Use of immunodiagnostic tests on an outbreak of scrapie in Latxa sheep: Pathogenetic and epidemiologic implications

Small Ruminant Research 72 (2007) 141–148

Use of immunodiagnostic tests on an outbreak of scrapie in Latxa sheep: Pathogenetic and epidemiologic implications N. Gomez a , L. Benedicto a , M.V. Geijo a , J.M. Garrido a , D. Garcia-Crespo a,1 , J.L. Korkostegi b , A. Hurtado a , R.A. Juste a,∗ a

b

Department of Animal Health, NEIKER (Basque Institute for Agricultural Research and Development), Berreaga, 1, 48160 Derio, Bizkaia, Spain Animal Husbandry Service, Diputacion Foral de Gipuzkoa, Plaza Gipuzkoa, s/n, 20004 Donostia-San Sebasti´an, Spain Received 24 March 2006; received in revised form 20 September 2006; accepted 4 October 2006 Available online 7 November 2006

Abstract A scrapie outbreak was detected in the year 2003 in a flock of 222 black-faced Latxa sheep when two suspect animals were submitted for laboratory diagnosis. After the two index cases were confirmed as scrapie positive by rapid tests and immunohistochemistry (IHC), two new clinical cases were recognized in the ante-mortem inspection during the flock destruction. Of the remaining 218 animals, 16 sheep were also positive to at least one of the rapid tests and IHC, and 6 more were negative by all rapid tests but positive by IHC in lymphoid and/or peripheral nervous tissues but not in central nervous system (CNS) tissues. Ten different genotypes were found in the flock, and, as expected, most of the positive sheep were of the ARQ/ARQ genotype (21/26, 80.7%), which was the most prevalent genotype in this flock and also in Latxa breed. In total, 26 positive ewes were identified in a flock with only 4 clinical cases (clinical/subclinical ratio of 1:5.5) indicating that the prevalence of infection in a positive flock might be considerably higher than clinical data indicates. The implications of the different patterns of PrPSc deposition in the pathogenesis and dissemination of scrapie within the host are discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Scrapie; PrP; Encephalopaties; Rapid tests; Immunohistochemistry; TSE; Genotype

1. Introduction Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE), chronic wasting disease in deer ∗

Corresponding author. Tel.: +34 944 034 300; fax: +34 944 034 310. E-mail address: [email protected] (R.A. Juste). 1 Present address: Montreal Children’s Hospital Research Institute, Department of Human Genetics, Room 242, 4060 Ste. Catherine St. West, H3Z 2Z3 Montreal, Quebec, Canada. 0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.10.007

(CWD) and various forms of Creutzfeldt–Jakob disease (CJD) in humans. These diseases are caused by an unidentified agent and are accompanied by the accumulation in the brain and other tissues of an insoluble, protease-resistant isoform (PrPSc ) of the host-encoded cellular prion protein (PrPc ) (Prusiner, 1998). Although the pathological mechanisms are not well defined, PrPSc deposited in tissues correlates with infectivity and PrPSc deposits are currently the only specific marker for TSE infections. Human and animal TSE PrPSc show different phenotypes that can be biochemically differentiated on the basis of its molecular mass and in the degree of

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glycosylation (Collinge et al., 1996; Somerville et al., 1997; Somerville, 1999; Kuczius et al., 2004). Depending on the host species and the TSE strain, proteinase K (PK) removes 55–70 residues form the N-terminal domain of PrPSc , yielding a product that consists mainly of three glycoforms: unglycosylated, monoglycosylated and diglycosylated fractions. In addition, there is a genetic relationship between the ovine PrP gene polymorphisms and the susceptibility to scrapie, notably in codons 136 (alanine (A) or valine (V)), 154 (arginine (R) or histidine (H)) and 171 (glutamine (Q), R, or H) (Goldmann et al., 1990; Hunter et al., 1996). In general, the ovine alleles VRQ and ARQ seem to be related to a higher susceptibility while the alleles ARR and AHQ are associated to lower susceptibility to scrapie. In natural scrapie the oral route of infection is the most relevant pathway for natural transmission (Pattison et al., 1972; Hadlow et al., 1982). The mechanisms of oral pathogenesis are not well established but the lymphoreticular (LRS), peripheral (PNS) and enteric nervous systems (ENS) are consistently involved (van Keulen et al., 1999, 2000, 2002; Andreoletti et al., 2000; Jeffrey et al., 2001; Ersdal et al., 2003). In the early phase of the disease, detection of scrapie infectivity in these tissues suggests that primary replication occurs in these locations. This replication usually occurs for many months before reaching the central nervous system (CNS) via the blood or nervous fibers innervating lymphoid tissues. A secondary scrapie replication then takes place in the spinal cord and brain. In this study, IHC was used to determine PrPSc distribution in CNS and LRS tissues of ewes with clinical and subclinical scrapie from a natural outbreak occurred in Latxa sheep. Patterns of PrPSc deposition are discussed in the context of TSE rapid tests performance and scrapie pathogenesis. 2. Materials and methods 2.1. Background The ovine population of the Basque Country includes about 320,000 sheep of Latxa, a dairy breed used for cheese production, of which two varieties are recognized, black-faced Latxa and fair-faced Latxa (Gabi˜na et al., 1993). Fourteen different genotypes have been reported in Latxa breed, the most frequently found being ARQ/ARQ (49.3%), followed by ARR/ARQ (32.6%) and ARQ/ARH (5.8%) (Garcia-Crespo et al., 2004). During the period comprised between 1982 and 1998 passive surveillance failed to detect any case of TSE in ruminants in the Basque Country. Since 1999 an active surveillance program was set in place. During the years 1999 and 2000 this

program was based only in histopathological and immunohistochemical methods and a total of 107 animals were tested. From 2001 onwards, the use of rapid tests allowed to increase the annual number of sheep examined, and therefore, a total of 2292 slaughtered sheep, 4 goats and 1144 farm fallen stock sheep and 108 goats have been screened by rapid tests. The first scrapie outbreak was detected in the year 2000 in a flock of 148 black-faced Latxa sheep when a suspect animal was submitted for laboratory diagnosis. Only 1 of the 12 ewes examined showed clinical signs and severe spongiosis of the cerebellar molecular layer was detected by immunolabelling with scrapie-specific antibodies (6H4 and L42). Another sheep showed positive immunolabelling only in LRS tissues (Peyer’s patches, tonsil, retropharyngeal ganglia and spleen) and in ENS structures (Meissner and Auerbach plexuses), but did not show clinical signs. The second outbreak, which is the subject of this report, was also detected after two ewes were brought to the laboratory with suspicious clinical signs. Finally, two more cases have been identified within the fallen stock surveillance program in 2004 and another one in slaughtered sheep in 2005. 2.2. Animals and tissues The outbreak was detected in the year 2003 in a flock of 222 black-faced Latxa and 2 goats (n = 224). After the two index cases were confirmed as scrapie positive by rapid tests and IHC, two new clinical cases were recognized in the ante-mortem inspection during the flock destruction. These four animals showed physical signs like hyper-responsiveness to external stimuli, abnormal/uncoordinated/exaggerated gait mainly in rear legs, fallings, teeth grinding, tremors, pruritus, recumbence and decreased body weight. All sheep were euthanatized by exanguination after anesthesia was applied by intravenous injection with sodium pentobarbital (Dolethal, Vetoquinol) in the four clinical cases and with T61 (Hoechst) in the remaining 220 animals. Blood samples and the whole head were collected from all the animals. Tissue samples collected from the clinical cases were brain and cervical spinal cord, trigeminal ganglion (TG), third eyelid, lymphoid organs (spleen, retropharyngeal/mandibular lymph nodes, mesenteric lymph nodes, caudal jejunal and ileocaecal lymph nodes, palatine tonsil), sections of digestive tract (ileocaecal valve, Peyer’s patches at level of jejunum and ileum) and a portion of masseter muscle. 2.3. Diagnostic rapid tests Four EU approved TSE rapid tests were performed following the manufacture’s instructions (i) Platelia (Bio-Rad), a colorimetric sandwich ELISA; (ii) Enfer TSE kit (Enfer Scientific), a chemiluminiscent direct ELISA; (iii) Prionics Check LIA (Prionics), a chemiluminiscent sandwich ELISA; and finally (iv) Prionics Check Western Blot (Prionics), an immunoblotting test based on a Western blotting procedure for the detection of PrPSc . These tests are all designed to be carried out on samples of obex that can be readily collected after slaughter.

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2.4. Modified Western blot

2.6. Genotyping

This Western blot was performed using the same reagents and test conditions as the Prionics Check WB with only two modifications: primary antibody P4 (1:5000) was used instead of the antibody included in the test kit (6H4) and band patterns were developed with NBT/BCIP (AP Conjugate Substrate Kit, Bio-Rad) on membranes instead of using auto-radiographic films. A sample of brainstem at the level of obex from a positive cow detected within the Basque Country TSE surveillance program was included in each gel as a positive control for band pattern and staining comparison.

Blood samples were used for the analysis of the PrP polymorphisms at codons 136, 154 and 171 by realtime PCR on a ABI PRISM 7000 thermocycler (Applied Biosystems) as previously described (Garcia-Crespo et al., 2004). Scrapie susceptibility was classified according to NSP (National Scrapie Plan, http://www.defra.gov.uk/animalh/bse/ othertses/scrapie/nsp/pdf/genotypes.pdf accessed 16 March 2006).

2.5. Immunohistochemical staining Fifty sheep were immunohistochemically processed. These included all the animals with clinical signs (n = 4) and 46 sheep that did not show clinical signs but were positive in the rapid tests (n = 16) or were randomly selected from genotypes belonging to risk groups types 3, 4 and 5 (n = 30) (UK National Scrapie Plan risk groups 2003 http://www.defra. gov.uk/animalh/bse/othertses/scrapie/nsp/pdf/genotypes.pdf ). Tissue samples were fixed on phosphate-buffered formalin (10%) for 3–4 days before being trimmed, post-fixed and paraffin-embedded. Tissue sections 3–4 ␮m thick were cut in triplicate on a microtome, mounted onto adhesive Polyl-Lysine (Sigma) glass slides and dried overnight at 37 ◦ C. Sections were deparaffinized and rehydrated by routine methods. One of the triplicates was stained with haematoxylin–eosin and used as reference control to determine severity and distribution of vacuolation when possible. Another one was archived and the last one was used for immunolabelling. IHC sections were then subjected to a retrieval procedure of formic acid 98% (Merck), PK (Gibco BRL) treatment and steam autoclaving. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in methanol. PrPSc detection was performed using mouse monoclonal antibody 6H4 (Prionics). Enzyme-conjugated HRP EnVisionTM + (DAKO) was used as visualization system and DAB (Vector) as chromogen. At least one positive and one negative control section, subjected to the same protocol were included in each IHC run. The positive control used was a previously confirmed clinical scrapie case. The negative control tissue was selected from a sheep (medulla oblongata) raised in a scrapie-free flock. Sections were examined in all their surface and IHC results for each examined tissue were graded from (−) to (+++) according to the number of positive fields observed, assigning (−) to negative samples, (+) to <10 positive fields, (++) to 10–30 positive fields and (+++) to >30 positive fields. Sections of all the samples taken from clinical cases were submitted to IHC, as well as CNS (brain stem and spinal cord), trigeminal ganglion and lymphoid tissues (tonsil, draining lymph node (LN) and third eyelid) from the other 46 sheep.

2.7. Statistical analysis A Chi square test was used for testing genotype and risk group distribution differences between the Latxa population, the whole flock and the positive animals. To better show the differences, risk levels were also grouped as Low (Type 1 and Type 2) and High (Type 3, Type 4 and Type 5). The Kappa index was calculated for each rapid test in relationship with IHC in order to evaluate agreement with the reference method.

3. Results The four clinically affected sheep were positive to all of the rapid tests and all of them had the ARQ/ARQ genotype. These sheep showed a tissue distribution of PrPSc throughout the CNS (except in cerebellum cortex), the ENS (submucosal Meissner and myenteric Auerbach plexuses), TG, the adrenal medulla and also the digestive-associated lymphoid tissues like tonsil, Peyer’s patches, ileocaecal valve and draining lymph node (caudal jejunal lymph node). All sheep that showed PrPSc accumulation in Peyer’s patches and in caudal jejunal lymph node were also positive in ENS. PrPSc was associated with enteric neurons, follicular dendritic cells and tingible body macrophages. Other affected tissues were spleen, third eyelid and retropharyngeal/mandibular lymph nodes. These locations showed in general a weaker labelling intensity when compared with CNS or digestive-associated lymphoid tissues. Sheep clinically affected showed variable vacuolar degeneration in the brain, more prominent in the brainstem, mainly in the dorsal vagus nucleus of the medulla oblongata (Fig. 1). Staining was particularly intense in neuronal cell bodies, neuropile and around blood vessels in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. In the cervical spinal cord, PrPSc deposits, which took the form of particulate neuropile labelling, were confined, bilaterally symmetric to the intermediolateral columns and around the ependymal conduct (Fig. 2). In clinically suspect sheep 1 and 3 deposits of PrPSc were also observed in temporal and frontal cortex, with a light vacuolization associated.

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Fig. 2. Clinical case: clinical affected sheep 1 IHC grade 3 (+++) positive at CNS (spinal cord). ARQ/ARQ genotype. Immunolabelling bilaterally symmetric to the intermediolateral columns and around the ependymal conduct, 10×.

Sixteen other sheep without clinical signs were also positive to at least one of the rapid tests, and six more were only positive by IHC in lymphoid and/or peripheral nervous system (TG) tissues, which are not routinely analyzed by rapid tests. In CNS only limited vacuolation and PrPSc accumulation were detected in the brain. The heaviest accumulation of PrPSc was observed at spinal cord (grey matter preferentially, and occasionally only 2–3 neurons per section, as shown in Fig. 3) and caudal medulla oblongata, mainly in dorsal motor nucleus of the vagus, solitarious nucleus tract and spinal tract of trigeminal nerve, located in neuronal perikarya and axons. The neuropile was also labelled in several cases, being particularly strong in four animals (Sh93, Sh198, Sh204

Fig. 1. Clinical case: clinical affected sheep 3 IHC grade 2 (++) positive at CNS (medulla oblongata). ARQ/ARQ genotype. Dorsal motor nucleus of the Vagus nerve (DMNVN) and spinal tract of Trigeminal nerve (STTN) positive immunolabelling, 20×.

Fig. 3. Subclinical case: sheep 54 IHC grade 1 (+) positive at CNS. ARQ/ARQ genotype. Arrows show positive neuron immunolabelling, 40×.

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Table 1 IHC results of the 50 sheep examined distributed by genotype Identification

Genotype

CNS

TG

Third eyelid

Tonsil

Retropharyngeal/mandibular LN

Sh 201 Sh 51 Sh 68 Sh 88 Sh 122 Sh 161 Sh 177 Clinical 1a Clinical 2a Clinical 3a Clinical 4a Sh 7 Sh 40 Sh 43 Sh 44 Sh 58 Sh 60 Sh 71 Sh 84 Sh 93 Sh 102 Sh 105 Sh 123 Sh 129 Sh 131 Sh 132 Sh 135 Sh 144 Sh 176 Sh 196 Sh 204 Sh 207 Sh 54 Sh 67 Sh 82 Sh 86 Sh 103 Sh 115 Sh 137 Sh 147 Sh 166 Sh 181 Sh 188 Sh 193 Sh 198 Sh 210 Sh 212 Sh 72 Sh 80 Sh 157

AHQ/VRQ ARQ/ARH ARQ/ARH ARQ/ARH ARQ/ARH ARQ/ARH ARQ/ARH ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARQ/VRQ ARR/VRQ ARR/VRQ ARR/VRQ

− − − − − − − +++ ++ ++ ++ − + ++ − + + ++ − +++ − − − + + + − + + − +++ +++ − − − − − − − − − − − − +++ − − − − −

− − − NA − − − + − ++ + − − − + − − − − − − NA − − − − + − − ++ − + − − NA − − NA − − − − − − +++ − − NA − +

− − − NA − − − + NA ++ + − +++ NA − NA NA NA − NA − NA − NA − − − − NA ++ − + − − + − − NA − − − − − − +++ − − NA − −

− − − NA − − ++ ++ NA ++ + − +++ + + + + + − ++ − + − + + + + + + +++ ++ + + − + − − NA − − − − − − +++ − − NA − −

− − − NA − − + ++ NA ++ + − NA NA − NA NA NA − NA − NA − NA − − + − NA − − + − − NA − − NA − − − − − − +++ − − NA − −

a All clinical cases were also positive in ENS, spleen, ileal Peyer’s patches and jejunal caudal lymph node; −, negative result; + to +++, positive result from grade 1 to 3; NA, not available. Text in bold corresponds to sheep negative in the rapid tests and positive only by IHC.

and Sh207). Immunolabelling was not in all the cases associated with morphological changes in the affected cell-types. The results from the LRS and PNS were comparable with those of the clinically affected animals.

PrPSc was deposited in a reticular pattern within germinal centers of the affected follicles, present in the dark zone to a lesser extent than in the light zone, both in third eyelid, tonsil and lymph nodes, located in the cyto-

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N. Gomez et al. / Small Ruminant Research 72 (2007) 141–148 Table 2 Genotype distribution in the present outbreak Risk groupa

Genotype

Flock n

Fig. 4. Subclinical case: sheep 196 IHC grade 3 (+++) positive at tonsil. ARQ/VRQ genotype, 4×.

plasm of the dendritic cells, and macrophages. However, only in the palatine tonsils was PrPSc present in a consistently high percentage of the lymphoid follicles (Fig. 4). In several sheep variable amounts of labelling were also observed in TG (in ganglion cells and occasionally also in satellite cells, as shown in Fig. 5) only (Sh157) or associated with lymphoid tissue, with no positive reaction at CNS level. These results are summarized in Table 1. Ten different genotypes were found in the present flock, with a distribution slightly biased towards susceptibility compared to the Latxa breed general population (Garcia-Crespo et al., 2004) (␹2 = 118.05 p < 0.0001). The NSP risk level distribution also significantly differed from the general Latxa population (␹2 = 13.75 p = 0.0082). Using the simplified grouping (i.e., Low vs. High risk), 71.6% of the sheep in the flock fell within the High risk category versus only 59.3% of

Fig. 5. Subclinical case: sheep 198 IHC grade 2 (++) positive at TG. ARQ/ARQ genotype. Arrows show satellite cells, 40×.

Positives %

n

%

Type 1

ARR/ARR

9

4.1

0

0.0

Type 2

ARR/AHQ ARR/ARQ ARR/ARH

0 53 0

0.0 23.9 0.0

0 0 0

0.0 0.0 0.0

Type 3

AHQ/AHQ ARQ/AHQ AHQ/ARH ARH/ARH ARQ/ARH ARQ/ARQ

2 18 1 0 6 114

0.9 8.1 0.4 0.0 2.7 51.3

0 0 0 0 1 21

0 0.0 0.0 0.0 3.9 80.7

Type 4

ARR/VRQ

3

1.4

1

3.9

Type 5

AHQ/VRQ ARH/VRQ ARQ/VRQ VRQ/VRQ

1 0 15 0

0.4 0.0 6.8 0.0

0 0 3 0

0.0 0. 11.5 0.0

Total a

222

26

Risk group according to NSP.

the general Latxa population (␹2 = 12.70 p = 0.0004). The distribution of genotypes in the flock is shown in Table 2. As expected, most of the positive sheep were of the ARQ/ARQ genotype (21/26, 80.7%), which is the most prevalent genotype in this flock and in Latxa breed (Garcia-Crespo et al., 2004). Other three positive animals had the ARQ/VRQ genotype, representing 11.5% of positive samples. One ARR/VRQ and one ARQ/ARH sheep were also positive. Altogether 16 (7.2%; mean age 4.2 years, with 5 animals less than 2 years old) of the sheep had Type 5 genotypes, 197 animals (88.7%; mean age 4 years, 62 ewes ≤ 2 years old) had intermediate genotypes and 9 (4.1%; mean age 3.9 years, 3 animals ≤ 2 years old) had Type 1 PrP genotypes. Compared to IHC results, the rapid tests detected 76.9, 61.5, 53.8 and 53.8% of the positives for BioRad, Enfer, Prionics-LIA and Prionics-WB, respectively. No IHC negative sheep was scored as positive by any of the rapid tests. Kappa values related to IHC were 0.762 ± 0.137, 0.606 ± 0.130, 0.528 ± 0.125 and 0.528 ± 0.125 for Bio-Rad, Enfer, Prionics-LIA and Prionics-WB, respectively. The electrophoretic pattern obtained upon replacing of MAb 6H4 with P4 fitted the scrapie pattern better than that of BSE, with the unglycosylated band slightly bigger in scrapie than in BSE. The signal obtained with P4 was stronger in ovine samples than in bovine, whereas the opposite occurred when using 6H4 as MAb, as shown in Fig. 6.

N. Gomez et al. / Small Ruminant Research 72 (2007) 141–148

Fig. 6. Comparison of immunoblot patterns generated with two different MAb: (A) 6H4 and (B) P4. M, Bio-Rad Precision Plus ProteinTM Standard molecular weight marker; CSh1, clinical suspect sheep 1; CSh3, clinical suspect sheep 3; Sh71, sheep 71; Sh198, sheep 198; Sh132, sheep 132; BSE, Cattle BSE.

4. Discussion Our investigations led us to detect 26 sheep with PrPSc positive immunolabelling in an outbreak of natural scrapie occurred in a flock of 222 Latxa sheep. Whereas only 20 positive animals were detected by the implementation of 4 EU approved TSE rapid tests, 6 other ewes were identified as positive when IHC was performed on 50 animals of the flock. However, these six animals showed only positive immunolabelling in peripheral or lymphoreticular tissues like trigeminal ganglion, palatine tonsil or retropharyngeal/mandibular lymph nodes. These sheep would have passed unnoticed by rapid tests due to sample selection since rapid tests are validated to be used on obex samples and these animals were not positive in CNS. This is concordant with results described by other authors (van Keulen et al., 1996; Andreoletti et al., 2000) who pointed out that the immune system is involved in the early phase of scrapie pathogenesis. However, the route of natural scrapie and its spread within the host are not entirely known. The kinetics of PrPSc accumulation in sheep organs have been determined by IHC by several authors (van Keulen et al., 1999, 2000, 2002; Maignien et al., 1999; Andreoletti et al., 2000; Jeffrey et al., 2001) with an apparent entry site at the ileal Peyer’s patches as well as its draining mesenteric lymph node. PrPSc has to travel through the bloodstream to reach secondary LRS targets such as spleen and lymph nodes associated with other organ systems. Moreover, the Peyer’s patches are in direct contact with the gut epithelium and are connected with plexuses of the ENS. This way, by infecting nerve endings of sympathetic or parasympathetic efferent neurons, the scrapie

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agent might then spread to the celiac nerve complex and intermediolateral column of the spinal cord (sympathetic case) or the dorsal motor nucleus of the vagus of the medulla oblongata (parasympathetic case). The detection of positive immunolabelling in the adrenal medulla in one ewe suggests that it was associated with processes of the medullar ganglion cells (Jeffrey et al., 2001) at a peripheral dissemination. Therefore, on the basis of the morphology and anatomical distribution of the deposits, distinct disease-associated patterns of PrPSc deposition were identified: the ‘early’ pattern, which would correspond to animals with IHC positive at gut-associated lymphoid tissues (GALT) or PNS and a ‘late’ pattern, which would be associated to animals affected also at CNS level. In the ‘early’ phase only the lymphoid tissue and peripheral nerves would be affected, and this would correspond to the six cases in our series that were negative by the rapid tests. In the ‘late’ phase the CNS would also be affected and both IHC and rapid tests would be positive as was observed in the 20 concordant cases. The genotype distribution in the studied flock indicates a slightly higher susceptibility than the average described for the Latxa breed (Garcia-Crespo et al., 2004). This could have favored the onset and spread of the infection in the flock. All IHC positive ewes belonged to risk groups Type 3, Type 4 and Type 5 (moderate to high susceptibility) with mean age 4.4 years and only one ewe below 2 years old. Interestingly, this animal had an ARR/VRQ genotype and was only reactive at trigeminal ganglion and not in lymphoid tissues. In addition to these patterns, the finding of sheep with IHC positive results only in third eyelid and/or trigeminal ganglion might point out an alternative route of entrance involving the head (terminal nervous endings of cranial nerves I (Olfactory nerve), V (Trigeminal nerve), X (Vagus nerve). These could account for negative results in the classical CNS locations and would be consistent with reports of higher levels of PrPSc deposition in cerebral and/or cerebellar cortex in atypical cases. Analysis of the protease cleavage of the pathological prion protein by Western blot has been shown to provide some potential to distinguish BSE and scrapie in sheep (Jeffrey et al., 2003; Thuring et al., 2004). A comparison of immunoblot patterns was made on a selected set of samples with both 6H4 and P4 MAbs in order to preliminarily assess the bovine or ovine type of the prion strain found in this outbreak. The immunoblot electrophoretic pattern of the analyzed sheep was barely distinguishable from that of BSE according to molecular size of the unglycosylated isoform. However, upon replacing of MAb 6H4 (near C-terminal site) with P4 (near N-terminal PrP

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cleavage site), the intensity of the signal generated with P4 was greater in samples of ovine origin than in bovine samples (Lezmi et al., 2004). In the absence of in vivo tests, these results highlight the necessity to adapt the available rapid tests for the detection of PrPSc in lymphoid tissues to prevent subclinical cases from passing unnoticed. These results also confirm the value of IHC as the reference method and they provided interesting data regarding scrapie pathogenesis. IHC results proved that the prevalence of PrPSc positive ewes in a sheep flock with few clinical scrapie cases (4 cases from 222 animals; 1.8%) might be considerably higher than clinical data indicates, with a clinical/subclinical ratio of 1/5.5. This suggests that depending on the time span since the onset of the agent within the flock and the individual genetic resistance, different patterns of PrPSc deposition can occur in individual sheep. Only after enough exposition time has elapsed in a flock would the full picture be completed. Acknowledgements Financial support was provided by the Department of Agriculture and Fisheries of the Basque Government. We would like to thank the staff from the TSE laboratory of Neiker (D. Nagore, G. Urrutia, A. Badiola, A. Etxezarreta, Z. P´erez, F. Garc´ıa, S. Ayuso and F. Goiri) for their contribution in sample processing. References Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L.J., Schelcher, F., Elsen, J.M., Lantier, F., 2000. Early accumulation of PrP(Sc) in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. J. Gen. Virol. 81, 3115–3126. Collinge, J., Sidle, K.C., Meads, J., Ironside, J.W., Hill, A.F., 1996. Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383, 685–690. Ersdal, C., Ulvund, M.J., Benestad, S.L., Tranulis, M.A., 2003. Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie. Vet. Pathol. 40, 164–174. Gabi˜na, D., Arrese, F., Beltr´an de Heredia, I., Arranz, J., 1993. Average milk yields and environmental effects on Latxa sheep. J. Dairy Sci. 76, 1191–1198. Garcia-Crespo, D., Oporto, B., Gomez, N., Nagore, D., Benedicto, L., Juste, R.A., Hurtado, A., 2004. PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR. Vet. Rec. 154, 717–722. Goldmann, W., Hunter, N., Foster, J.D., Salbaum, J.M., Beyreuther, K., Hope, J., 1990. Two alleles of a neural protein gene linked to scrapie in sheep. PNAS 87, 2476–2480.

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