The Neuropathology of Experimental Bovine Spongiform Encephalopathy in the Pig

J. Comp. Path. 2000, Vol. 122, 131–143 doi:10.1053/jcpa.1999.0349, available online at http://www.idealibrary.com on

The Neuropathology of Experimental Bovine Spongiform Encephalopathy in the Pig S. J. Ryder, S. A. C. Hawkins, M. Dawson and G. A. H. Wells Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK

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

Introduction Bovine spongiform encephalopathy (BSE) is one of the transmissible spongiform encephalopathies (TSEs), or prion diseases, a group of progressive fatal nervous disorders of man and animals. The pathology of these diseases is characterized by a triad of morphological features, namely, spongiform change, neuronal loss and gliosis; the latter is predominantly an astrocytosis but also includes a microgliosis (Fraser, 1976). Essential also to the disease definition is the accumulation of the abnormal isoform PrPSc of the cellular protein PrP (Prusiner, 1991). Up to 1985, the TSEs, which include scrapie of sheep, appeared to be sporadic diseases affecting a small number of species. In 1985, however, a similar disease emerged in the British cattle population (Wells et al., 1987) and was subsequently recognized as a food-borne epidemic (Wilesmith et al., 1988). Since the original description of BSE in cattle, novel spongiform encephalopathies have been recorded in captive exotic ruminants (Kirkwood and Cunningham, 1993), domestic (Wyatt et al., 1991) and captive exotic cats (Kirkwood et al., 1995), and human beings (Will et al., 1996). 0021–9975/00/020131+13 $35·00

The origin of these novel diseases is now considered to be the same as that of BSE (Bruce et al., 1997), i.e., ingestion of the BSE agent in contaminated feed. It is notable that there is no evidence to date of naturally occurring TSE in the pig, a species which, in the UK, must have been exposed to contaminated feed before 1996, at which time the inclusion of meat and bone meal was banned from all animal feeds under the Bovine Spongiform Encephalopathy Order 1996. Furthermore, before the advent of BSE, attempts to transmit a TSE to the pig had been confined to the experimental challenge of pigs with material from cases of kuru, a human TSE; this study failed to produce disease (Gibbs et al., 1979). It would seem that the species barrier (Bruce et al., 1994) has prevented transmission of BSE to the pig under natural conditions. However, pigs are normally slaughtered at less than 6 months of age; in view of this and of the probably lengthy incubation period of a BSE-like disease in this species, it is unlikely that many pigs would develop a clinically detectable spongiform encephalopathy. There is, nevertheless, still the possibility that breeding stock, exposed to infection via meat and bonemeal before

132

S. J. Ryder et al.

the cessation of its use in animal feed, may, if infected, have lived long enough to accumulate significant concentrations of the disease agent, if not develop clinical disease. Experimental studies were initiated at this laboratory in 1989 to investigate the transmissibility of BSE to the pig. Transmission was achieved by multiple-route parenteral exposure to the agent and preliminary reports were published earlier (Dawson et al., 1990, 1994). The purpose of this report is to describe the pathology of the central nervous system (CNS) lesions found in the experimentally infected pigs. Details of the clinical signs and results of bioassay for infectivity in a wide range of tissues will be reported elsewhere. Materials and Methods Experimental Challenge with BSE Ten Landrace cross Large White pigs aged 1–2 weeks from the Central Veterinary Laboratory herd were inoculated simultaneously under general anaesthesia by three routes. As already described by Dawson et al. (1990), each pig received 0·5 ml intracranially, 1–2 ml intravenously and 8–9 ml intraperitoneally of an inoculum consisting of a 10% homogenate in saline of pooled bovine brain stem from four confirmed natural cases of BSE. Eleven pigs were similarly inoculated with normal saline as controls. Two challenged animals and two control animals were lost due to intercurrent disease early in the study and are therefore excluded from the present report. Post-mortem Examination and Histology Groups of challenged and control animals were killed at 24–25 and 60 months post-inoculation (p.i.) or when showing clinical signs of neurological disease. Pigs were sedated with azaperone (Stresnil; Janssen, High Wycombe, UK) before being killed by an intravenous injection of pentobarbitone. After sampling small portions of tissue for bioassay of infectivity, the brains and spinal cords were removed and fixed in 10% formol saline for 3 weeks before routine processing and paraffin wax embedding. Sections were cut at 5 lm thickness and stained with haematoxylin and eosin (HE) for routine histopathological examination. Full coronal sections were examined from the medulla oblongata at the levels of the obex and the cerebellar peduncles, from the midbrain at the levels of the rostral colliculi and the red nucleus, and from the thalamus at the level of the mamillary body. Half

coronal sections were examined from the occipital cortex including the hippocampus, from the thalamus, hypothalamus and parietal cortex, and from the basal nuclei and frontal cortex.

Immunocytochemistry Anti-PrP immunocytochemistry was carried out on wax sections as described by Haritani et al. (1994). Briefly, sections were de-waxed and re-hydrated to water, pre-treated with 96% formic acid for 20 min and by hydrated autoclaving at 122°C for 30 min. Initially, sections from a single affected animal (no. 58/90) were examined by means of five primary antisera and “detection” with streptavidin-biotinhorseradish peroxidase (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine. The five anti-PrP primary antisera, known to have broad species cross-reactivity, were: rabbit antiserum 971F (dilutions 1 in 4000 to 1 in 15,000) raised against a bovine PrP peptide fragment from amino acids 230–244 (Wells and Wilesmith, 1995); rabbit antiserum 1B3 (1 in 1000 to 1 in 4000), raised against murine ME7 scrapie-associated fibrils (Farquhar et al., 1989); rabbit antiserum SP40 (1 in 250 to 1 in 2000), raised against the ovine PrP peptide sequence 219–232 (Lantos et al., 1992); 3F4 monoclonal antibody (1 in 4000 to 1 in 16,000), which recognizes an epitope between amino acids 109– 112 of the hamster PrP molecule (Kascsak et al., 1987); and the KG9 monoclonal antibody (1 in 20 to 1 in 160) which recognizes a linear epitope within the proteinase-resistant part of the bovine and human PrP molecules (C. Birkett, Institute for Animal Health, Compton, UK). Normal rabbit serum was used as a control. After evaluation of the immunolabelling results given by the five antisera, five coronal sections from each pig (medulla at the level of the obex; cerebellum and rostral medulla at the level of the cerebellar peduncles; midbrain at the level of the rostral colliculi; the thalamus with parietal cortex; and the forebrain, including the basal nuclei and frontal cortex) were immunolabelled with antibody 1B3 at a dilution of 1 in 1000 as described above.

Results The numbers of animals killed at each time point, the presence of clinical signs, and the immunocytochemical and histopathological results are summarized in Table 1.

133

Neuropathology of Experimental BSE in the Pig Table 1 Results of immunocytochemistry and routine histopathology Pig no.

Status

Time killed after challenge (months)

Clinically affected

Immunolabelling

Lesions of TSE∗

58/90 36/91 38/91 42/91

Challenged Challenged Challenged Challenged

17 25 25 25

Yes No No No

Pos Pos Pos Neg

++ + + –

44/91 40/91 37/91 33/91

Control Control Control Control

24 24 24 24

No No No No

Neg Neg Neg Neg

– – – –

341/91 42/92 66/92 310/92

Challenged Challenged Challenged Challenged

33 35 35 38

Yes Yes Yes Yes

Pos Pos Pos Pos

++++ ++++ ++++ ++++

129/92 193/94 402/94 425/94 433/94

Control Control Control Control Control

36 60 60 60 60

No No No No No

Neg Neg Neg Neg Neg

– – – – –

∗ Vacuolation: −, none; +, mild to moderate in most areas; ++, moderate to severe, widespread; ++++, severe and extensive, many areas affected.

Histopathology of Pigs with Clinical Disease Five animals developed neurological signs (principally behavioural changes and ataxia) and were killed in the terminal stages of disease. The incubation periods ranged from 17 to 38 months. In all five animals there was severe neuropil vacuolation of the telencephalon, diencephalon and mesencephalon (Fig. 1). Vacuolar changes were least severe in the caudal brainstem. Occasional neuronal vacuoles were seen within neurons of the dorsal nucleus of the vagus nerve (up to three in any one nuclear column). A few neuropil vacuoles were seen in the nucleus of the spinal tract of the trigeminal nerve. In addition, neuropil vacuoles were seen in the nucleus of the solitary tract and in the reticular formation in two animals (nos 310/92 and 341/91) and in the accessory cuneate nucleus in one (341/91). No changes were seen in other nuclei, including the dorsal nuclei of the trapezoid body. The cerebellar cortex was severely affected in four of the five animals. The molecular and granulecell layers of all lobes showed severe vacuolar change. Pig 58/90 showed only mild vacuolation of the cerebellum, the vacuolation being located mainly in the molecular and granule-cell layers of the vermis. No changes were seen in the cerebellar nuclei. In all five animals the severest vacuolation of the

midbrain was seen in the tectum (Fig. 2) and periaqueductal grey matter. Marked vacuolation was also found in the substantia nigra. The red nucleus and oculomotor nucleus were unaffected. In four pigs extensive and severe vacuolar changes affected all areas of the thalamus, including the geniculate nuclei. Only the habenular nuclei were spared. Pig 58/90 showed extensive but relatively mild vacuolar change in this area. The hypothalamus was only moderately affected by fine neuropil vacuolation; however, several large vacuoles, which were bilaterally distributed but not symmetrical, were seen in the hypothalamus of pig 310/92. In four pigs the entire cerebral cortex showed severe neuropil vacuolation. In pig 58/90, however, only the frontal cortex was severely affected, other areas showing no more than moderate changes. The piriform lobe was the least affected area of cortex in all five pigs. In all five animals, the amygdala showed a clearly demarcated, severe vacuolation of the dorsal area but only moderate changes in the ventral part. Vacuolar changes in the hippocampus were mild in all five pigs. Areas CA1–3 (Pearson and Pearson, 1976) showed slight to moderate neuropil vacuolation. The dentate gyrus showed minimal changes in four animals but none in pig 58/90. In all five pigs the severest changes were found

134

S. J. Ryder et al.

Fig. 1. Severe spongiform encephalopathy in the putamen. Pig 310/92, killed 38 months after infection. HE. ×256.

in the basal nuclei. Neuropil vacuolation was extremely severe in the caudate nucleus and putamen, and slightly less so in the globus pallidus and septum. In the spinal cord, occasional neuronal and neuropil vacuoles were seen in the ventral horns in pig 341/91 and in the dorsal horns in pig 58/ 90. Throughout the neuraxis, the principal lesion was vacuolation of the grey matter. Significant white matter changes were not observed. In full coronal sections of the medulla, cerebellum, midbrain and thalamus the lesions were found to be bilateral but not symmetrical in respect of the intensity of vacuolation. Swollen and pleomorphic astrocyte nuclei, suggestive of astrocytosis, were noted in severely affected areas of the thalamus, basal nuclei and cerebral cortex of all pigs, but gliosis was not a prominent feature. Histopathology of Pigs with Pre-clinical Disease Three animals (nos 36/91, 38/91 and 42/91) were killed 25 months p.i. In two of these pigs, widespread vacuolar changes, similar to those described in clinically affected animals, were observed.

One pig (36/91) showed a few neuropil vacuoles in the nucleus of the spinal tract of the trigeminal nerve; the other (38/91) showed a single vacuolated neuron in the dorsal nucleus of the vagus nerve. Otherwise no changes were seen in the medulla oblongata of these two animals. In the cerebellum, occasional vacuoles were seen, confined to the granule-cell and molecular layers of the vermis. Moderate vacuolation of the superficial layers of the rostral colliculi was observed in both pigs, but in the peri-aqueductal grey matter this change occurred in only one animal (36/91). No changes were seen in the reticular formation, red nucleus or oculomotor nucleus of either pig. In the thalamus, moderate to severe vacuolation affected all areas except the habenular nucleus. In the hypothalamus, large neuropil vacuoles were seen in one pig (38/91). No lesions were observed in the hippocampus or amygdala and only occasional vacuoles were seen in the cerebral cortex, basal nuclei, caudate nucleus and putamen. In the third animal (42/91), a single vacuole was seen in the olivary nucleus; moderate vacuolation was observed in the superficial layers of the rostral

Neuropathology of Experimental BSE in the Pig

135

Fig. 2. Spongiform change affecting all layers of the rostral colliculus. Pig 42/92, killed 35 months after infection. HE. ×128.

colliculi, but nowhere else in the midbrain, thalamus, cerebral cortex or basal nuclei. Several large vacuoles were observed in the hypothalamus. No lesions were observed in the spinal cords of these three animals. Histopathology of Control Animals There was no evidence of the marked neuropil vacuolation described in challenged animals in the brains of any of the control animals. However, in all control pigs in this study there was vacuolation of the superficial layers of the grey matter of the rostral colliculi (Fig. 3). These vacuoles were of variable size but indistinguishable, except in number, from the vacuoles found at this site in affected animals. Unlike that in infected animals, the vacuolation did not extend into the deeper layers of the rostral colliculi or into other areas of the midbrain or diencephalon. Vacuolated neurons in the dorsal nucleus of the vagus nerve (up to three in any one nucleus) were found in five control animals.

Vacuolation of the hypothalamus was also observed in seven control animals (Fig. 4). The vacuoles were, in general, larger than those observed in the infected animals. In addition, vacuolar change was seen in the internal capsule and occasionally in other white matter tracts. Occasional pleomorphic and paired astrocyte nuclei accompanied by some rod cells, suggesting moderate astrocytosis and microgliosis, were found in many of the controls, especially in one animal killed at 3 years (no. 129/92), which also showed a mild non-suppurative encephalitis. Immunocytochemistry Of the five antisera tested initially on the single infected pig (no. 58/90) only the antiserum 1B3 was found to give positive immunolabelling. No immunolabelling was obtained with the antisera 971F and SP40, or with normal rabbit serum, or with monoclonal antibodies 3F4 and KG9. The results given are therefore those obtained with antiserum 1B3.

136

S. J. Ryder et al.

Fig. 3. Small numbers of neuropil vacuoles in the superficial layers of the rostral colliculus. Pig 33/91, control animal killed 24 months after inoculation with saline. HE. ×128.

Challenged Animals Several patterns of immunopositivity were identified. Fine particulate and punctate neuropil labelling and stellate foci of labelling apparently associated with glial cells (Fig. 5), which were the most commonly found patterns, were found in all seven pigs in which widespread vacuolar changes were observed. Coarse granular neuronal cytoplasmic labelling was also commonly seen (Fig. 6). Intense peri-neuronal immunolabelling was seen to outline some perikarya in the globus pallidus of one pig (341/91) (Fig. 7). In two of the clinically normal animals killed 24 months p.i., granular cytoplasmic labelling was found in neurons of the brain stem nuclei. Neuropil staining was found in the brain stem, dorsal thalamus, corpus striatum (particularly the caudate nucleus and putamen) and deeper layers of the cerebral cortex. The cerebellum showed increased labelling of the granule-cell and molecular layers. Mild labelling was found in the medial geniculate nucleus and the rostral colliculi.

Pig 42/91, which did not show widespread vacuolar change, showed fine granular neuronal cytoplasmic labelling in the medulla and fine granular perineuronal labelling in the dorsal nuclei of the trapezoid body, as described below for control animals. No evidence of neuropil immunolabelling was found in this animal. At the end-stage of disease, immunolabelling was similar but much more intense and widespread, particularly so in the substantia nigra, thalamus, lentiform nuclei and to a lesser extent in the brainstem and cerebral cortex. It was, nevertheless, extremely mild in the medial geniculate nucleus and the rostral colliculi. In the dorsal nucleus of the trapezoid body, neurons were uniformly diffusely labelled throughout the cytoplasm and did not show distinct peri-neuronal staining. Control Animals Immunopositivity, found mainly in neuronal cell bodies in the brain stem, appeared as fine granular

Neuropathology of Experimental BSE in the Pig

137

Fig. 4. Numerous large vacuoles in the hypothalamus. Note the difference in size of vacuoles compared with Fig. 2. Pig 33/ 91, control animal killed 24 months after inoculation with saline. HE. ×128.

cytoplasmic labelling, with or without nuclear labelling. There was marked individual variation in the intensity and location of labelling. Various brain stem nuclei showed positively labelled cells, including the dorsal nucleus of the vagus nerve, hypoglossal nucleus, accessory cuneate nucleus, nucleus of the spinal tract of the trigeminal nerve, nucleus ambiguus, olivary nucleus, facial nucleus, vestibular nucleus and cochlear nucleus. The cerebellum showed weak labelling of both granulecell and molecular layers. In the midbrain, the oculomotor and red nuclei were similarly labelled. In three of the control animals (129/92, 40/91 and 44/91) there was bilaterally symmetrical, weak to moderate, peri-neuronal labelling in the dorsal nuclei of the trapezoid body (Fig. 8). There was no labelling of neurons in the tectum of the midbrain, the thalamus, basal nuclei or cerebral cortex. There was no neuropil immunolabelling in any brain area. Discussion The pathological changes described were consistent with a diagnosis of TSE, showing characteristic

neuropil and neuronal vacuolation and PrP accumulation (Wells and McGill, 1992; Wells et al., 1994). This further confirms the earlier preliminary reports (Dawson et al., 1990, 1994) of the susceptibility of the pig to BSE when exposed to infected brain tissue by multiple parenteral routes. The lesions in the pig consisted predominantly of neuropil vacuolation, affecting areas rostral to the medulla oblongata, in particular the midbrain, thalamus, striatum, cerebral and cerebellar cortices. A slight to moderate astrocytic response was found in most of the pigs examined, but there was considerable overlap in the severity of this change between affected and control animals. Astrocytosis and mild microgliosis appeared to be variable and were certainly not striking features of experimental BSE in this species. Amyloid plaques, which are rarely found in BSE (Wells et al., 1991), were not found in any of the animals; although Congo red staining, a specific stain for amyloid, was not employed to confirm this, no concentrations or plaquelike aggregations were detected by PrP immunohistochemistry. The lesion pattern of experimental BSE in cattle

138

S. J. Ryder et al.

Fig. 5. Widespread multi-focal PrP labelling in neuropil, with a stellate configuration, apparently around glial cells. Pig 341/ 91, killed 33 months after infection. Immunolabelling. ×256.

infected intracranially resembles that found in the natural disease (Wells et al., 1992). Vacuolation is most severe in the brain stem nuclei, particularly the spinal nucleus of the trigeminal nerve and the nucleus of the solitary tract, and also in the periaqueductal grey matter of the midbrain. In naturally occurring cases of BSE, lesions more rostral to the midbrain are generally less severe, and are minimal in the cerebral and cerebellar cortices. However, in experimental BSE produced by parenteral (including intracranial) inoculation more intense lesions occur in the thalamus than are found in natural disease. This is consistent with the differences in lesion profile found in rodent scrapie produced by intracerebral inoculation as opposed to inoculation by other parenteral routes (Kimberlin and Walker, 1986). The lesions in the pig did not reflect this pattern but showed the severest lesions rostral to the brain stem; this more closely resembled the pattern in feline spongiform encephalopathy (FSE) (Wyatt et al., 1991). One challenged animal (58/90; Table 1) developed clinical signs after an incubation period of 17 months, compared with a mean of c.35 months

for the other four clinical cases. The reasons for this finding are unclear. It may have been due to relative differences between the three inoculation routes in terms of spread of the agent, the short incubation period reflecting dominance of the intracerebral inoculum. However, the lesions found in pig 58/90 were similar in distribution to those of all the other infected animals, appearing intermediate in severity between the preclinical cases and the other four clinical cases. This suggests a common pathogenesis in all of the pigs, with little or no influence from possible different effects of the individual components of the multiple-route method of inoculation. A further possible explanation for the incubation period difference is differences in PrP genotype. The PrP gene of the pig has been sequenced (Martin et al., 1995); little is known, however, about gene polymorphism and it is therefore not possible to assess the role of PrP genotype in controlling the incubation period in this species. In fact, for all of the pigs in this study, the PrP genes were sequenced, but no coding region polymorphisms were found (unpublished observation).

Neuropathology of Experimental BSE in the Pig

139

Fig. 6. Coarse granular PrP labelling of the neuronal cytoplasm and mild neuropil labelling in the accessory cuneate nucleus. Pig 38/91, killed 25 months after infection. Immunolabelling. ×512.

The control animals in this study showed vacuolation in several areas of the brain to a degree that can probably be considered normal for this species. However, such vacuolation has not been reported previously in pigs and the possibility of some incipient disease cannot be ruled out. Neuronal vacuolation in certain neuroanatomical locations has been reported in cattle (McGill and Wells, 1993), sheep (Zlotnik and Rennie, 1958) and dogs (Kortz et al., 1997) as an incidental finding. The neuropil vacuolation of the rostral colliculi and the hypothalamus reported here in control animals and the otherwise unaffected challenged animal was almost certainly a similar incidental finding. Preliminary studies of archival material from several geographically diverse locations outside the UK support this view (G. A. H. Wells, unpublished data). The nature of the PrP deposits found in the brains of affected animals was broadly similar to that described in other species, including cattle, with BSE (Wells et al., 1994) and in sheep with scrapie (Foster et al., 1996). The coarse granular neuronal cytoplasmic and perineuronal labelling

and glial and neuropil labelling found in the affected pigs in this study have all been associated with disease-specific PrPSc accumulation in cattle and sheep. PrP immunocytochemistry served to confirm the diagnosis of TSE in pigs experimentally exposed to BSE but did not identify any of the abnormal patterns of PrP mentioned above in the one exposed pig (no. 42/91) found by routine histopathology to be unaffected. Similarly, this pattern of PrP accumulation was not observed in any of the control animals. Most important, PrP accumulation was not associated with the pattern of incidental vacuolar change. Nevertheless, PrP was consistently detected in control animals in the form of neuronal cytoplasmic labelling in neurons of the brain stem nuclei and of perineuronal labelling in the dorsal nuclei of the trapezoid body. With the antiserum 971F, we have observed similar smooth or finely granular cytoplasmic labelling within (but never around) neurons in similar sites (particularly the accessory cuneate nucleus) in both normal sheep and cattle (unpublished observations). This study suggests that in the pig the peri-neuronal distribution may be

140

S. J. Ryder et al.

Fig. 7. Intense peri-neuronal and particulate neuropil PrP labelling accompanying spongiform change in the globus pallidus. Pig 341/91, killed 33 months after infection. Immunolabelling. ×512.

another form of normal PrP (PrPc) expression. The presence of a consistent pattern of PrP immunolabelling has been reported in normal mice (Bruce et al., 1989) but not other species, despite the known presence of PrPc (the normal and antigenically almost identical form of the prion protein) in normal brain (Taraboulos et al., 1992). The consistency of this finding in different species and with two different antisera suggests that it represents PrPc, rather than non-specific labelling. In previous reports of PrP immunocytochemistry vacuolation has invariably been associated with PrP accumulation in cattle (Wells et al., 1994), sheep (Foster et al., 1996) and other species; however, PrP has been detected in the absence of vacuolation ( Jeffrey et al., 1998). In the present study it was therefore perhaps surprising to find (1) severe and extensive vacuolation in the absence of correspondingly intense PrP accumulation, and (2) little relation in many areas (e.g., cerebral cortex, rostral colliculi and medial geniculate nuclei) between the intensity of PrP immunolabelling and the extent of vacuolation. Of the antisera used to study the material initially, only 1B3 (raised against

a murine scrapie-associated fibril preparation) resulted in positive labelling. The reason for this is unclear. The antiserum 971F (raised against a bovine PrP peptide fragment from between residues 230 and 244) failed to label porcine PrP, despite 100% homology between the bovine and porcine PrP amino-acid sequences in this region (Groschup et al., 1997). Similarly, the monoclonal antibody 3F4 recognizes the three amino-acid epitope 109– 112 of hamster PrP, which is identical with the same region in the pig PrP amino-acid sequence. Clearly, amino-acid sequence differences between PrP of the pig and that of other species do not account for the failure of these reagents to detect porcine PrP. Technical factors affecting the ease with which epitopes can be unmasked in the pig may reflect the low intensity of labelling. However, there are reports of disease occurring in mice infected with BSE in the absence of any detectable PrP on the first passage (Lasmezas et al., 1997). Furthermore, in mice infected with fatal familial insomnia (FFI), PrP was undetectable, despite the presence of severe vacuolation (Collinge et al., 1995).

Neuropathology of Experimental BSE in the Pig

141

Fig. 8. Peri-neuronal labelling of neurons of the dorsal nucleus of the trapezoid body. Pig 40/91, control animal killed 24 months after inoculation with saline. ×512.

The nature of the disease that the BSE agent would produce in a foreign species (e.g., the pig) on first passage cannot necessarily be assumed to resemble the disease in its natural host. Intracranial challenge of cattle with transmissible mink encephalopathy (TME) or scrapie produced neurological disease in both cases, but only cattle challenged with TME showed marked neuropil vacuolation; scrapie produced a pathological change which, although similar, was much milder (Cutlip et al., 1994; Robinson et al., 1995). It may, therefore, be speculated that BSE has occurred in the pig already, without showing the expected diagnostic findings, especially as the clinical signs may overlap with those of other nervous system diseases. This study, however, indicates that should infection occur naturally it is likely to resemble natural BSE in respect of the lesions but not necessarily their distribution. To date no such disease has been reported in the pig under natural conditions; attempts to transmit BSE to pigs by means of large oral doses have

proved unsuccessful (Bradley, 1996) but have yet to be reported in detail. Acknowledgments The antiserum 1B3 was a gift from Dr C. Farquhar, Institute for Animal Health, Edinburgh. The monoclonal antibody KG9 was a gift from Dr C. Birkett, Institute for Animal Health, Compton. The antiserum SP40 was a gift from Dr B. Anderton, Institute of Psychiatry, London. The monoclonal antibody 3F4 was a gift from Dr R. Rubenstein, Institute for Basic Research in Developmental Disabilities, New York, USA. This study was funded by the Ministry of Agriculture, Fisheries and Food.  2000 Crown References Bradley, R. (1996). Bovine spongiform encephalopathy: distribution and update on some transmission and

142

S. J. Ryder et al.

decontamination studies. In: Bovine Spongiform Encephalopathy: the BSE Dilemma, C. J. Gibbs, Ed., Springer-Verlag, New York, pp. 11–27. Bruce, M. E., Chree, A., McConnell, I., Foster, J., Pearson, G. and Fraser, H. (1994). Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philosophical Transactions of the Royal Society of London B, 343, 405–411. Bruce, M. E., McBride, P. A. and Farquhar, C. F. (1989). Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neuroscience Letters, 102, 1–6. Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., Cousens, S., Fraser, H. and Bostock, C. J. (1997). Transmissions to mice indicate that “new variant” CJD is caused by the BSE agent. Nature, 389, 498–501. Collinge, J., Palmer, M. S., Sidle, K. C. L., Gowland, I., Medori, R., Ironside, J. W. and Lantos, P. L. (1995). Transmission of fatal familial insomnia to laboratory animals. Lancet, 346, 569–570. Cutlip, R. C., Miller, J. M., Race, R. E., Jenny, A. L., Katz, J. B., Lehmkuhl, H. D., DeBey, B. M. and Robinson, M. M. (1994). Intracerebral transmission of scrapie to cattle. Journal of Infectious Diseases, 169, 814–820. Dawson, M., Wells, G. A. H., Parker, B. N. J., Francis, M. E., Scott, A. C., Hawkins, S. A. C., Martin, T. C., Simmons, M. M. and Austin, R. (1994). Transmission studies of BSE in cattle, pigs and domestic fowl. In: Transmissible Spongiform Encephalopathies. A Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities held in Brussels, September 14–15, 1993. R. Bradley and B. Marchant, Eds, Document VI/4131/94-EN, European Community, Agriculture, Brussels, pp. 161–167. Dawson, M., Wells, G. A. H., Parker, B. N. J. and Scott, A. C. (1990). Primary parenteral transmission of bovine spongiform encephalopathy to the pig. Veterinary Record, 127, 339–338. Farquhar, C. F., Somerville, R. A. and Ritchie, L. A. (1989). Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. Journal of Virological Methods, 24, 215–222. Foster, J. D., Wilson, M. and Hunter, N. (1996). Immunodetection of the prion protein (PrP) in the brains of sheep with scrapie. Veterinary Record, 139, 512–515. Fraser, H. (1976). The pathology of natural and experimental scrapie. In: Slow Virus Diseases of Animals and Man, R. H. Kimberlin, Ed., North-Holland Publishing Company, Amsterdam and Oxford, pp. 267–305. Gibbs, C. J., Gajdusek, D. C. and Amyx, H. (1979). Strain variation in the viruses of Creutzfeldt-Jakob disease and kuru. In: Slow Transmissible Diseases of the Nervous System, S. B. Prusiner and W. J. Hadlow, Eds, Academic Press, London, pp. 87–110. Groschup, M., Harmeyer, S. and Pfaff, E. (1997). Antigenic features of prion proteins of sheep and of other mammalian species. Journal of Immunological Methods, 207, 89–101.

Haritani, M., Spencer, Y. and Wells, G. A. H. (1994). Hydrated autoclave pre-treatment enhancement of prion protein immunoreactivity in formalin-fixed bovine spongiform encephalopathy-affected brain. Acta Neuropathologica, 87, 86–90. Jeffrey, M., Goodsir, C. M., Holliman, A., Higgins, R. J., Bruce, M., McBride, P. A. and Fraser, J. R. (1998). Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminal-specific PrP antibodies in scrapieaffected sheep and mice. Veterinary Record, 142, 534– 537. Kascsak, R. J., Rubenstein, R., Merz, P. A., TonnaDeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M. and Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie associated fibril protein. Journal of Virology, 61, 3688–3693. Kimberlin, R. H. and Walker, C. A. (1986). Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. Journal of General Virology, 67, 255–263. Kirkwood, J. K. and Cunningham, A. A. (1993). Spongiform encephalopathy in captive wild animals in Britain: epidemiological observations. In: Transmissible Spongiform Encephalopathies. A Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities held in Brussels, September 14–15, 1993. R. Bradley and B. Marchant, Eds, Document VI/4131/94-EN, European Community, Agriculture, Brussels, pp. 29–47. Kirkwood, J. K., Cunningham, A. A., Flach, J., Thornton, S. M. and Wells, G. A. H. (1995). Spongiform encephalopathy in another captive cheetah (Acinonyx jubatus): evidence for variation in susceptibility or incubation periods between species? Journal of Zoo and Wildlife Medicine, 26, 577–582. Kortz, G. D., Meier, W. A., Higgins, R. J., French, R. A., McKiernan, B. C., Fatzer, R. and Zachery, J. F. (1997). Neuronal vacuolation and spinocerebellar degeneration in young Rottweiler dogs. Veterinary Pathology, 34, 296–302. Lantos, P. L., McGill, I. S., Janota, I., Doey, L. J., Collinge, J., Bruce, M., Whatley, S. A., Anderton, B. H., Clinton, J., Roberts, G. W. and Rosser, M. N. (1992). Prion protein immunocytochemistry helps to establish the true incidence of prion diseases. Neuroscience Letters, 147, 67–71. Lasmezas, C. I., Deslys, J., Robain, O., Jaegly, A., Beringue, V., Peyrin, J., Fournier, J., Hauw, J., Rossier, J. and Dormont, D. (1997). Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science, 275, 402–405. Martin, T., Hughes, S., Hughes, K. and Dawson, M. (1995). Direct sequencing of PCR amplified pig PrP genes. Biochimica et Biophysica Acta, 1270, 211–214. McGill, I. S. and Wells, G. A. H. (1993). Neuropathological findings in cattle with clinically suspect but histologically unconfirmed bovine spongiform encephalopathy. Journal of Comparative Pathology, 108, 241–260. Pearson, R. and Pearson, L. (1976). The Vertebrate Brain, Academic Press, London.

143

Neuropathology of Experimental BSE in the Pig

Prusiner, S. B. (1991). Molecular biology of prion diseases. Science, 252, 1515–1521. Robinson, M. M., Hadlow, W. J., Knowles, D. P., Huff, T. P., Lacy, P. A., Marsh, R. F. and Gorham, J. R. (1995). Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie. Journal of Comparative Pathology, 113, 241–251. Taraboulos, A., Jendroska, K., Serban, D., Yang, S., DeArmond, S. and Prusiner, S. B. (1992). Regional mapping of prions in brain. Proceedings of the National Academy of Sciences of the USA, 89, 7620–7624. Wells, G. A. H., Hawkins, S. A. C., Hadlow, W. J. and Spencer, Y. I. (1992). The discovery of bovine spongiform encephalopathy and observations on the vacuolar changes. In: Prion Diseases of Humans and Animals, S. B. Prusiner, J. Collinge J. Powell and B. Anderton, Eds, Ellis Horwood, New York, pp. 256–274. Wells, G. A. H. and McGill, I. S. (1992). Recently described scrapie-like encephalopathies of animals: case definitions. Research in Veterinary Science, 53, 1–10. Wells, G. A. H., Scott, A. C., Johnson, C. T., Gunning, R. F., Hancock, R. D., Jeffrey, M., Dawson, M. and Bradley, R. (1987). A novel progressive spongiform encephalopathy in cattle. Veterinary Record, 121, 419– 420. Wells, G. A. H., Spencer, Y. I. and Haritani, M. (1994). Configurations and topographic distribution of PrP in the central nervous system in bovine spongiform encephalopathy: an immunohistochemical study. In:

Slow Infections of the Central Nervous System, J. Bjornsson, R. I. Carp, A. Love and M. Wisniewski, Eds, Annals of the New York Academy of Sciences, 724, 350–352. Wells, G. A. H. and Wilesmith, J. W. (1995). The neuropathology and epidemiology of bovine spongiform encephalopathy. Brain Pathology, 5, 91–103. Wells, G. A. H., Wilesmith, J. W. and McGill, I. S. (1991). Bovine spongiform encephalopathy: a neuropathological perspective. Brain Pathology, 1, 69–78. Wilesmith, J. W., Wells, G. A. H., Cranwell, M. P. and Ryan, J. B. M. (1988). Bovine spongiform encephalopathy: epidemiological studies. Veterinary Record, 123, 638–644. Will, R. G., Ironside, J. W., Zeidler, M., Cousens, S. N., Estibeiro, K., Alperovich, A., Poser, S., Pochiari, M., Hoffman, A. and Smith, P. G. (1996). A new variant of Creutzfeldt-Jakob disease in the UK. Lancet, 347, 921–925. Wyatt, J. M., Pearson, G. R., Smerdon, T. N., GruffydJones, T. J., Wells, G. A. H. and Wilesmith, J. W. (1991). Naturally occurring scrapie-like spongiform encephalopathy in five domestic cats. Veterinary Record, 129, 233–236. Zlotnik, I. and Rennie, J. C. (1958). A comparative study of the incidence of vacuolated neurons in the medulla from apparently healthy sheep of various breeds. Journal of Comparative Pathology, 68, 411–415.



Received, April 29th, 1999 Accepted, September 13th, 1999