inoculation of the TSE agent may be a more efficient route though. what the threshold from sub-clinical to clinical disease, well, that's still another million dollar question that is unanswered for humans. ...tss
Research Letters
Up to 400 000 cows with undiagnosed bovine spongiform
encephalopathy (BSE) infection are estimated to
have been slaughtered for food before brain and spinal
cord were banned from human consumption in 1989.
More restricted exposure to BSE could have continued
through 1995 from consumption of processed meat
products containing mechanically recovered meat
contaminated with central nervous system (CNS) tissue
and spinal ganglia.1 The discovery of BSE in Canada and
the USA, where consumption of brain and other viscera
was allowed until 2003, and of secondary cases of variant
Creutzfeldt-Jakob disease (vCJD) in the UK, possibly
attributable to contaminated blood donated by people
with pre-clinical primary infection, reinforces the need
for an experimental assessment of the risk of oral
exposure to BSE. We therefore investigated oral
transmission of BSE to non-human primates.
We chose cynomolgus macaques for the study because
these old-world monkeys have a digestive physiology
similar to that of human beings, are methionine
homozygous at codon 129 of the PRNP gene, and have a
BSE neuropathology similar to that of vCJD.2,3 We gave
two 4-year-old adult macaques a 5 g oral dose of brain
homogenate from a BSE-affected cow. We tested for
proteinase-resistant prion protein (PrPres) in this
homogenate with a commercial BSE-testing ELISA kit
(Bio-Rad, Marnes-la-Coquette, France). A sample of the
100% homogenate brain paste inoculum that was fed to
the primates was rehomogenised at 20% weight-pervolume
in the kit buffer. Serial dilutions were made with
a pool of 20% weight-per-volume BSE-negative brain
homogenate in the same buffer. Testing was done
according to the manufacturer’s instructions and results
were confirmed by a western blot test (Bio-Rad) with a
similar process of PrPres dilution. With both methods,
dilutions of up to 1 in 300 provided a positive signal
(figure A).
One macaque developed neurological disease
60 months after exposure and was killed at 63 months
because of recumbency. Histopathological examination
of the brain of this animal showed the typical pathology
of vCJD (figure B) and an accumulation of PrPres
associated with the follicular dendritic cells in tonsils
(figure C), spleen, and intestine. A western blot showed
similar patterns of PrPres in a brain sample from the
macaque and the BSE-infected bovine inoculum
(figure D). The other macaque remained free of clinical
signs 76 months after exposure, and a tonsil biopsy done
at 72 months was negative (figure E).
In a previous study, two macaques orally dosed with
5 g of brain from a macaque with terminal clinical BSE
became ill after 44 and 47 months.4 The results of the
present study suggest that the incubation period for
interspecies transmission of BSE can be considerably
Published online
January 27, 2005
http://image.thelancet.com/
extras/05let1056web.pdf
Commissariat à l’Energie
Atomique/Direction des
Sciences du Vivant/Départment
de Recherche Médicale,
18 Route du Panorama, 92265
Fontenay-aux-Roses, France
(C I Lasmézas DrMedVet,
E Comoy DrMedVet,
C Herzog DipBiol,
F Mouthon DipBiol, F Auvré,
E Correia,
N Lescoutra-Etchegaray DipBiol,
Prof N Salès PhD, J-P Deslys MD);
Veterinary Laboratories
Agency, New Haw, Addlestone,
UK (S Hawkins MIBiol,
T Konold DrMedVet,
G Wells BVetMed); and 7815
Exeter Road, Bethesda, MD
20814, USA (P Brown PhD)
Correspondence to:
Dr Jean-Philippe Deslys
e-mail:
[email protected]
www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf 1
Risk of oral infection with bovine spongiform
encephalopathy agent in primates
Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia,
Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys
The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)—which can lead to variant
Creutzfeldt-Jakob disease (vCJD)—is compounded by incomplete knowledge about the efficiency of oral infection
and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral
transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a
BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the
other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a
preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public
health measures can prevent transmission of BSE to man.
B
C
E
A
Dilution
D
3·215
1·989
0·984
0·302
0·131
0·065
0·052
1/10
1/30
1/100
1/300
1/1000
1/3000
Neg
36 kDa
36 kDa
22 kDa
22 kDa
16 kDa
1 2 3 4
ELISA detection of PrPres (absorbance units)
Figure: PrPres content of brain homogenate and histopathological assessment of macaque tissues
(A) Results of in-vitro testing for PrPres in BSE-infected inoculum by ELISA and western blot. Neg=normal bovine
brain material. (B) Typical florid plaque in the occipital cortex of the macaque that developed disease.
PrPres detected by proteinase K treatment with SAF32 anti PrP monoclonal antibody (kindly provided by Jacques
Grassi, CEA Saclay). The dense core of PrPres is surrounded by several vacuoles in a fibrillar proteinaceous corona;
bar=10 m. (C) Positive PrPres staining in tonsil (80% of follicules stained positive) of the macaque that developed
disease; bar=50 m. (E) Negative PrPres staining in tonsil of the macaque that did not develop disease; bar=50 m.
(D) Western blot showing similar PrPres patterns in samples from a patient with vCJD (lane 1), the macaque that
developed disease (lane 3), and the bovine BSE inoculum (lane 4). By contrast, a macaque inoculated intracerebrally
with material from a patient with sporadic CJD showed a different PrPres pattern (lane 2).
For personal use. Only reproduce with permission from Elsevier Ltd
Research Letters
longer than that of intraspecies transmission (60 months
vs 44 and 47 months, representing 36% and 28%
increases, respectively). The interval between the period of
peak exposure to infectious BSE tissue and the hitherto
peak incidence of vCJD is about 10–15 years, but
incubation periods of up to 40 years have followed oral
infection with kuru between human beings.5 Therefore,
maximum incubation periods might exceed 50 years in
cases of oral transmission of BSE from cattle to man.
The present data do not provide a definitive minimum
infective dose for transmission of cattle BSE to primates,
but they do give enough information for a preliminary
assessment of the adequacy of existing measures to
protect the human food chain. Results of ongoing
experiments provide a rough estimation of the intraspecies
transmission rates in cattle. The BSE brain
inoculum to which the cattle were exposed had an
infectivity titre of 103·5 mouse infectious (intracerebral
and intraperitoneal) units ID50 per g (ID50 is the dose at
which 50% of animals become infected). Interim results
at 6 years after exposure suggest that the oral ID50 in
cattle may be between 100 mg and 1 g (table 1; S A C
Hawkins, T Konold, G A H Wells, unpublished data).
Since the brain of a cow weighs 500 g and a spinal cord
200 g, CNS tissues from a cow with clinical signs of BSE
could contain enough infective agent to transmit disease
orally to 490–1400 cows (70% of 700 g if 1g is needed, or
20% of 700 g if 100 mg is sufficient), or to 70 primates
(50% of 700 g if 5 g represents the oral ID50).
The accuracy of estimates of the oral ID50 for man will
not be improved until completion, several years from
now, of a large dose-response European study (QLK1-
2002-01096) in macaques, in which the minimum dose
is 50 mg. However, because similar inocula were used in
both the cattle and macaque studies,6 a tentative comparison
can be made between the efficiency of oral infection
in cattle and that in primates. On this basis, a factor of
7–20 could be considered as the range of magnitude of a
bovine-to-primate species barrier for oral BSE infection
(70 primates infected compared with 490 or 1400 cows,
with a similar dose).
Elimination from the human food chain of CNS
tissues from cows with clinical BSE is estimated to have
reduced the risk of human exposure to the disease by
about 90%.7 Risk was further reduced in continental
Europe by systematic screening for the diagnostic
presence of PrPres in the brainstem of all cattle older than
30 months, and in the UK by the total interdiction of
cows older than 30 months. In an oral exposure study to
assess the pathogenesis of BSE in cattle, in which the
same European Union-evaluated test as we used in the
present study was applied to CNS tissues, some
preclinical cases of the disease were diagnosed.8
Using the same test, pooled brainstem from cows with
clinical BSE has yielded a endpoint titre of PrPres
corresponding to a 1-in-300 to 1-in-1000 dilution of
positive brainstem.6,9 If people were to eat CNS tissues
from a cow with preclinical BSE with a concentration of
PrPres just below the test detection limit of 1 in 300, they
would need to ingest at least 1·5 kg to reach the degree
of exposure equivalent to that in the 5 g of brain used for
oral transmission to the macaque in the present study. If
the oral ID50 for man was one log below this dose (ie,
similar to that in cattle, and not accounting for any
species barrier between cattle and man; see table), 150 g
of CNS tissue that tested falsely negative could represent
an infective dose. Because use of cattle brain and spinal
cord for human consumption is prohibited, and in view
of the existing mechanically recovered meat regulations,
a person would be very unlikely to ingest this amount of
cattle CNS tissue.
The minimum sensitivity of screening tests to detect
100% of BSE-infected animals has yet to be ascertained.
However, our results provide reassurance that BSE
screening procedures combined with CNS removal are
effective measures to protect the human food chain.
Contributors
J-P Deslys, C Lasmézas, and E Comoy were responsible for design and
management of this study. G Wells, S Hawkins, and T Konold were
responsible for the pathogenesis study in ruminants. C Lasmézas,
C Herzog, and N Lescoutra-Etchegaray were in charge of the primate
experiments. F Auvré undertook the biochemical analyses. N Salès was
responsible for the immunohistochemical analyses, which were done
by E Correia. C Lasmézas, E Comoy, F Mouthon, G Wells, P Brown, and
J-P Deslys drafted the manuscript.
Conflict of interest statement
Commissariat à l’Energie Atomique owns a patent covering the BSE
diagnostic test commercialised by Bio-Rad. All authors had full access to
all data and had responsibility to submit for publication. The funding
sources had no role in the collection, analysis, and interpretation of
data, writing of the report, or decision to submit the paper for
publication.
2 www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf
BSE bovine brain inoculum
100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg
Primate (oral route)* 1/2 (50%)
Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)
RIII mice (icip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)
PrPres biochemical detection
The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was
inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of
bioassays is generally judged to be about plus or minus 1 log. icip=intracerebral and intraperitoneal.
Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula
Research Letters
Acknowledgments
We gratefully acknowledge the expert care of the primate animals
provided by René Rioux, Sébastien Jacquin, and Anthony Fort, and the
technical expertise of Dominique Marcé, Capucine Dehen,
Sophie Freire, and Aurore Jolit Charbonnier. This work has received
financial support from the French Ministry of Research (GIS Prion). It is
now continued within the framework of the EU consortium QLK1-2002-
01096 and the European network of Excellence NeuroPrion. Ongoing
studies by the Veterinary Laboratories Agency in cattle are funded by the
UK Department for Environment, Food, and Rural Affairs.
References
1Anderson RM, Donnelly CA, Ferguson NM, et al. Transmission
dynamics and epidemiology of BSE in British cattle. Nature 1996;
382: 779–88.
2 Lasmézas CI, Deslys JP, Demaimay R, et al. BSE transmission to
macaques. Nature 1996; 381: 743–44.
3 Lasmézas CI, Fournier JG, Nouvel V, et al. Adaptation of the bovine
spongiform encephalopathy agent to primates and comparison with
Creutzfeldt-Jakob disease: implications for human health. Proc Natl
Acad Sci USA 2001; 98: 4142–47.
4 Herzog C, Salès N, Etchegaray N, et al. Tissue distribution of bovine
spongiform encephalopathy agent in primates after intravenous or
oral infection. Lancet 2004; 363: 422–28.
5 Klitzman RL, Alpers MP, Gajdusek DC. The natural incubation
period of kuru and the episodes of transmission in three clusters of
patients. Neuroepidemiol 1984; 3: 3–20.
6 Deslys JP, Comoy E, Hawkins S, et al. Screening slaughtered cattle
for BSE. Nature 2001; 409: 476–78.
7 European Commission. Opinion of the Scientific Steering
Committee on the Human Exposure Risk via food with respect to
BSE. Adopted on 10 December 1999. http://europa.eu.int./comm/
food/fs/sc/ssc/out67_en.pdf (accessed Jan 17, 2004).
8 Grassi J, Comoy E, Simon S, et al. Rapid test for the preclinical
postmortem diagnosis of BSE in central nervous system tissue.
Vet Rec 2001; 149: 577–82.
9 Moynagh J, Schimmel H. Tests for BSE evaluated. Bovine
spongiform encephalopathy. Nature 1999; 400: 105.
www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf 3
Vol. 96, Issue 7, 4046-4051, March 30, 1999
Neurobiology
Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents
Nöelle Bons*,, Nadine Mestre-Frances*, Patrick Belli, Françoise Cathala§, D. Carleton Gajdusek¶, and Paul Brown
* Ecole Pratique des Hautes Etudes, Laboratoire de Neuromorphologie Fonctionnelle, Université Montpellier II, 34095-Montpellier cedex 5, France; Centre National d'Etudes Veterinaires et Alimentaires, Pathologie Bovine, 31 Av. Tony Garnier, 69342-Lyon cedex 07, France; § 68 Bd Saint-Michel, 75006-Paris, France; ¶ Institut Alfred Fessard, Centre National de la Recherche Scientifique, 91198-Gif-sur-Yvette, France; and Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
Contributed by D. Carleton Gajdusek, December 21, 1998
ABSTRACT
Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.
MATERIALS AND METHODS
Epidemiological Study. A detailed study was undertaken of 61 primates belonging to 11 species housed in the Montpellier Zoological Park to evaluate the possible role of diet on the longevity of the animals. The animals live in very large cages spread out in a natural garrigue (Mediterranean forest). Depending on animal size, no more than three simians or five lemurians live in any one cage. A questionnaire also was mailed to other zoos and primate breeding facilities in France, asking for information about neurological or unexplained primate deaths and dietary practices. In the course of this inquiry, we were informed that a number of apparently healthy lemurs in the Besançon zoo and the Strasbourg breeding facility were going to be euthanized because of a new French regulation concerning hybrid primates, and so we obtained an additional group of 18 animals (six from Besançon and 12 from Strasbourg).
These 79 animals were all large-sized, long-lived monkeys and lemurs (over 1,000 g in body weight and more than 20 years longevity), who were fed a daily diet of vegetables and fruits supplemented by 20-40 g/kg of commercial food products containing animal-derived proteins (Singe 107, MP, or Marex). According to the manufacturers, this food contained various items, including gross protein (19.2-25.4%), fats (5.7-7.5%), corn, soya, carob bean, alfalfa, mineral, yeasts, vitamins A, C, D3, and E, and cracklings (the so-called "fifth quarter of beef" suitable for human consumption).
Experimental Study. This study involved a group of five lemurs belonging to the small-sized and short-lived species Microcebus murinus (around 100 g in body weight, 8-10 years longevity). These animals, from a colony housed at the Center for Laboratory Animals of the Montpellier University of Science, were 1-year-old adults and had never been fed commercial food containing meat. Three lemurs (control animals nos. 538, 593, and 655) were allowed to remain in the colony. Two lemurs (nos. 654 and 656) were reared in a locale protected under French law, one animal (no. 654) having been fed a single 0.5-g dose of a BSE-infected cattle brain (obtained from Centre National d'Etudes Veterinaires et Alimentaires, Lyon, France), and the other (no. 656) having been fed two 0.5-g doses, spaced 2 months apart, of the same cattle brain. The brain fragments were mixed with apple compote and given to the animals before their customary daily diet.
Immunohistology. Animals were anaesthetized by an i.p. injection of pentobarbital (0.5 ml/kg). The various organs were dissected, and samples were fixed by immersion in paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) and Carnoy's liquid. After routine histological protocols, 6-µm microscopic sections of different parts of the gastrointestinal tract, spleen, tonsil, thymus, spinal cord, and brain were prepared for PrP immunohistological study as follows: sections were immersed in 85% formic acid for 45 min, washed in distilled water, immersed in 5% hydrogen peroxide for 10 min, immersed in distilled water, and autoclaved for 10 min at 121°C.
The sections then were rinsed in Tris-buffered saline (TBS) before overnight incubation at 4°C with either of two mouse monoclonal primary antibodies: anti-PrP106-126 (dilution 1:2) or anti-PrP 3F4 (dilutions 1:200, 1:500, or 1:1,000). Sections then were incubated for 1 h with a secondary anti-mouse IgG antibody coupled to peroxidase (Boehringer Mannheim). Color was developed with 0.2% diaminobenzidine (Sigma) in TBS containing 0.02% hydrogen peroxide and counterstained with Harris' hematoxylin. Histological sections of brain, spleen, and gastrointestinal tract from several different Eulemur spp. were independently studied in the laboratory of P. Belli, using the laboratory's own rabbit polyclonal antibody RS1 and revealed by the kit Duet (Dako) according to the protocol of Tagliavini et al. (3).
Selected brain and spinal cord sections also were treated with the polyclonal antibody 961S28T (4) (1:200 dilution for 5 days), which stains abnormal neuronal Tau proteins, and the polyclonal glial fibrillary acidic protein antibody (GFAP) (Dako, 1:100 dilution overnight), which stains reactive astrocytes. The protocol was identical to that used for anti-PrP antibodies, except for the omission of formic acid and autoclaving pretreatment. Quantitative studies were performed on brain sections chosen with reference to the microcebe brain atlas (5); the distribution of cortical neurons containing abnormal aggregated Tau proteins was mapped with an image analysis computer (Biocom Histo 200, Paris).
Because no anti-PrP antibody is capable of distinguishing between the normal and pathological isoforms of PrP in fixed tissue, and because discrimination by proteinase K partial digestion also is rendered ineffective by fixation, it is essential that a number of methodological criteria be met for a proper interpretation of immunostaining results. These criteria include: unequivocal staining having a characteristic morphological appearance, with little or no background noise; and the absence of such staining in parallel sections treated with (i) preimmune serum from the animal in which the primary antibody was raised, (ii) immune serum preabsorbed with its corresponding PrP antigen, (iii) secondary antibody without previous incubation with the anti-PrP antibody, and (iv) at least one other antibody unrelated to PrP. In addition, staining must not occur in identically prepared sections from tissues of healthy control animals, and the results should be duplicated by an independent laboratory using the same or different immunohistochemical techniques and antibodies. Our study meets all of these criteria, and we therefore have accepted positive staining results as representing the presence of the pathological isoform of PrP.
snip...
Epidemiological Study. Among the primates in the Montpellier zoo, 26 deaths were recorded between 1989-1998, of which 23 occurred between 1989 and 1993 (Table 1). The date of arrival of each primate at the zoo was always known, but the date and the locality of its birth were often unknown (many animals came from other zoological parks). Although detailed clinical information rarely was recorded in the zoo registers, clinical signs were observed before death in 14 animals, of which 12 were characterized as having had serious neurological abnormalities.
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Table 1. Epidemiological summary of primates housed in the Montpellier Zoological Park during the period 1989-1998
In view of the multiple geographic origins of the animals dying at the Montpellier zoo, it is not possible to state that infection in all animals occurred in this locale. However, three animals dying from spongiform encephalopathy must certainly have been infected in Montpellier: two lemurs (nos. 481 and 586) came directly from Madagascar to Montpellier in 1974 and 1979, well before the era of BSE, and one animal (no. 474) was born and raised in the Montpellier zoo.
We received nine responses (representing only about a 10% response rate) from our mailed questionnaire to other primate holding facilities: one respondent zoo had no primates, and of the eight respondent zoos with primates, seven denied any suspicious or neurological deaths, and one (Lille) noted three deaths in January 1996 in primates after neurological illnesses similar to those seen in the Montpellier primates.
All of the primates in Lille, Strasbourg, Besançon, and Montpellier, as well as animals in the seven zoos that reported no neurological deaths, had diets that included nutritional supplements containing meat meal, sold under the names Singe 107, MP, or Marex. The supplements are produced by two different companies (one of which is based in the United Kingdom), which distribute them through a French company to zoos and animal breeding facilities. It is highly likely that British beef was included in the source of meat powder, especially as the British manufacturer announced that as of June 1996 it ceased to use beef meal in its nutritional supplements.
Immunohistological Studies. We studied two lemurs (microcebes) that were experimentally fed with BSE-infected brain tissue and three unexposed control lemurs. After the killing of one of the BSE-fed lemurs (no. 654) by its cage mates, we sacrificed one of the two remaining BSE-fed animals (no. 656) to have optimally preserved tissues for examination from at least one animal during the incubation phase of disease (5 months postinfection). Other animals are being held under observation until such time as they may show signs of neurological disease.
We also studied two additional symptomatic lemurs in the Montpellier zoo (nos. 456 and 586), and 18 asymptomatic lemurs (nos. 700-717) in captivity in either Besançon or Strasbourg. All of these animals were 6-16 years of age (except for two animals 25 years of age), with body weights of 1,500-1,800 g. The presence and distribution of PrP immunoreactivity described in the following paragraphs was similar in the captive lemurs and in the two microcebes that had been experimentally infected with BSE (Tables 2 and 3). Uninfected control animals showed no PrP immunoreactivity.
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Table 2. PrP immunostaining in non-nervous system tissues of spontaneous cases of spongiform encephalopathy in eulemurs and in microcebes fed with BSE-infected brain tissue (nos. 654 and 656)
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Table 3. PrP, Tau, and GFAP immunopositivity, and micro-vacuolation in nervous system tissues of spontaneous cases of spongiform encephalopathy in eulemurs, and in microcebes fed with BSE-infected brain tissue (nos. 654 and 656)
In the tonsils, PrP was seen in the peripheral epithelium, lymphoid nodules, and in scattered cells inside the glands. In the esophagus, PrP was present in the stratified epithelial cells, but not in the mucigen-secreting esophageal glands. Immunoreactive lymphocytes were scattered throughout the connective tissue of the lamina propria and infiltrating the muscularis mucosae and the submucosa. An abrupt transition between the esophagus and the stomach was conspicuous by a different PrP distribution starting at the cardia: the gastric columnar epithelium bordering the lumen and the gastric pits were PrP-negative but the gastric glands were positive. The underlying lymphoreticular tissue in the lamina propria also was labeled (Fig. 1 E and F).
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Fig. 1. (A) Zoo lemur no. 703. PrP deposits in large vacuolated fibers of the ventral funiculus of the cervical spinal cord. Arrows point to fiber membranes. Anti-PrP 3F4, 1:200. (B) Zoo lemur no. 712. Nerve fibers showing PrP immunoreactivity (brown) in layer IV of the cerebral cortex. Anti-PrP 3F4, 1:200. (C) Experimental BSE-infected microcebe no. 656. Microvacuolation in the neuropil of the parietal cortex (layer V). Hematoxylin and eosin. (D) Experimental BSE-infected microcebe no. 656. Abnormal Tau proteins inside pyramidal neurons of the parietal cortex layer III. Anti-tau 961S28T, 1:200. (E) Experimental control microcebe no. 593. High magnification of the stomach wall: no PrP immunoreactivity is detected in the epithelium, secretory glands, or various lymphoreticular tissue elements (arrows). Star indicates luminal surface. Anti-PrP 3F4, 1:200. (F) Experimental BSE-infected microcebe no. 656. PrP distribution in the stomach wall. Arrows point to reticulolymphatic elements; star indicates luminal surface. Anti-PrP 3F4, 1:500. (G) Experimental BSE-infected microcebe no. 656. PrP localization in an intestinal villus. Note the interrupted epithelium at the level of M cells containing a lymphocyte, and the immunoreactivity of lymphoid reticular structures. Stars indicate luminal surfaces. Anti-PrP106-126, 1:2. (H) Experimental BSE-infected microcebe no. 656. Peyer's patch with PrP immunoreactive lymphoid structures. Anti-PrP106-126, 1:2. (I) Experimental BSE-infected microcebe no. 656. PrP labeling in splenic red pulp. Anti-PrP 3F4, 1:500. (J) Experimental BSE-infected microcebe no. 656. Small intestine. Anti-PrP 3F4, 1:200, pre-adsorbed with PrP antigen.
In the small intestine, including the duodenum, finely particulate PrP was spread throughout the cytoplasm of the epithelial cells (except in goblet cells), located close to the lumen as well in the villi. The PrP was located within the striated border cells, the glandular cells located at the base of the villi, and the specialized M cells associated with lymphocytes infiltrating the epithelium (Fig. 1 G and J). The lamina propria and the submucosa contained labeled lymphocytes as did the wall of the lymph and blood vessels. In these areas, PrP-labeled cellular elements also were observed at the periphery of both lymphoid structures associated with the intestine: the elongated Peyer's patches (Fig. 1H) and the spherical lymph nodes. In the colon, PrP immunoreactivity was noted in the columnar epithelial cells near the lumen but not in the crypts. The tunica muscularis of the different regions of the gastrointestinal tract never exhibited immunoreactivity. The spleen showed an obvious staining of numerous cells located in the red pulp (Fig. 1I) and, in lower number, at the periphery of the white pulp.
In the central nervous system of large-size lemurs in the preclinical stage of disease, we observed PrP particles in both dorsal and ventral roots of the spinal cord in the cervical region and scattered along vacuolated fibers in the spinal cord (Fig. 1A). PrP was also visible as dust-like particles in layer IV of the cerebral cortex near PrP-labeled fibers originating from the corpus callosum (Fig. 1B). Moreover, clearly degenerative central nervous system processes were seen in both the zoo eulemurs and the experimental microcebes. This degeneration was manifested by three abnormalities, which were never detected in the brains of control animals.
First, numerous aggregated Tau-containing neurons were present throughout the cerebrum, particularly in the cerebral cortex, the brain stem, the superior colliculus, and the thalamus (Fig. 1D). As the evolution of Tau proteins in the cortical pyramidal neurones is well studied in microcebes (6, 7), we were able to compare their number to those in the experimental microcebe with optimally preserved tissue (the condition of the tissue from the lemur killed by his cage mates was not good enough for quantitative study). The BSE-infected lemur had more than 10 times as many degenerating neurones as aged normal lemurs (8-13 years), and nearly 300 times as many as young lemurs of comparable age (1-2 years). In particular, degeneration of the pyramidal cortical neurones in healthy young adult microcebes begins in the occipital cortex, and aggregated Tau-containing neurones are never observed in the parietal and frontal cortices, whereas, on average, 280 and 269 abnormal neurones were found in these areas of the BSE-infected lemur.
Second, innumerable small vacuoles were present in the cortical parenchyma (Fig. 1C), often in close contact with the hyperphosphorylated Tau-containing neurones. In the brains and spinal cords of all animals, a majority of large nerve tract fibres exhibited vacuolation, and in some large bundle tracts, such as the reticular formation and corpus callosum, it was possible to distinguish between discrete vacuolated and nonvacuolated tracts.
Third, astrocytic gliosis was evident in the large increase of reactive astrocytes showing GFAP immunoreactivity, particularly well developed in the white matter of the brain, in layers I, V, and VI of the cortex, and in proximity to blood vessels. Blood vessesls in the pia matter also were surrounded by reactive astrocytes. In the spinal cord, GFAP-labeled astrocytes were very numerous in the white matter but also scattered in the central gray matter. Aggregated Tau proteins were seen in fibers of the spinal cord tracts and in the axoplasm of myelinated fibers in peripheral nerves near the spinal cord.
DISCUSSION
Pathogenesis has been a continuing subject of importance in the study of transmissible spongiform encephalopathies, having been first addressed systematically by Hadlow et al. (8-10) in a landmark set of experiments in which the sequential appearance of infectivity in different organs was determined in both naturally and experimentally acquired disease, continued by Kimberlin and Walker (11, 12) in a series of experiments on orally infected mice, and most recently extended by Beekes et al. (13, 14) to include parallel studies of PrP in tissues after oral infection and by Klein et al. (15) with particular attention to the role of B cells in neuroinvasion. All of these studies were undertaken by using scrapie as the model of infection, but preliminary investigations also have been reported on BSE in naturally and experimentally infected cattle (16).
From the ensemble of these studies it has become clear that, after oral infection, infectivity and pathologic PrP first appear in the digestive tract and its contained or proximate lymphoid tissues (tonsils, lymph nodes, Peyer's patches, and spleen), before moving, presumably through autonomic nervous system fibers, to the spinal cord and up to the brain. Natural and experimental BSE in bovines is notable in the comparatively limited distribution of infectivity outside the central nervous system, having been demonstrated only in the trigeminal and dorsal root ganglia, distal ileum, and (possibly) bone marrow and retina.
The present study, which extends our earlier investigations of two lemurs and one monkey dying with spongiform encephalopathy in the Montpellier zoo (1, 2), contributes two additional pieces of information about oral infection by transmissible spongiform encephalopathy agents. First, the immunohistochemical results of our experimental study of BSE-fed lemurs has precisely defined the distribution and localization of PrP within a variety of tissues early in the incubation period of disease. PrP (and by implication, the infectious agent) evidently is taken up by epithelial cells lining the lumen of the digestive tract (including those of the tonsil), initiating a reaction of the M cells and lymphocytes within the tissues of the digestive tract and in their lymphatic drainage system (including lymph nodes and spleen). Our observations also show that even before PrP can be detected in the central nervous system in the pattern typical of terminal illness, it can be traced along nerve pathways from ventral and dorsal root ganglia through the spinal cord into the brain cortex. These results are consistent with the observed distribution and progression of infectivity and PrP during the evolution of scrapie, as measured by infectivity assays (12) and Western blots of extracted PrP (14).
Second, the similar neuropathology and distribution of PrP in orally infected experimental lemurs and spontaneously affected zoo lemurs, together with the epidemiological observations confirming the occurrence of spongiform encephalopathy in animals fed a diet supplemented with meat protein that until 1996 had very likely included rendered British beef, leave little room for doubt that cases of spongiform encephalopathy in French primates resulted from infection by BSE-contaminated meat, just as in felines and ungulates in zoos elsewhere. Our unexpected finding that the same patterns of PrP distribution and brain degeneration were present in asymptomatic lemurs from two other French primate facilities suggests that BSE-contaminated diets may have been far more widespread than appreciated and mandates continued surveillance of primates in European zoos and breeding facilities.
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http://www.pnas.org/cgi/content/full/96/7/4046
J Infect Dis 1980 Aug;142(2):205-8
Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.
Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.
Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.
PMID: 6997404
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract
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