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##################### Bovine Spongiform Encephalopathy ##################### Subject: PrP(d) accumulation in organs of ARQ/ARQ sheep experimentally infected with BSE by peripheral routes Date: June 19, 2006 at 8:11 am PST PrP(d) accumulation in organs of ARQ/ARQ sheep experimentally infected with BSE by peripheral routes. Lezmi S, Ronzon F, Bencsik A, Bedin A, Calavas D, Richard Y, Simon S, Grassi J, Baron T. Agence Francaise de Securite Sanitaire des Aliments (AFSSA), Lyon cedex 07, France. 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. snip... Results and Discussion For all samples analysed from the ARR/ARR sheep, no PrPd was detected by any of the three PrPd 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been reported in sheep with this genotype (Buschmann et al., 2004); (Ikeda et al., 1995) (French surveillance program; unpublished data). Furthermore, BSE has been transmitted to ARR/ARR sheep by the intra-cerebral route (Houston et al., 2003b). 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 PrPd accumulation (table 1 ; figure 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 (Jeffrey et al., 2001; van Keulen et al., 1999). In the CNS, the quantities of abnormal PrP, expressed as equivalent in recPrP, were estimated by ELISA at up to 13000 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 PrPd/g of CNS tissues. Lymphoid organs accumulated lower levels of PrPd and large quantities of material were required to detect a signal by WB in the mandibular or iliac medial Copyright @ Acta Biochimica Polonica, Paper in Press, No. 1273 - 5 - lymph nodes (LN) of SB3 (Figure 1). In the spleen of SB1 and SB3, 46 and 2 ng equivalent of PrPd/g of tissue were detected, respectively. In the ileum, 232 ng equivalent of PrPd /g of tissue was detected and correlated with a higher number and size of germinal centres when compared to spleen or iliac LN. The mean quantity of PrPd in the CNS was 187 and 36 fold greater than the quantities determined respectively in spleen and in the intestine. Qualitatively, different types of PrPd deposits in the brain were identified from the frontal cortex to the lumbar spinal cord. These PrPd deposits were mainly identical to those previously identified in scrapie- or BSE-affected sheep (Gonzalez et al., 2002; Ryder et al., 2001). In the retina, PrPd accumulation was mainly detected in the ganglionar layer (1), intern (2) and extern (4) plexiform layers (numbers corresponding to the different layers in the retina, figure 2a). Interestingly, in the enteric nervous system of ARQ/ARQ sheep, PrPd was detected associated with neurons (figure 2g) as well as in the coeliac ganglia in which intraand peri-neuronal PrPd deposits were visualized (figure 2h). In the adrenal gland, two types of PrPd accumulation were observed as dense intracellular or synaptic-like deposits (figure 2i). In lymphoid organs, PrPd was detected in germinal centres of secondary lymphoid follicles, in follicular dendritic cells and in tingible body macrophages (figure 2j). PrPd was also detected in cells with a morphology consistent with macrophages in the subcapsular sinus of some lymph nodes (figure 2j, arrowhead, table 1*). These observations are in agreement with previous results obtained both in sheep naturally affected with scrapie (Ersdal et al., 2005); (Jeffrey et al., 2000); (Lezmi et al., 2001) and in experimentally BSE-infected sheep (Lezmi et al., 2001); (Jeffrey et al., 2001). Interestingly, not all germinal centres were labelled for PrPd; 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 PrPd. 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 Copyright @ Acta Biochimica Polonica, Paper in Press, No. 1273 - 6 - 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, PrPd was detected by IHC in all motor nerves and associated with Schwann’s cells (figure 2d, e). PrPd 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 (data not shown) and two previous articles report similar data in sheep with natural scrapie (Archer et al., 2004); (Groschup et al., 1999). PrPd 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 (figure 2k, l) and are a specialized subset of myocytes implicated in proprioception. In the tongue of sheep, the accumulation of PrPd in these structures was less evident. Only one study reported the PrPd 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 PrPd 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 rodentadapted 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 Copyright @ Acta Biochimica Polonica, Paper in Press, No. 1273 - 7 - ARQ/ARQ sheep. The distribution of PrPd 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 PrPd 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. ... snip...end...TSS http://www.actabp.pl/pdf/Preprint/20061273.pdf TSS #################### https://lists.aegee.org/bse-l.html ####################