Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents, PNAS, March 30, 1999

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tent/full/96/7/4046

The National Academy of Sciences, 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*, Belli, Françoise Cathala§, D.

Carleton Gajdusek¶, and 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.

    INTRODUCTION

In previous papers (1, 2), we reported that a rhesus monkey and two lemurs

housed in the Zoological Park in Montpellier, France, died of neurological

illnesses associated with spongiform encephalopathy and the presence of

proteinase-resistant protein (prion protein, or PrP). In this paper, we

bolster the presumption that the zoo animals had been infected with the

agent of bovine spongiform encephalopathy (BSE) with epidemiological and

experimental observations describing spongiform encephalopathy and PrP in an

additional 20 lemurs that had been exposed to beef protein dietary

supplements in three different primate facilities (Montpellier, Besançon,

and Strasbourg, France), and show that the distribution of PrP in the

tissues of these lemurs was similar to that seen in two experimental lemurs

fed with BSE-infected brain tissue.

    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 ' 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.

    RESULTS

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. ( Zoo lemur no. 712. Nerve fibers showing

PrP immunoreactivity (brown) in layer IV of the cerebral cortex. Anti-PrP

3F4, 1:200. © 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 (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.

    ACKNOWLEDGEMENTS

We are extremely grateful to J. Bons, C. Hovette, C. Legrand, and J. Scordo

(Montpellier); L. Chapuis (Dijon), J.Y. (Besançon), F. Haelewyn

(Lille), Y. Rumpler (Strasbourg), and D. on (Geneva), for helpful

discussions and/or the gift of animals. We also acknowledge Y. Charnay

(Geneva), A. Delacourte (Lille), and R. stein (New York) who provided

anti-PrP106-126, anti-Tau 961S28T, and anti-PrP 3F4; and C. Cohen-Solal,

V. Jallageas, E. , and S. Rouland (Montpellier) for technical

assistance. This work was supported in part by the Region

Languedoc-Roussillon, the Ministry of National Education, Research and

Technology, and the Mediterranean Association for the Study on Cerebral

Aging.

    ABBREVIATIONS

PrP, proteinase-resistant protein, or prion protein; BSE, bovine spongiform

encephalopathy; GFAP, glial fibrillary acidic protein.

    FOOTNOTES

To whom reprint requests should be addressed. e-mail:

ephemcb@....

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