Vol. 53 No. 2/2006, 399–405 Regular paper on-line at: www.actabp.pl

To study the pathogenesis of bovine spongiform encephalopathy infection in small ruminants, two Lacaune sheep with the AA136RR154QQ171 and one with the AA136RR154RR171 genotype for the prion protein, were inoculated with a brain homogenate from a French cattle BSE case by peripheral routes. Sheep with the ARQ/ARQ genotype are considered as susceptible to prion diseases contrary to those with the ARR/ARR genotype. The accumulation of disease-associated prion protein (PrP(d)) was analysed by biochemical and immunohistochemical methods. No PrP(d) accumulation was detected in samples from the ARR/ARR sheep 2 years post inoculation. In the two ARQ/ARQ sheep that had scrapie-like clinical symptoms, PrP(d) was found in the central, sympathetic and enteric nervous systems and in lymphoid organs. Remarkably, PrP(d) was also detected in some muscle types as well as in all peripheral nerves that had not been reported previously thus revealing a widespread distribution of BSE-associated PrP(d) in sheep tissues.


INTRODUCTION
The bovine spongiform encephalopathy (BSE) agent, linked to the variant Creutzfeldt-Jakob disease in humans (Bruce et al., 1997), has been experimentally transmitted to sheep (Foster et al., 1993), without clinical distinction from natural scrapie.The possible presence of this agent within sheep flocks used for human consumption is therefore of considerable concern.It has recently been demonstrated in France that a major risk factor for introduction of transmissible spongiform encephalopathies (TSE) in a flock is linked to the use of proprietary concentrates and milk replacers (Philippe et al., 2005), which have been implicated in the BSE epidemic in cattle (Wilesmith et al., 1992).Furthermore, the BSE agent was recently identified in a naturally infected French goat (Eloit et al., 2005) and this reinforces the possible presence of the BSE agent in the sheep and goat flocks.
In sheep, the development of prion disease is complex and depends on several factors such as the genotype and breed of animals as well as the nature of the infectious agent.Thus, polymorphism of the prion protein PrP gene that predominantly determines scrapie or BSE susceptibility are linked to variations of codons 136, 154 and 171.The V 136 R 154 Q 171 /V 136 R 154 Q 171 genotypes are associated with a very high susceptibility to scrapie (Hunter, 1997) whereas the ARR/ARR animals are more resistant to scrapie (Hunter, 2003) even though cases of scrapie have been reported in this genotype (Ikeda et al., 1995;Buschmann et al., 2004).The ARQ/ ARQ genotype, largely represented in flocks, and associated with scrapie susceptibility, would lead to an increased susceptibility to BSE (Houston & Gravenor, 2003).
To study the pathogenesis of BSE in small ruminants, Lacaune sheep were inoculated with the BSE agent.The accumulation of the disease-associ-S.Lezmi and others ated prion protein, PrP d , the most specific marker of the disease (Bolton et al., 1982), was investigated using three complementary methods that allowed us to obtain both qualitative and quantitative results (Madec et al., 2004) allowing the identification of the tissues that may represent a risk for consumption.

MATERIAL AND METhODS
Animals.In our study, two ARQ/ARQ Lacaune sheep were inoculated either by intra-peritoneal (SB1 sheep) or intra-splenic (SB3 sheep) routes with brain homogenate from a French BSE-affected cow.One sheep naturally died at 672 days post inoculation (dpi) and the other was euthanized 1444 dpi.Both sheep had clinical signs of neurological disorders and had molecular characteristics of PrP d consistent with BSE infection (Houston & Gravenor, 2003;Lezmi et al., 2004).The other Lacaune sheep with the ARR/ARR genotype (SB2) was inoculated by the intra-peritoneal route and was sacrificed at 673 dpi.
Samples from healthy sheep 2 years of age having the ARR/ARR and ARR/ARQ genotypes were used as negative controls.The negative status of these animals was confirmed by checking the absence of PrP d in the central nervous system (CNS) and tonsils.
Immunohistochemistry (IhC).IHC allowing the identification of PrP d at the cellular level with high sensitivity was performed as described in previous studies (Lezmi et al., 2003); PrP d deposits appeared in situ as brown or black deposits using DAB alone or intensified with NiCl 2 .
Western-blot (WB).Following a WB procedure previously described (Biacabe et al., 2004), PrP d detection was performed using anti-PrP mAbs Bar233 and peroxidase-conjugated anti-mouse IgG (Southern Biotechnology Associates).This method has the advantage of identifying with high specificity the proteinase K resistant form of PrP d .However, the Western Blot analysis is less sensitive than the ELISA method, and as all samples were not in sufficient quantities for both biochemical analysis, samples were tested by ELISA in priority, as this method allows quantification of PrP d .
ELISA.ELISA was performed with the extraction kit 'Platelia BSE Bio-Rad' currently used for TSE diagnosis (Grassi et al., 2001).For each plate an internal standard was used, i.e. ovine recombinant prion ( rec PrP) purified as previously described (Betemps & Baron, 2001).ELISA detection was performed using SAF34 anti-PrP mAbs as capture antibodies and acetylcholinesterase-conjugated Bar224 anti-PrP mAbs as a tracer.

RESULTS AND DISCUSSION
For all samples analysed from the ARR/ARR sheep, no PrP d was detected by any of the three PrP d detection methods used (Table 1).This result correlates with the higher genetic resistance to TSE associated with this genotype naturally affected with scrapie (Elsen et al., 1999) or orally infected with the BSE agent (Jeffrey et al., 2001).However, resistance of the ARR/ARR sheep challenged with TSE infection is not considered complete since natural scrapie cases have been reported in sheep with this genotype (Ikeda et al., 1995;Buschmann et al., 2004;French surveillance program, unpublished data).Furthermore, BSE has been transmitted to ARR/ARR sheep by the intra-cerebral route (Houston et al., 2003).
In both ARQ/ARQ sheep, the CNS (including retina), the lymphoid system and the autonomous nervous system were identified by each method as major sites of PrP d accumulation (Table 1, Fig. 1) and were also described earlier by other groups in experimentally BSE affected sheep (Foster et al., 2001;Jeffrey et al., 2001) as well as in naturally scrapie-affected sheep (van Keulen et al., 1999;Jeffrey et al., 2001).In the CNS, the quantities of abnormal PrP, expressed as equivalent in rec PrP, were estimated by ELISA at up to 13 000 ng/g of brainstem tissue.Comparatively, the levels found in 13 ARQ/ARQ or ARQ/VRQ sheep clinically affected with natural scrapie averaged 40 000 ± 20 000 ng of PrP d /g of CNS tissues.Lymphoid organs accumulated lower levels of PrP d and large quantities of material were required to detect a signal by Westren blot in the mandibular or iliac medial lymph nodes (LN) of SB3 (Fig. 1).In the spleen of SB1 and SB3, 46 and 2 ng equivalent of PrP d /g of tissue were detected, respectively.In the ileum, 232 ng equivalent of PrP d /g of tissue was detected and correlated with a higher number and size of germinal centres when compared to spleen or iliac lymph nodes.The mean quantity of PrP d in the CNS was 187-and Qualitatively, different types of PrP d deposits in the brain were identified from the frontal cor-tex to the lumbar spinal cord.These PrP d deposits were mainly identical to those previously identified in scrapie-or BSE-affected sheep (Ryder et al., 2001;Gonzalez et al., 2002).In the retina, PrP d accumula- In the enteric nervous system of ARQ/ARQ sheep, PrP d was detected associated to neurons (g, black deposits, × 200) as well as in the coeliac ganglia in which intra and peri-neuronal PrP d deposits were visualized (h, brown deposits, × 200; higher magnification in the inset).In the adrenal gland, two types of PrP d accumulation were observed, dense intracellular and synaptic-like (i, brown deposits, × 400).In lymph nodes, the PrP d accumulation was mainly detected in germinal centers (arrow) (j, black deposits, × 100).In the muscle PrP d accumulation was observed associated to neuro-muscular spindle (k-l, brown deposits, × 100 and × 400).

2006
S. Lezmi and  For ELISA measurements, samples were stated positive or negative by reference to a cut-off value calculated as the mean of the measurements made on the negative controls plus 3-fold the value of the standard deviation calculated on negative controls.A "grey area" was defined between the value of the cut off and the mean of the negative controls plus 2-fold the value of the standard variation (Table 1, '+/-' results).PrP d accumulation was also quantified (values in brackets, ng/g of tissue).For each plate, at least three negative controls chosen in function of the studied tissue were deposited in duplicate.tion was mainly detected in the ganglionar layer (1), intern (2) and extern (4) plexiform layers (numbers corresponding to the different layers in the retina, Fig. 2a).Interestingly, in the enteric nervous system of ARQ/ARQ sheep, PrP d was detected associated with neurons (Fig. 2g) as well as in the coeliac ganglia in which intra-and peri-neuronal PrP d deposits were visualized (Fig. 2h).In the adrenal gland, two types of PrP d accumulation were observed as dense intracellular or synaptic-like deposits (Fig. 2i).

Samples
In lymphoid organs, PrP d was detected in germinal centres of secondary lymphoid follicles, in follicular dendritic cells and in tingible body macrophages (Fig. 2j).PrP d was also detected in cells with a morphology consistent with macrophages in the subcapsular sinus of some lymph nodes (Fig. 2j, arrowhead, Table 1*).These observations are in agreement with previous results obtained both in sheep naturally affected with scrapie (Jeffrey et al., 2000;Lezmi et al., 2001;Ersdal et al., 2005) and in experimentally BSE-infected sheep (Lezmi et al., 2001;Jeffrey et al., 2001).Interestingly, not all germinal centres were labelled for PrP d ; this partial absence of labelling in germinal centres (as in tonsils) was not observed in samples from 13 natural scrapie-infected sheep in which all lymphoid germinal centres were positively labelled for PrP d .This agreed with data describing an early and systematic immune system involvement in lambs affected with scrapie (Andreoletti et al., 2000) which was not a feature of BSE agent infection in sheep during the first passage (Jeffrey et al., 2001;Martin et al., 2005).
In our study, as opposed to previous published results, in both ARQ/ARQ sheep, PrP d was detected by IHC in all motor nerves and associated with Schwann's cells (Fig. 2d, e).PrP d deposits were similarly detected in all other tissue samples containing peripheral nerves, most notably in nerves in muscle samples.This observation was not reported in other studies with sheep BSE (Foster et al., 2001;Jeffrey et al., 2001).However, we observed the same type of deposits in two other sheep (ARQ/VRQ) naturally affected with scrapie (not shown) and two previous articles report similar data in sheep with natural scrapie (Groschup et al., 1999;Archer et al., 2004).
PrP d presence was also identified in striated muscles for both ARQ/ARQ sheep.These deposits were associated with neuromuscular spindles that are highly innervated structures made of groups of myocytes surrounded by a thin fibrous capsule (Fig. 2k, l) and are a specialized subset of myocytes implicated in proprioception.In the tongue of sheep, the accumulation of PrP d in these structures was less evident.Only one study reported the PrP d presence in the muscle of sheep affected with scrapie using IHC and ELISA (Andreoletti et al., 2004).Here, sampling and analysis of different muscles were not systematic and thus the ELISA/IHC results were not correlated.However, the accumulation in muscle tissue of PrP d in sheep affected with scrapie is not systematic (Andreoletti et al., 2004).Recently, pathological prion protein was detected in muscles of hamsters and mice infected with rodent-adapted BSE or vCJD (Thomzig et al., 2006).Previously, other studies failed to detect prion in nerves and muscles of BSE-or scrapie-infected sheep (Foster et al., 2001;Hamir et al., 2004) possibly relying on the use of different pre-treatments and antibodies.
In conclusion, we have shown that the inoculation of the BSE agent of French origin by peripheral routes to Lacaune sheep lead to the development of the clinical disease only in ARQ/ARQ sheep.The distribution of PrP d in ARQ/ARQ sheep infected with BSE was very similar to that described in natural scrapie.Overall, we demonstrated for the first time the presence of PrP d in muscles and nerves of sheep infected experimentally with BSE agent, which stresses the potential risk for humans related to consumption of sheep products from sheep naturally infected with BSE.

Figure 1 .
Figure 1.Detection of PrP d by Western blot -ARQ/ ARQ sheep.Representative pattern of PrP d observed in different tissues.Lanes 1 and 2: mandibular and iliac-medial lymph nodes, respectively; lane 3: adrenal gland; lane 4: cortex.All homogenates were prepared from sheep SB3.MW, molecular mass.

Figure 2 .
Figure 2. Immunodetection of PrP d in various tissues.PrP d accumulation was detected in the retina (a, black deposits, × 200) and in the cerebellum of both ARQ/ARQ sheep (b, brown deposits) but not in the ARR/ARR sheep (c).PrP d was also detected in the ARQ/ARQ sheep in sciatic nerve (d-e, black deposits, × 400, × 200) but neither in the ARR/ARR sheep nor in healthy controls (f, × 200).In the enteric nervous system of ARQ/ARQ sheep, PrP d was detected associated to neurons (g, black deposits, × 200) as well as in the coeliac ganglia in which intra and peri-neuronal PrP d deposits were visualized (h, brown deposits, × 200; higher magnification in the inset).In the adrenal gland, two types of PrP d accumulation were observed, dense intracellular and synaptic-like (i, brown deposits, × 400).In lymph nodes, the PrP d accumulation was mainly detected in germinal centers (arrow) (j, black deposits, × 100).In the muscle PrP d accumulation was observed associated to neuro-muscular spindle (k-l, brown deposits, × 100 and × 400).

Table 1 . Detection of PrP d by immunohistochemistry (IhC), ELISA and Western blot (WB).
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