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From: TSS ()
Subject: PATHOGENESIS OF EXPERIMENTAL BSE: PRECLINICAL INFECTIVITY TONSIL AND OBSERVATIONS DISTRIBUTION OFO LINGUAL TONSIL IN SLAUGHTERED CATTLE
Date: April 20, 2005 at 10:38 am PST
-------- Original Message --------
Subject: PATHOGENESIS OF EXPERIMENTAL BSE: PRECLINICAL INFECTIVITY TONSIL AND OBSERVATIONS DISTRIBUTION OFO LINGUAL TONSIL IN SLAUGHTERED CATTLE
Date: Tue, 19 Apr 2005 14:35:21 -0500
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTS.UNI-KARLSRUHE.DE
##################### Bovine Spongiform Encephalopathy #####################
Papers & Articles
Pathogenesis of experimental bovine
spongiform encephalopathy: preclinical
infectivity in tonsil and observations on
the distribution of lingual tonsil in
slaughtered cattle
G. A. H. Wells, J. Spiropoulos, S. A. C. Hawkins, S. J. Ryder
The infectivity in tissues from cattle exposed orally to the agent of
BSE was assayed by the intracerebral
inoculation of cattle. In addition to the infectivity in the central
nervous system and distal ileum at stages of
pathogenesis previously indicated by mouse bioassay, traces of
infectivity were found in the palatine tonsil
of cattle killed 10 months after exposure. Because the infectivity may
therefore be present throughout the
tonsils in cattle infected with BSE, observations were made of the
anatomical and histological distribution of
lingual tonsil in the root of the tongue of cattle. Examinations of
tongues derived from abattoirs in Britain
and intended for human consumption showed that macroscopically
identifiable tonsillar tissue was present
in more than 75 per cent of them, and even in the tongues in which no
visible tonsillar tissue remained,
histological examination revealed lymphoid tissue in more than 90 per
cent. Variations in the distribution of
the lingual tonsil suggested that even after the most rigorous trimming
of the root of the tongue, traces of
tonsillar tissue may remain. The Veterinary Record, March 26, 2005
BOVINE spongiform encephalopathy (BSE) is a foodborne
transmissible spongiform encephalopathy or prion disease of
domestic cattle (Wilesmith 1991, 1998,Wilesmith and others
1991) in which the pathogenetic importance of the alimentary
tract has been demonstrated experimentally by a sequential
study of calves orally exposed to the BSE agent (Wells and
others 1994, 1996, 1998, Terry and others 2003). A summary
of the previously reported findings is given in Fig 1. The
pathogenesis of BSE appears to differ from that of scrapie in
sheep in that the distribution of infectivity (Wells and others
1998, 1999, EC 2002) and disease-related prion protein
(PrP) (Somerville and others 1997,Wells and others 1998,
Terry and others 2003) in the tissues of the lymphoreticular
system (LRS) is relatively limited. The examination of tissues
of the central nervous system (CNS) (Wells and others 1998)
and of the distal ileum (Terry and others 2003) has shown
that the sensitivities of the detection of disease-specific PrP
by immunohistochemistry and the detection of infectivity by
mouse bioassay, which has a previously calculated limit of
detectability of 101·4 LD50/g (Kimberlin 1996), are comparable.
The analytical sensitivity of a rapid method for the detection
of PrPSc in CNS tissues from this study of cattle exposed
orally to the agent of BSE (CEA test, later marketed as the BSE
Bio-Rad test) has also been shown to be similar (Grassi and
others 2001). In contrast, mouse bioassays of all the remaining
large range of tissues, representing principally the LRS, the
peripheral nervous system, the CNS, alimentary tract, striated
muscles and major viscera (Wells and others 1996), taken at
all sequential kill time points in the same experimental oral
exposure study, have shown no evidence of infectivity (Wells
and others 1999; G. A. H.Wells, S. A. C.Hawkins, unpublished
observations).
A further experiment (data not shown) has shown that the
titre of infectivity of BSE in tissues, when titrated across a
species barrier in mice, was underestimated by a factor of 500
(Wells and others 1999, Hawkins and others 2000, EC 2002;
G. A. H.Wells, S. A. C. Hawkins, unpublished observations).
To improve the sensitivity of the assay of infectivity in tissues
from the oral exposure study, additional assays of selected tissues
have therefore been conducted by the intracerebral inoculation
of cattle. These assays provide an assessment of the
infectivity in a tissue, by using the host species and the most
efficient route of inoculation. On December 29, 2004, the
assays had been in progress for between 65 and 99 months,
and they have confirmed infectivity in the tissues that were
found to be positive by the mouse bioassay (EC 2002). This
paper describes these interim results and provides preliminary
evidence of traces of infectivity in the palatine tonsil of
cattle killed 10 months after being exposed orally; this infectivity
had not been detected previously by the mouse bioassay
(Wells and others 1998) and its presence raises the
possibility that infectivity may be present throughout the tonsils,
the independent lymphatic organs of the pharynx.
In cattle, the anatomically named components of the
tonsils comprise the palatine, lingual, pharyngeal and tubal
tonsils (Schummer and Nickel 1979).Under EU legislation to
prevent human exposure to the agent of BSE, before October
1, 2003 (EC 2001), the entire head, including the tonsils but
excluding the tongue, was designated specified risk material
(SRM) from six months of age in cattle from the UK and
Portugal, and the skull, including the brain, eyes and tonsils,
was designated SRM from 12 months of age in cattle from
other EU countries. From October 2003, as a result of the
preliminary findings of this study, the tonsils from bovine animals
of all ages slaughtered throughout the EU were designated
SRM (EC 2003). All of the tonsillar structures are within
the head, but the tongue is not classified as SRM and can be
removed from the head for human consumption. Because the
lingual tonsil is located at the root of the tongue, it is the one
part of the tonsillar structures that has the potential, depending
upon the precise butchering practices, to enter the human
food chain inadvertently. In view of this possibility, the
anatomical and histological distribution of tonsillar tissue in
the root of the tongue of cattle, as supplied for processing and
human consumption in Britain, were investigated.
MATERIALS AND METHODS
Tissue samples and bioassays
For the assay of infectivity by the intracerebral inoculation
of cattle, two-week-old Holstein-Friesian calves were assem-
Papers & Articles
Pathogenesis of experimental bovine
spongiform encephalopathy: preclinical
infectivity in tonsil and observations on
the distribution of lingual tonsil in
slaughtered cattle
G. A. H. Wells, J. Spiropoulos, S. A. C. Hawkins, S. J. Ryder
The infectivity in tissues from cattle exposed orally to the agent of
BSE was assayed by the intracerebral
inoculation of cattle. In addition to the infectivity in the central
nervous system and distal ileum at stages of
pathogenesis previously indicated by mouse bioassay, traces of
infectivity were found in the palatine tonsil
of cattle killed 10 months after exposure. Because the infectivity may
therefore be present throughout the
tonsils in cattle infected with BSE, observations were made of the
anatomical and histological distribution of
lingual tonsil in the root of the tongue of cattle. Examinations of
tongues derived from abattoirs in Britain
and intended for human consumption showed that macroscopically
identifiable tonsillar tissue was present
in more than 75 per cent of them, and even in the tongues in which no
visible tonsillar tissue remained,
histological examination revealed lymphoid tissue in more than 90 per
cent. Variations in the distribution of
the lingual tonsil suggested that even after the most rigorous trimming
of the root of the tongue, traces of
tonsillar tissue may remain.
Veterinary Record (2005)
156, 401-407
G. A. H.Wells, BVetMed,
FRCPath, DipECVP,
DipACVP, MRCVS,
J. Spiropoulos, DVM, PhD,
MRCVS,
S. A. C.Hawkins, MIBiol,
S. J. Ryder, MA, VetMB,
CertVR, PhD, MRCVS,
Veterinary Laboratories
Agency Weybridge,
New Haw, Addlestone,
Surrey KT15 3NB
The Veterinary Record, March 26, 2005
bled from herds free of clinical BSE and with a history of
no exposure to meat and bone meal. The PrP genotype of
the calves with respect to the octarepeat polymorphism
(Goldmann and others 1991) was determined from EDTA
blood samples, to ensure that as far as possible representative
genotypes were allocated to the experimental groups,
given that the calves were acquired for the programme of
inoculations over a period of three years (1996 to 1999). The
PrP coding region of the DNA extracted was amplified by PCR
and the octarepeat number, detected by the length of the
amplimer generated, was determined by gel electrophoresis.
Only 6:6 and 6:5 genotypes were detected among the 320
calves used in the study.
Inocula were freshly prepared from frozen tissue samples
as 10 per cent homogenates in saline, as described for the
assay of infectivity in mice by Wells and others (1998). The
inocula consisted of tissue pools from exposed cattle killed
at selected sequential time points of the oral exposure study
(Wells and others 1996) (Table 1). Groups of five calves aged
four to six months were inoculated intracerebrally with
1·0 ml of inoculum, using a semistereotaxic technique which
ensured that the inoculum was deposited in the brain in an
anatomically reproducible pattern. A spinal hypodermic needle
(Yale 9 cm) was inserted to a calculated depth through a
paramedian trephine in the left frontal bone and directed
through the left parietal cortex of the brain, traversing the
midline, into the contralateral mesencephalon. The inoculum
was deposited along the entire needle tract while the needle
was being slowly withdrawn. Two groups of five calves were
similarly inoculated with saline to serve as procedural controls.
After they had been inoculated the experimental groups
were housed separately, to avoid nose-to-nose contact and
prevent possible contact with excreta between groups.
Husbandry procedures ensured the integrity of each group by
the rigorous use of dedicated equipment and protective clothing.
The cattle were monitored clinically for signs of disease,
and on the development of clinical signs indicative of BSE they
were killed using an intravenous injection of pentobarbitone
sodium and exsanguinated.A sample of caudal brainstem was
taken fresh for the detection of PrP by the Prionics Check
Western immunoblotting method probed with monoclonal
antibody (mAb) 6H4 (Cooley and others 2001), and sections
of the brain (medulla at the level of the obex,medulla at the
level of the caudal cerebellar peduncles and mesencephalon)
were examined by histopathology and by immunohistochemistry
for PrP, as described by Terry and others (2003).
The original histopathological assessment of the brains of
mice inoculated with pooled palatine tonsil from cattle killed
10 months after having been exposed orally was supplemented
with an immunohistochemical examination for the
presence of disease-specific PrP. This approach, described by
Wells and others (1999, 2003), was used because it has the
potential to provide greater specificity and sensitivity in the
detection of disease in experimental transmissions of TSE
agents. The immunohistochemical method used was that
Papers & Articles
FIG 1: Summary of significant observations from a sequential time point
kill study of the
pathogenesis of bovine spongiform encephalopathy (BSE) after the oral
exposure of
calves to the BSE agent (Wells and others 1998, Terry and others 2003;
G. A. H. Wells,
S. A. C. Hawkins, unpublished observations). Bars indicate the range of
intervals at which
the tissues of the exposed cattle were positive for each of the
observational parameters.
* Mouse bioassays completed subsequent to Wells and others (1998)
extended the
confirmation of infectivity to 40 months. IHC Immunohistochemistry, DRG
Dorsal root
ganglion, ND Not done
Months after exposure 2 6 10 14 18 22 26 32 36 38 40
Onset of clinical signs
Infectivity and PrP (IHC) in distal ileum ND ND
Infectivity and PrP (IHC) in CNS
Infectivity in DRG
Infectivity in trigeminal ganglion
Infectivity in bone marrow
Diagnostic spongiform changes
*
*
Time of death of orally exposed donor cattle (months)
Tissue (derived from orally exposed cattle) 6 10 18 22 26 32 36
Caudal medulla/spinal cord* (pooled) 83 81 23
(22-23)
Caudal medulla 72 81 71 70
Spinal cord* 72 80 71 70
Skeletal muscle§ 76 99 71 98
Sciatic/radial nerves 74 72 71 97
Parotid/submandibular salivary 72 71
glands
Distal ileum 27 22 24 71 83
(23-30) (22-23) (24-25)
Liver 76 99 71 98
Spleen 73 78 71 70
Thymus 81 77
Tonsil 73 45! 71 70
(45)
Mesenteric lymph node 73 72 70
Superficial cervical/popliteal lymph nodes 74 72 71
Buffy coat 73 72 71 97
Bone marrow 70 69 70 69
Skin 72 70 81
Kidney 77 99 71 98
Urine 65
* Spinal cord levels C2-C3, T10-T11, L3-L4
Mean incubation period of five of five cattle affected
! One of five cattle affected, with the remaining four animals in the
group surviving 76 months to December 29, 2004
§ Pool of masseter/semitendinosus/longissimus dorsi
No assay
TABLE 1: Status, on December 29, 2004, of intracerebral assays of
infectivity of tissue pools from cattle killed at sequential time kill
points between six and 36 months after experimental oral exposure to
BSE. Positive tissue infectivity assay results are given as the
mean (range) incubation period in months. Interim results of as yet
negative assays are expressed as survival period after
inoculation in months
The Veterinary Record, March 26, 2005
described by Wells and others (2003), but the antiserum was
applied at a dilution of 1/2000.
Samples of formalin-fixed palatine tonsil and retropharyngeal
lymph node taken from the cattle at all sequential kill
time points throughout the experimental oral exposure study
(Wells and others 1998) were also examined immunohistochemically
for disease-specific PrP, by using the R145 mouse
mAb (Terry and others 2003). This examination was conducted
on the palatine tonsil and retropharyngeal lymph
node contralateral to that sampled fresh for the assay of infectivity.
Anatomical examination of the tongue
Initially, 100 bovine heads were purchased after meat inspection
from a single abattoir. The tongues were removed and
examined macroscopically for the presence of the focal epithelial
invaginations (fossulae tonsillares) that denote the location
of lingual tonsillar lymphoid follicles (Schummer and
Nickel 1979). In this region, three positions were sampled from
each tongue for histological examination (Fig 2): the arrays
of fossulae indicating the main area of the lingual tonsils (position
1), the caudal border of the filiform papillae (position 3)
and a position intermediate between these sites (position 2).
At each level, a 2 to 3 mm thick transverse slice was taken to
include the full depth of the mucosa and submucosa extending
to the striated muscle. From this slice two blocks of tissue
were cut, each representing a 3 cm wide cross-section of the
dorsum of the tongue, inclusive of each lateral aspect of the
tongue but excluding the middle area of the slice, as it had previously
been determined that the rows of fossulae radiated rostrolaterally
(Fig 3), so that to detect the most rostral visible
fossulae, the site sampled must include the lateral border of the
tongue. In addition, a fourth position was sampled from 15
of the tongues, to include areas observed where solitary
fossulae-like structures suggested that there might be underlying
tonsillar tissue. The selected blocks of tissue were placed
in histology cassettes, immersed in 10 per cent buffered
formol saline for three days, processed to paraffin wax by standard
methods, sectioned at 5 µm and stained with haematoxylin
and eosin. Each section was examined by bright-field
light microscopy for the presence of either lymphoid follicles
associated with fossulae or diffuse lymphoid tissue.
After this exercise 251 bovine tongues were purchased
from 15 abattoirs after meat inspection and trimming for dispatch
to a processor. The abattoirs included large, medium
and small plants, located throughout Great Britain, and each
supplied between seven and 20 tongues. Each tongue was
examined macroscopically to identify the presence of fossulae
of the lingual tonsil close to the cut border at the root of
the tongue, and they were then classified into the following
three categories: tongues with abundant macroscopically
identifiable fossulae (group 1) (Fig 4); tongues with minimal
macroscopically identifiable fossulae, detected only after
detailed examination (group 2); and tongues with no macroscopically
identifiable fossulae (group 3) (Fig 4). On the basis
of indications from the initial study of 100 tongues, the lateral
borders of each tongue at the level of the torus linguae
were also examined for fossulae-like structures.
The tongues in which obvious lingual tonsil was identified
(group 1) were not examined further. From a random selection
of approximately three-quarters of the tongues in groups
Papers & Articles
FIG 2: Dorsal view of
the cut root of a bovine
tongue showing the
areas examined for
lingual tonsil.
Transverse cuts 1, 2 and
3 indicate the sampling
sites. Bar=2 cm
FIG 3: Dorsal view of the root of a bovine tongue showing
rostrolateral radiation of rows of fossulae in epithelial
grooves (scalpel blade tip), corresponding to position 1
shown in Fig 2; rostral to these are additional fossulae
arranged irregularly (blue arrowhead), and further rostral are
the vallate papillae (arrowheads) and the most caudal of the
filiform papillae (arrows), corresponding to position 3 shown
in Fig 2. Bar= 2 cm
FIG 4: Examples of two
bovine tongues
purchased from
abattoirs after meat
inspection and
trimming. The tongue
on the left shows no
visible lingual tonsil
(group 3); the tongue on
the right shows the
complete retention of
visible lingual tonsil
(group 1)
1
2
3
The Veterinary Record, March 26, 2005
2 and 3 a transverse block of tissue 2 to 3 mm thick was taken
from the cut surface at the root of the tongue by cutting parallel
to the slaughtermans cut. This tissue was fixed, as previously
described, and examined histologically for lymphoid
tissue.
RESULTS
Bioassay
The current status of the assays by the intracerebral inoculation
of cattle with tissues from cattle killed sequentially in the
previous oral exposure pathogenesis study (Wells and others
1998) is given in Table 1. One of the five cattle injected intracerebrally
with pooled palatine tonsil tissue from the three
cattle killed 10 months after oral exposure showed clinically
progressive signs of BSE at 45 months after inoculation, and
the diagnosis of BSE was confirmed by histopathological
examination, immunohistochemistry and Western blotting.
On December 29, 2004, the remaining four animals in this
group and the cattle inoculated intracerebrally with palatine
tonsil from cattle killed at other times after exposure
remained alive (Table 1). All of the remaining assays by the
intracerebral inoculation of cattle with tissues from the oral
exposure study have so far provided no evidence of transmission.
Of the 20 mice inoculated with palatine tonsil from cattle
killed 10 months after inoculation in the oral exposure study,
12 survived to the final kill at 650 days after inoculation. The
application of immunohistochemistry for the detection of
PrP in the mouse brains which were diagnosed negative by
histological examination (Wells and others 1999) confirmed
the negative results.
The immunohistochemical examination of samples of
palatine tonsil and retropharyngeal lymph node from cattle
killed at all sequential time points throughout the oral
exposure study provided no evidence of disease-specific
immunostaining.
Distribution of lingual tonsil
The macroscopic examination of the initial 100 tongues suggested
that there were large variations in the distance between
the rostral border of the visible fossulae of the lingual tonsil
and the caudal extent of the papillae of the dorsum of the
tongue. In some animals the borders of these structures were
separated by an area of smooth epithelium free of visible
papillae extending up to 1 cm rostrocaudally, in approximately
15 per cent of the tongues there was no apparent separation
between them, and in the remainder the separation
varied between these extremes.
The results of the histological examinations of these
tongues are summarised in Table 2. Lymphoid tissue, almost
entirely represented by lymphoid follicles, was present in all
the samples from position 1 (Fig 5); in 68 per cent of the samples
lymphoid tissue was present at position 3, that is, at the
level of the most caudal papillae, and was evident either as
lymphoid follicles (43 per cent) or as diffuse aggregations of
lymphoid cells (25 per cent).
The macroscopic observations of tonsillar tissue in the 251
tongues obtained from 15 different abattoirs are summarised
in Table 3. In 127 of the tongues (50·6 per cent) the cut edge
at the root of the tongue was caudal to most of the macroscopically
visible fossulae of the lingual tonsil, and in 65 (25·9
per cent) the cut was rostral to the majority of the fossulae
of the lingual tonsil, but clearly identifiable fossulae remained;
lingual tonsil fossulae were therefore identified in 76·5 per
cent of the tongues. In addition, in 141 of the tongues (56·2
per cent) occasional fossulae-like structures were observed on
the lateral aspect of the tongue, at the level of the vallate and
filiform papillae of the torus linguae (Fig 6).
The results of the histological examination of 96 tongues
selected randomly from the total of 124 tongues from groups
2 and 3 (Table 3) are given in Table 4. Lymphoid tissue was
found in 44 of 45 sampled in group 2 (97·8 per cent) and in
46 of 51 sampled in group 3 (90·2 per cent).
Papers & Articles
FIG 5: Histological sections of mucosa of tongue from
position 1 showing lymphoid follicles (arrows); bracketed
areas indicate confluent follicles. Haematoxylin and eosin.
Bar=1 cm
Sampling Number of Lymphoid Diffuse No lymphoid
position* tongues follicles lymphoid tissue tissue detected
1 100 99 (99·0) 1 (1·0) 0 (0·0)
2 99 89 (89·8) 5 (5·1) 5 (5·1)
3 100 43 (43·0) 25 (25·0) 32 (32·0)
4 15 3 (20·0) 0 (0·0) 12 (80·0)
* 1 Main concentration of fossulae, 2 Intermediate position between 1
and 3, 3 Caudal border of
filiform papillae, 4 Solitary fossulae-like structures
TABLE 2: Frequency of occurrence (%) of histologically observed lymphoid
tissue according
to sampling position/criterion in the root of the tongue
Percentage
Macroscopic classification* (number) of tongues
Group 1 50·6 (127)
Group 2 25·9 (65)
Group 3 23·5 (59)
* Group 1 Visible lingual tonsil, Group 2 Minimal amount of visible
lingual tonsil, Group 3 No visible lingual tonsil
TABLE 3: Percentages and numbers of tongues grouped by the
macroscopic classification of the occurrence of lingual tonsil
fossulae among 251 bovine tongues purchased from abattoirs
FIG 6: Bovine tongue
showing fossulae-like
structures (arrow) on
the lateral aspect at the
level of the vallate
papillae (arrowheads).
Inset: Histological
section showing
lymphoid follicles
(arrow) associated with
the fossulae-like
structures
The Veterinary Record, March 26, 2005
DISCUSSION
The assay by intracerebral inoculation of cattle confirmed
the presence of infectivity in a pool of caudal medulla and
spinal cord taken from cattle 32 months after oral exposure
to the BSE agent, and in samples of distal ileum taken from
cattle six, 10 and 18 months after exposure (Table 1). These
results are consistent with the previous results of the bioassay
of tissues from the oral exposure study (Wells and
others 1998). In each of these groups all five inoculated
cattle succumbed to disease. The mean incubation periods
recorded for the distal ileum groups were 27, 22, and 24
months, respectively. These results are consistent with the
RIII mouse bioassay, in which the mean incubation period of
mice inoculated with distal ileum taken from cattle six
months to 14 months after exposure gradually decreased,
indicating an increasing titre of infectivity, and the incubation
period of mice inoculated with distal ileum taken from
cattle 18 months after exposure reached a plateau (Wells and
others 1996, 1998).
Among the intracerebrally inoculated cattle that succumbed
to disease there was no apparent effect of differences
in the PrP gene octarepeat polymorphism on their susceptibility
to the infection.The single animal which developed disease
in the group inoculated with palatine tonsil was of the
6:6 genotype. Among the cattle which developed disease, the
ratio of 6:6 to 6:5 octarepeat genotypes was closely similar to
that reported in cattle naturally infected with BSE, irrespective
of breed (Hunter and others 1994).
Studies of infectivity in the tonsil of naturally occurring
cases of BSE (Fraser and Foster 1994) and of cattle experimentally
infected orally, by means of assays in conventional
inbred mouse strains (Wells and others 1998, EC 2002), have
not detected the BSE agent. This is therefore the first report
of the detection of infectivity in the tonsil of cattle infected
orally with the BSE agent. However, only one of the five
animals inoculated with the tissue has so far developed the
disease.
In studies of this nature the potential for such findings to
be the result of experimental error or artefact has to be considered.
The observation is unlikely to have been due to the
persistence of the inoculum by its entrapment in the palatine
tonsil after dosing in the experimental oral exposure
study: first, because of the lack of evidence for this phenomenon
in relation to earlier time points in the study at either
this or other anatomical sites where there could have been
residues of inoculum; and secondly, because of the absence of
any evidence of PrP immunostaining by immunohistochemistry.
Contamination of the source tissue at postmortem
examination is also unlikely because of the undetectable levels
in all other tissues except the distal ileum in the animals
killed 10 months after exposure in the oral exposure study. In
theory, one other possible reason for this animal succumbing
to BSE could be that it was naturally exposed to the infection
on its farm of origin, a potential hazard in the past when
obtaining calves for experimental BSE exposure from Great
Britain.However, this is also unlikely because no cases of BSE
have been traced either before or since on the farm from
which this animal was obtained.
It would be valuable to try to estimate the amount of infectivity
in the tonsil tissue taken from cattle 10 months after
their oral exposure to a 100 g dose (103·5 mouse intracerebral/
intraperitoneal LD50/g) of BSE-infected brain tissue, but
at present, given only a single animal incubation period, there
are insufficient data. However, the single value, when compared
with data derived from the titration of BSE-affected
brain tissue in cattle (Hawkins and others 2000; G. A. H.
Wells, S. A. C. Hawkins, unpublished observations) suggests
that the titre is low, even in terms of cattle intracerebral ID50/g.
The assays of cattle inoculated intracerebrally with pooled
palatine tonsil taken from cattle killed six, 18 and 26 months
after exposure in the oral exposure study, surviving (as of
December 29, 2004) at 73, 71 and 70 months after exposure,
respectively (Table 1), may still provide evidence of infectivity,
but the survival times may be approaching or even have
exceeded the limit of detection of BSE infectivity by the assay,
given the volume of inoculum and the numbers of animals
inoculated.
None of the remaining assays in cattle of tissues from the
oral exposure study (Table 1) includes tissues that were shown
to contain infectivity by the mouse assay.
The route by which the palatine tonsil became infected
cannot be determined from the present data but it is most
likely that it was infected primarily by direct exposure to the
orally administered inoculum and perhaps, because of rumination,
on several later occasions. The infection is postulated
to occur by mechanisms, as yet poorly understood, similar
to those which are thought to occur in relation to the primary
lymphoid tissue of the distal ileum (Ghosh 2002, Okamoto
and others 2003). There is no evidence from studies of the
pathogenesis of BSE that there is, at any stage of the disease,
widespread lymphatic or haematogenous spread of the agent.
The intracerebral inoculation of cattle with either pooled
lymph nodes (superficial cervical, mesenteric, retropharyngeal
and popliteal) or spleen, from five naturally infected BSE
cases (Wells and others 1999, Hawkins and others 2000, EC
2002; G. A. H.Wells, S. A. C.Hawkins, unpublished observations),
has not detected infectivity. The inoculated cattle did
not develop clinical signs, and when their brains were examined
86 months after inoculation there was no evidence of
transmission.
The presence of infectivity in tonsil is also a feature of naturally
occurring scrapie in sheep. It has long been established
that infectivity may occur and persist in the tonsil, and in
other alimentary lymphoid tissue, from the early preclinical
phase of scrapie (Hadlow and others 1979). In contrast with
BSE in cattle, scrapie infection is well recognised in lymphoid
tissues of sheep throughout much of the incubation period
(Hadlow and others 1979, 1982). In sheep, the tissue distribution
of PrPSc and presumably the scrapie agent is influenced
by their PrP genotype. PrPSc has been detected in palatine
tonsil of naturally infected sheep from three months of age in
VRQ homozygous Romanov sheep (Andreoletti and others
2000), from five months of age in VRQ homozygous Texel
sheep (van Keulen and others 2000) and from eight months
of age in ARQ homozygous Suffolk sheep (Jeffrey and others
2001a). In experimental BSE infection in ARQ homozygous
Romney sheep, PrPSc was detected in tonsil from 16 months
after infection, and rarely from four months of age in lymph
nodes draining the tonsil (Jeffrey and others 2001b). In contrast,
some sheep infected with scrapie, in particular those of
the VRQ/ARR genotype, do not have detectable PrPSc in tonsil
or in any other lymphoid tissue (Andreoletti and others
2000). In sheep, the palatine tonsil may in some respects be
a sentinel structure for the detection of PrPSc because it has
been found to be involved more frequently than many other
lymphoid tissues in natural scrapie (van Keulen and others
Papers & Articles
Macroscopic Lymphoid Diffuse No lymphoid tissue
classification* follicles lymphoid tissue detected
Group 2 64·5 (29) 33·3 (15) 2·2 (1)
Group 3 58·8 (30) 31·4 (16) 9·8 (5)
* Group 2 Minimal amount of visible lingual tonsil, Group 3 No
visible lingual tonsil
TABLE 4: Percentages and numbers of tongues with
histologically observed lymphoid tissue in 45 tongues from
group 2 and 51 tongues from group 3
The Veterinary Record, March 26, 2005
1996). However, in cattle, no genotype susceptibility to BSE
that might influence the tissue distribution of the infectivity
has been demonstrated.
The association of infectivity in BSE-infected cattle with the
distal ileal Peyers patch (Terry and others 2003) and, as
reported here, the palatine tonsil, clearly has pathogenetic
importance. Both tissues have a role in the sampling of antigens
from the lumen of the digestive system, and the presence
of infectivity at both sites from early in the incubation period
suggests that the early events in the pathogenesis of BSE and
scrapie may be similar. The major difference between the two
diseases in the involvement of the LRS appears to be a hostdetermined
quantitative difference in the levels of PrP that
accumulate in lymphoid tissues.
The continuation of the cattle bioassay studies and the
application of more sensitive methods for detecting the BSE
agent and PrP may provide further evidence of the distribution
of the agent in BSE-infected cattle, but the present findings
reinforce the idea that the involvement of the LRS in BSE
in cattle is highly restricted. The results of studies of BSE and
transmissible mink encephalopathy (TME) have been compared
with respect to possible similarities in pathogenesis that
contrast with that of scrapie (Wells and others 1996). In TME,
the involvement of extraneural tissues is also reported to be
confined to low concentrations of the agent in the lymphoreticular
tissues immediately before its detection in the CNS,
but the pathogenetic significance of this is unclear. In general,
extensive lymphoreticular involvement, especially in the alimentary
tract, has been suggested to be of possible significance
in relation to the potential for the shedding of the agent,
and therefore its horizontal transmission.
Anatomical texts give a clear description of the distribution
of the lingual tonsil in cattle (Schummer and Nickel
1979), but possible variations in its distribution were examined
because a knowledge of the extent of the distribution of
lymphoid tissue in the tongue is essential if such tissue is
to be excluded from materials for human consumption.
Furthermore, depending upon the method used to remove
the tongue from the head, it is possible that lingual tonsillar
tissue might be present in the part of the tongue which is
removed and intended for human consumption.
The tonsils form a ring of lymphatic tissue around the
nasopharynx and the oropharynx that consists of organised
accumulations of lymphoid tissue, the lymphoid follicles, or
diffuse, unorganised, lymphocytic infiltrations which may be
a transient anatomical feature (Schummer and Nickel 1979).
The latter, unless they show other pathological changes, are
generally indistinguishable from inflammatory foci. The specific
tonsillar tissue being assayed for infectivity in the oral
exposure study is the palatine tonsil; in domestic cattle this
is a discrete, bilateral, spherical structure, embedded in the
lateral wall of the oropharynx,which communicates with the
lumen of the pharynx via a sinus. In contrast, the lingual tonsil
consists of numerous tonsillar follicles distributed in the
mucosa of the root of the tongue, each with a distinct fossula
opening on to the dorsal mucosal surface of the root of the
tongue.
According to standard anatomical texts (Schummer and
Nickel 1979) the lingual tonsil, as indicated by the presence of
fossulae, lies in the caudal part of the root of the tongue and
is separated from the most caudal papillae, so that the tongue
can easily be dissected free of lingual tonsil. However,
lymphoid tissue was detected at the caudal border of the
papillae (position 3), and in the majority of the samples the
lymphoid tissue at position 3 was located at the lateral border
of the dorsum of the tongue.
Examinations of the tongues obtained from different abattoirs
showed that not all the lingual tonsil had been removed
from the majority of the tongues sold for human consumption,
and macroscopically identifiable tonsillar tissue was
retained at the root of more than 75 per cent of the tongues
(Table 3). Although 23·5 per cent of the tongues had no visible
lingual tonsil fossulae, only 12 per cent of those examined
histologically showed no evidence of lymphoid tissue (Table
4). There was histological evidence of foci of diffuse lymphoid
tissue in 31 of the 96 tongues examined from groups 2 and 3
(32·3 per cent). Because of the possibility of tangential planes
of section these foci could have indicated the proximity of
lymphoid follicles, but this was not confirmed; alternatively,
they could have been inflammatory reactions infiltrated with
lymphocytes. Such inflammatory foci may be formed in
subepithelial locations, as might be expected in any mucosal
site, but they would not be of significance in relation to BSE
infectivity, given the general lack of widespread involvement
of lymphoid tissue in BSE.
The degree to which the tongues were trimmed varied and
there was no consistency in the amount of tonsillar tissue
remaining at the root of the tongue. The variations in the
extent of the lingual tonsil may indicate that even in the most
rostrally trimmed tongues part of the lingual tonsil may
remain, because in some animals lingual tonsil was found at
the level of the last vallate papillae (Fig 3). Furthermore, the
fossulae-like structures observed on the lateral aspects of
the tongues, lateral to the torus, were rarely removed by the
trimming. The high frequency (90·2 per cent) with which
lymphoid tissue was detected histologically in the group 3
tongues (that is, those which lacked visible lingual tonsil) that
were examined (Table 4) suggests that the removal of all
macroscopic tonsillar tissue does not mean that the tongues
are necessarily free of lymphoid tissue.
No attempt was made to measure the amount of tonsillar
lymphoid tissue, relative to its precise anatomical location in
the tongue, but the qualitative results suggest that in order to
minimise the retention of lingual tonsillar lymphoid tissue in
tongues intended for human consumption, it would be necessary
to trim off the entire root of the tongue caudal to the
papillae of the dorsum. However, the trace level of infectivity
so far detected in tonsillar tissue and the localisation of the
lingual tonsillar lymphoid tissue, together with the current
SRM legislation for the removal of tonsil from cattle carcases
and the low and diminishing prevalence of BSE in the UK, suggest
that the risk of human exposure to infected tonsil is now
remote. It seems likely that under these circumstances any
additional trimming of the tongue would result in an immeasurable
reduction in the risk; nevertheless, the potential risk
needs to be assessed.
ACKNOWLEDGEMENTS
The word-processing skills of Mrs L. P. Cooper are gratefully
acknowledged. The authors thank Dr Trevor Martin, VLA, for
determining the PrP genotype of the calves in the study. The
study was funded by the former Ministry of Agriculture,
Fisheries and Food and by the Food Standards Agency.
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