2016 Volume 4 Issue 4 Pages 121-129
After the detection of the first cases of atypical bovine spongiform encephalopathy (BSE) more than ten years ago, the etiology, pathogenesis and agent distribution of these novel BSE forms in cattle were completely unknown. Many studies have been performed in the meantime to elucidate the pathogenic mechanisms of these diseases. A wealth of data has been accumulated regarding the distribution of the abnormal isoform of the prion protein, PrPSc, in tissues of affected cattle, confirming the general restriction of the PrPSc and agent distribution to the central and peripheral nervous system, albeit at slightly higher levels as compared to classical BSE. However, due to lack of data, the assumptions regarding the spontaneous etiology of both atypical BSE forms (H-BSE and L-BSE) and also the origin of the classical BSE epidemic are still mainly speculative. By performing subpassage experiments of both the atypical BSE forms in a variety of conventional and transgenic mice and Syrian Gold hamsters, we aimed to improve our understanding of the strain stability of these BSE forms. It turned out that under these experimental conditions, both the atypical BSE forms may alter their phenotypes and become indistinguishable from classical BSE. Information about the classical and atypical BSE strain characteristics help to improve our understanding of the correlation between all three BSE forms.
The pathogenesis of classical (C-) BSE in cattle has been analyzed in various oral challenge studies1,2,3,4,5). These studies confirmed the general restriction of the PrPSc and infectivity accumulation in the central nervous system (CNS), with only a minor involvement of the peripheral nervous system (PNS) at the late stage of the disease4,6,7,8,9,10). In contrast to the situation of scrapie in sheep11) and Chronic Wasting Disease (CWD) in cervids12), the lymphoreticular system (LRS) is not involved during the BSE pathogenesis in cattle6,7). PrPSc or infectivity have never been detected in the spleen or in lymph nodes of BSE affected cattle6,13). The only exceptions are the detection of BSE infectivity in the lingual tonsils in one study14), and various reports on the detection of PrPSc and BSE infectivity in the Peyer’s patches mainly of the ileum and jejunum15,16,17,18,19). However, these tissues most probably only act as entry ports for the agent after oral exposure14). From these gut-associated lymphatic tissues (GALT), the agent is transferred to the enteric nervous system, from where the coeliac and mesenteric ganglion complex, the splanchnic nerves and the lumbal/caudal thoracic spinal cord, or alternatively the vagus nerve, are reached3). The centripetal spread of BSE prions seems to mainly occur along the autonomic nervous system to the central nervous system, starting already halfway in the incubation time20).
Atypical BSE cases seem to occur independently of the classical BSE situation in a country (Table 1), and almost exclusively in cattle over eight years of age. A total number of 111 atypical BSE cases have been detected worldwide so far (as of May 2016). A retrospective study performed in the European Union in 2015 revealed a number of atypical BSE cases that had initially been identified as classical BSE. This underlines the importance of reliable and harmonized discriminatory methods applied in all National Reference Laboratories (NRL).
| Country | Number ofH-BSE cases | Number ofL-BSE cases | Total number ofatypical BSE cases | Total number of BSE cases |
Percentage of atypical BSE cases /total BSE cases |
| Austria | 1 | 2 | 3 | 8 | 37.5 |
| Brazil | 1 | 1 | 2 | 2 | 100 |
| Canada | 1 | 1 | 2 | 20 | 10.5 |
| Czech Republic | 1 | 0 | 1 | 30 | 3.3 |
| Denmark | 0 | 1 | 1 | 16 | 12.5 |
| France | 16 | 16 | 32 | 1030 | 3.1 |
| Germany | 2 | 3 | 5 | 415 | 1.2 |
| Ireland | 5 | 0 | 5 | 1651 | 0.3 |
| Italy | 0 | 5 | 5 | 145 | 3.4 |
| Japan | 0 | 1 | 1 | 36 | 2.8 |
| Poland | 2 | 12 | 14 | 75 | 18.7 |
| Portugal | 2 | 0 | 2 | 1091 | 0.2 |
| Spain | 7 | 7 | 14 | 812 | 1.7 |
| Sweden | 1 | 0 | 1 | 1 | 100 |
| Switzerland | 2 | 0 | 2 | 467 | 0.4 |
| The Netherlands | 1 | 3 | 4 | 88 | 4.5 |
| United Kingdom | 6 | 9 | 15 | 184.637 | 0.008 |
| United States of America |
2 | 1 | 3 | 3 | 100 |
| Total | 49 | 62 | 111 | 190.488 | 0.048 |
After the first detection of H-BSE21) and L-BSE22) in 2004, it was unclear whether the pathogenesis of these TSE forms would mirror the pattern observed in classical BSE in cattle, or whether a more lymphoid transmission with an earlier involvement of the PNS would be present in these forms. Due to the initial lack of positive brain material for challenge experiments, challenge studies were performed as intracerebral inoculations23,24,25,26,27). Later it turned out that this route of inoculation is by far more efficient than the oral challenge. The PrPSc distribution in intracerebrally challenged cattle has been analyzed in depth by a number of groups, revealing a distribution pattern similar to that of classical BSE in cattle. However, PrPSc and also infectivity were detectable at earlier time points after infection and in higher amounts as compared to classical BSE. Iwamaru and colleagues were the first to report a more pronounced involvement of the PNS in the pathogenesis of L-BSE as compared to classical BSE28). These results were complemented by an Italian study, where the accumulation of BSE infectivity in skeletal muscle samples of experimentally challenged as well as of natural L-BSE cases was identified29). Subsequently, the Japanese colleagues performed immunohistochemical analyses of PNS tissues collected from cattle experimentally challenged with H-BSE, and were able to confirm that also in this BSE form, the PNS is involved in the pathogenesis30).
In experiments performed by other workgroups, subpassage experiments of atypical BSE in cattle31,32) revealed no differences in the subsequent incubation times, in the biochemical characteristics of the accumulated PrPSc or in the neuropathological findings as compared to cattle that had been challenged with atypical BSE field cases, arguing for the stability of these TSE strains when passaged in cattle. These findings contradicted the hypothesis that classical BSE might have evolved from atypical BSE after one or more subpassages in cattle via the feed chain. However, the emergence of a novel TSE strain from H-BSE affected cattle has been described very recently after several subpassages in bovine PrP transgenic mice33), resulting in shorter incubation times and a western blot pattern reminiscent of the classical BSE, which can however be clearly distinguished from classical BSE using antibodies binding to the core region of PrP. This shows that the stability of a TSE strain is highly dependent of the respective host species.
In one of our earlier studies, we addressed the anatomical distribution of PrPSc in different brain regions of affected cattle. The results obtained during this study confirmed what had also been noticed during the analysis of the first case of L-BSE in Italy22). While the brainstem, and especially the obex region, are most severely involved in cattle affected with classical BSE, both atypical BSE forms are marked by a much broader PrPSc distribution in the brains of affected cattle34). This finding might imply an underdetection of atypical BSE cases, especially of L-BSE cases, because the obex region is the only region that is routinely analyzed in the frame of BSE surveillance programs. However, since these data were based on samples collected from cattle experimentally challenged by the intracranial route, it remained questionable if these findings mirrored the situation in natural cases. We therefore determined the quantity of PrPSc depositions in different brain regions of natural H- and L-BSE cases in comparison to a natural C-BSE case by quantitative Western blot analysis. In the natural case of classical BSE, the obex region was most severely affected, while the levels determined for the other brainstem regions and the midbrain as well as the cerebellum only reached levels between 20 and 53% of the value determined for the obex. The levels obtained for the cortex regions varied between 2 and 14% of those determined for the obex level. On the other hand in both the natural and the experimental H-BSE cases, the brainstem (cranial medulla, pons) and thalamus regions reached generally distinctly higher levels than the obex, (up to 146%). Interestingly, the cortex regions were much more involved in the experimental case (22–48%) while in the natural case, the values were with up to 12% even a little bit lower that those determined for the natural C-BSE case (Fig. 1A). When we compared the intensities of PrPSc depositions in the brains of natural H-BSE, L-BSE and C-BSE cases, it became clear that the involvement of cortex regions was clearly predominant in L-BSE, low in C-BSE and almost completely absent in H-BSE, while the thalamus was with 118% of the obex value most severely affected in the H-BSE case (Fig. 1B). These results generally confirm the distribution patterns that have been described for cattle experimentally challenged with atypical BSE, however, it seems that the very high PrPSc quantities seen in the brainstem and cortex regions of experimentally challenged animals may not mirror the situation in natural cases of atypical BSE.
Anatomical distribution of PrPSc depositions in different brain regions of natural and experimental cases of atypical BSE in cattle as determined by a quantitative Western blot.
A)PrPSc distribution pattern in brain areas of experimental H-BSE (i.c. inoculation), a German H-BSE field case and a German C-BSE field case. Samples were analyzed by PTA precipitation followed by immunoblot, as described earlier9). Each sample was analyzed at least in triplicate, banding signals were visualized and quantified using the Quantity One software (Biorad, Munich). Signal intensities obtained for the cranial medulla sample were set as 100%.
B) PrPSc distribution in selected brain areas of a German C-BSE field case, a German H-BSE field case, and an Italian L-BSE field case. Analysis and quantification was performed as described under A).
After the detection of atypical BSE cases, transmission experiments into other species were performed, in order to better understand the species barriers and specific characteristics of these strains. After intracerebral challenge of a sheep carrying the TSE susceptible genotype ARQ/ARQ with L-BSE, the animal succumbed to the disease after more than 1500 days35). This incubation time is considerably longer as compared to challenge of sheep carrying the same genotype with C-BSE, where incubation times of 537–608 days were observed36). Interestingly, although the peripheral nervous system was involved as described for scrapie and C-BSE in sheep, the LRS displayed no involvement in the pathogenesis of L-BSE in this animal35).
In order to decipher the zoonotic potential of L-BSE, nonhuman primates (cynomolgus macaques) were challenged intracerebrally with L-BSE37,38). The animals developed disease after a distinctly shorter incubation time than the control animals inoculated with C-BSE (e.g. 26 instead of 40 months post infection). These findings confirm what has been published after transmission of L-BSE to transgenic mice expressing human PrPC, where a higher attack rate was observed than for C-BSE challenged animals39,40). A higher zoonotic potential must therefore be assumed for L-BSE.
For a better understanding of the nature and the characteristics of both atypical BSE forms, subpassage experiments were performed in different wild-type and transgenic mice as well as Syrian Gold hamsters. Earlier experiments had shown that especially the L-BSE strain may adopt the classical BSE phenotype after passage through transgenic mice41,42,43). Capobianco and colleagues showed that challenge of inbred wild-type mouse lines (SJL, C57Bl/6, RIII, and VM mice) did not induce disease in these mice. However, after several passages, these mice developed a TSE which was indistinguishable from C-BSE as determined by Western blot analysis and lesion profile scoring41). When bovine PrP transgenic mice were challenged with L-BSE, they succumbed to the disease after shorter incubation times of 178 days as compared to 216 days after challenge with C-BSE, and the biochemical characteristics were those of L-BSE. These findings were confirmed in a different line of bovine PrP transgenic mice and using Japanese L-BSE prions shortly afterwards42). Beringue and colleagues43) transmitted L-BSE to ovine PrP transgenic mice, and observed a 100% attack rate and an incubation time of 423 days, which is distinctly shorter than >700 days after challenge with C-BSE. Subpassage of L-BSE in ovine PrP transgenic mice resulted in reduced incubation times of 140 (2nd passage) and 143 days (3rd passage). Interestingly, the Western blot profile in these ovine PrP transgenic mice was again reminiscent of that related to C-BSE, even after the first passage. Finally, transmission of L-BSE into Syrian Gold hamsters induced disease and revealed a molecular weight of the unglycosylated PrP fraction, but a glycosylation pattern similar to C-BSE, with a clearly prominent diglycosylated PrP band44,45).
When H-BSE was transmitted to bovine PrP transgenic mice, the biochemical and histological characteristics of H-BSE were maintained46). On the other hand when wild-type C56Bl/6 mice were challenged with H-BSE, the strain characteristics were maintained only during the first passage. In the second passage, a small proportion of the mice developed a TSE after distinctly shorter incubation times, which was indistinguishable from C-BSE by its biochemical characteristics47,48). Likewise, transmission of H-BSE to hamster PrP transgenic mice resulted in a TSE that was indistinguishable from C-BSE49). Interestingly, passage of this TSE into bovine PrP transgenic mice revealed again the transmission and biochemical characteristics of H-BSE.
We performed systematic passage and subpassage experiments with L-BSE and H-BSE in conventional RIII and VM mice, in transgenic mice overexpressing the bovine PrP (Tgbov XV)6) or ovine PrP (TgshpXI)50) expressing the PrPARQ allele, as well as tg338 mice51) expressing the PrPVRQ allele, as well as in Syrian Gold hamsters under identical experimental conditions. Brains were analyzed using PTA precipitation followed by a quantitative Western blot. Our results obtained for L-BSE confirmed that the L-BSE profile was preserved after passage through Tgbov XV mice. Passage in TgshpXI mice resulted in an intermediate BSE pattern, i.e. the molecular mass of the unglycosylated PrP band (16.7 kDa) concurred with the value determined for L-BSE, while the glycosylation pattern only displayed a minor predominance of the diglycosylated band of 44.3% on average, as compared to 38.2% for L-BSE and 55.4% for C-BSE in this assay. On the other hand, transmission of L-BSE to Syrian Gold hamsters resulted in a Western blot pattern that was indistinguishable from C-BSE. As observed before, wild type mice did not develop disease after first passage of L-BSE. When we performed subpassage experiments from the first passage hosts (Tgbov XV mice, TgshpXI mice and Syrian Gold hamsters into Tgbov XV and TgshpXI mice as well as Syrian Gold hamsters), the same TSE pattern as after first passage in the same host was preserved (Fig. 2A).
Subpassage experiments of L-BSE and H-BSE in bovine and ovine PrP transgenic mice, in Syrian Gold hamsters and in wild-type mice
A) Subpassage of L-BSE in laboratory rodents. Brains of mice and hamsters were analyzed by PTA precipitation and Western blot as described above. At least four separate gel runs per brain sample were analyzed and the mean values were interpreted. Quantification of Western blot signals was performed using the Quantity One software (Biorad, Munich).
B) Subpassage of H-BSE in laboratory rodents. Brains of mice and hamsters were analyzed by PTA precipitation and Western blot as described under A).
When we performed the respective passage experiments with H-BSE, only Tgbov XV mice developed diseases in the first passage, showing the H-BSE pattern upon Western blot analysis. In contrast, TgshpXI mice, Syrian Gold hamsters and wild type mice did not succumb to the disease. Upon subpassage in the same host, TgshpXI mice again did not develop disease, while Syrian Gold hamsters developed a TSE indistinguishable from C-BSE, and wild-type mice developed characteristics of H-BSE.
These experiments show that both atypical BSE strains are not completely stable and can therefore under certain circumstances be transformed into a different phenotype, mostly resembling that of C-BSE. Through further subpassages, it became obvious that the L-BSE and H-BSE strain characteristics are maintained even in a host where biochemical analysis reveals the C-BSE pattern. It therefore seems that the phenotype of a BSE strain is determined both by the strain itself, but also by the host.
During the last 10–15 years, an impressive gain of knowledge about the pathogenesis of classical and atypical BSE has been achieved through a variety of transmission experiments and pathogenesis studies. It could be shown that the pathogenesis of all three BSE forms is restricted to the nervous system, while the LRS (except the Peyer’s patches of the small intestine and the tonsils, acting as entry ports after oral uptake of the C-BSE agent) plays no role in the propagation of the agent. As opposed to C-BSE where infectivity and PrPSc are only detectable in the late stage of the clinical phase, tissues of the PNS and skeletal muscular system may contain infectivity and PrPSc already at slightly earlier stages of the disease. Given that the screening of healthy slaughtered cattle using BSE rapid tests has been ceased in a number of European Union member states, it cannot be excluded that tissues of animals affected with atypical BSE may enter the food chain. This is of more concern regarding L-BSE, since it has been shown that the zoonotic potential of this BSE form exceeds that of classical BSE.
Transmission experiments and subpassages of both atypical BSE forms into laboratory rodents revealed that the phenotypes of both atypical BSE strains are not fully stable. This has been interpreted as a possible explanation for the origin of C-BSE which might have emerged after passages of one of the atypical BSE strains. However, a transmission from L-BSE or H-BSE to C-BSE has never been observed after transmission to cattle or sheep, rendering this assumption rather unlikely.
