Progression of prion infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy challenge

¹ Centro de Investigación en Sanidad (CISA-INIA), Valdeolmos, Madrid, Spain
² School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland

Correspondence
Juan María Torres
jmtorres@inia.es

출처 : J Gen Virol 88 (2007), 1379-1383; DOI 10.1099/vir.0.82647-0
http://vir.sgmjournals.org/cgi/content/full/88/4/1379

The presence of BSE prion infectivity in asymptomatic cattle and its tissue distribution are important concerns for both human and veterinary health and food safety. In this work, a collection of tissues from asymptomatic cattle challenged orally with BSE and culled at 20, 24, 27, 30 and 33 months have been used to inoculate intracerebrally BoPrP-Tg110 mice expressing bovine PrP to assess their infectivity. Results demonstrate that BSE infectivity in asymptomatic cattle is essentially restricted to the nervous system, Peyer’s patches and tonsils, as reported previously for terminally BSE-diseased cattle. BSE infectivity was detectable in Peyer’s patches and tonsils at all time points analysed, but infectivity in nervous tissues (brainstem and sciatic nerve) was only detectable after 27 months from inoculation. Infectivity in brainstem increased markedly at 33 months after inoculation. All other investigated tissues or fluids (spleen, skeletal muscle, blood and urine) revealed no detectable infectivity throughout the time course studied.

	MAIN TEXT

Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with the accumulation of abnormal PrP^Sc conformer in the brain and subsequent neurodegeneration (Prusiner, 2004). The mechanism of conversion of PrP^C to PrP^Sc is not well understood, but it may occur by direct interaction of PrP^Sc with PrP^C, promoting refolding of the latter to produce additional PrP^Sc. The PrP^Sc conformer can be recognized by its partial resistance to proteinase K treatment.

Most TSEs are transmitted naturally by peripheral routes, either orally or transcutaneously. The mechanism(s) of spread from the periphery to the central nervous system (CNS) is an important issue. It is not clear how prions pass through the intestinal mucosa after oral uptake. M cells, which are portals for antigens and pathogens (Hathaway & Kraehenbuhl, 2000), may be involved in the transepithelial transport of prions (Heppner et al., 2001). Thus, the infectious agent may penetrate the mucosa through M cells and reach the Peyer’s patches. Although prion diseases are neurological disorders, critical events in their pathogenesis take place in restricted sites outside the nervous system, especially in peripheral lymph organs (Aucouturier et al., 2000).

Bovine spongiform encephalopathy (BSE) was recognized as a cattle prion disease during the 1980s (Wilesmith et al., 1988) in the UK. Ingestion of foods contaminated with BSE is the likely cause of the new variant Creutzfeldt–Jakob disease in humans (Bruce et al., 1997; Hill et al., 1997).

Several studies indicate that, to date, the BSE agent has been found only in the brain, spinal cord and retinal (eye) tissue of BSE-diseased cattle. Infectivity assessment in several tissues from orally inoculated cattle, using bioassays based on RIII mice (Wells et al., 1994, 1998), revealed BSE infectivity in the CNS, all brain regions, the spinal cord, the optic nerve, the retina (neuronal cells) and the facial and sciatic nerves, as well as in distal ileum and bone marrow. The skeletal muscles, spleen and other lymphatic tissues were shown to be free of detectable infectivity. More recently, Wells et al. (2005) showed infectivity in tonsil tissue from cattle killed 10 months after oral BSE challenge by intracerebral inoculation in cattle. These finding are in contrast to the spreading of the scrapie agent in infected sheep, mice and hamsters in tissues such as spleen, other lymphatic tissues, muscles etc., even during the preclinical stage (Bosque et al., 2002; Heggebo et al., 2003; Thomzig et al., 2003, 2004). In addition, PrP^Sc can be found in the lymphoreticular system and is not restricted to the nervous system following oral inoculation of sheep and primates with the BSE agent (Bons et al., 1999; Jeffrey et al., 2001; Herzog et al., 2004; Andreoletti et al., 2006). Recently, experiments in transgenic mice overexpressing bovine PrP confirmed the essential restriction of infectivity to the nervous system in terminally BSE-diseased cattle (Buschmann & Groschup, 2005).

The distribution of BSE infectivity in asymptomatic cattle incubating the disease and its progression through the silent period from inoculation to the appearance of clinical signs is of particular interest in relation to food safety. In the present work, we have used a highly sensitive bioassay based on transgenic mice overexpressing bovine PrP (BoPrP-Tg110 mice) (Castilla et al., 2003) to assess the infectivity in a panel of tissues from asymptomatic cattle at different times (20–33 months) after oral challenge.

Tissues from asymptomatic cattle after oral BSE challenge were prepared and kindly provided by the Veterinary Laboratory Agency (VLA), New Haw, Addlestone, Surrey, UK, as part of the VLA Project SE1736. Cattle (4–6 months of age) were inoculated orally with 100 g doses of BSE-infected brainstem material derived from a homogenate of about 150 clinically sick and pathologically confirmed cases of BSE. Control animals were maintained under the same conditions, but not infected. Clinical signs were assessed monthly by veterinarian experts from the VLA (New Haw, Addlestone, Surrey, UK). At different times post-inoculation, three infected and one control animal were culled and a panel of tissues and fluids were sampled aseptically. The tissue and fluids were stored at –70 °C. In this study, tissues from animals culled at 20, 24, 27, 30 and 33 months post-infection were investigated. Homogenates (10 % in PBS) of each tissue or fluid from asymptomatic cattle sampled at the indicated times were used for infectivity assessment in BoPrP-Tg110 mice. Samples from the three inoculated cows at each time point were used as pools.

All pools, containing each tissue sampled from three different cattle at the same time after challenge, were tested for the presence of PrP^Sc before inoculation of BoPrP-Tg110 mice. PrP^Sc was analysed by Western blotting in brain tissues collected and homogenized in PBS. One hundred microlitres of 10 % (w/v) brain homogenate was pre-cleared by centrifugation at 2000 g for 5 min in 5 % sarcosyl. Samples were treated with 20 µg proteinase K ml^–1 (Roche) at 37 °C for 60 min and insoluble fractions were obtained by centrifugation at 25 000 g for 30 min. SDS sample loading buffer was added to all samples, boiled for 10 min and loaded on an SDS/12 % polyacrylamide gel. For the immunoblotting experiments, mAbs 2A11 (Brun et al., 2004) and Sha31 (Feraudet et al., 2005) were used at a 1 : 1000 dilution from ascitic fluid. Immunocomplexes were detected by horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences). The immunoblots were developed under conditions of enhanced chemiluminescence (Amersham Biosciences).

None of the samples was scored positive by Western blotting using mAb 2A11 (Table 1 and data not shown). In addition, samples were analysed by using the highly sensitive Bio-Rad ELISA TeSeE test (Grassi et al., 2001) according to the manufacturer’s recommendations. Only brainstem sampled at 33 months post-inoculation was scored positive with this test (Table 1). The presence of infectivity in the different samples was analysed by intracerebral inoculation of BoPrP-Tg110 transgenic mice. These mice overexpress bovine PrP (around eightfold) under the control of the murine Prnp gene promoter from the MoPrP.Xho vector (Borchelt et al., 1996) and are highly susceptible to BSE infection (Castilla et al., 2003, 2004). Groups of six mice (6–7 weeks old, weighing around 20 g) were inoculated with 20 µl of the appropriate tissue pool in the right parietal lobe by using a 25-gauge disposable hypodermic needle. After inoculation, the mice were observed daily and their neurological status was assessed twice weekly. Mice showing at least three of ten signs of neurological dysfunction (Scott et al., 1989, 1993) over several consecutive days were sacrificed and samples were collected for diagnostic evaluation. Survival times were calculated as the time between inoculation and death. All mice in an experiment were tested for PrP^Sc accumulation in their brains, and only those positive for PrP^Sc were included in the calculation of survival times.

The PrP^Sc electrophoretic profiles found in the brain of BoPrP-Tg110 mice inoculated with the different tissues showed no apparent differences when positive tissues were compared with each other or with PrP^Sc from the brain of a natural BSE case (Fig. 1).

View larger version (53K): [in this window] [in a new window]   Fig. 1. Western blot of brain homogenates from selected BoPrP-Tg110 mice inoculated with a pool of tissues from BSE-infected asymptomatic cattle (33 months after oral challenge). Bovine tissues used for the inoculation of BoPrP-Tg110 mice are indicated above each line, with the exception of BSE2, which is a BoPrP-Tg110 mouse inoculated with BSE2 derived from a natural case of BSE in cattle (Castilla et al., 2003). mAb 2A11 was used as indicated in the text. Molecular mass is given on the right in kDa.

In brainstem, the detection of infectivity was only achieved 27 months after oral challenge. The infectivity found in brainstem at 27 or 30 months after challenge was low, as only one-third of the BoPrP-Tg110 mice inoculated were scored positive and with long incubation times. Previous work using the RIII mouse bioassay failed to detect infectivity in the CNS before 32 months after inoculation (Wells et al., 1998). In contrast, the pool of brainstem material from cattle sampled 33 months after oral challenge contained a high titre of infectivity, as shown by the maximum percentage of infected BoPrP-Tg110 mice found. This indicates a marked accumulation of the infectious agent in at least one of the three pooled animals during the last few months (from 30 up to 33 months) after challenge. This increased infectivity is confirmed by the detection of PrP^Sc by the Bio-Rad ELISA TeSeE test in this brainstem pool. As only one of the three animals used for each pool needs to be positive to transmit the disease to the BoPrP-Tg110 mice, we cannot be sure that the differences detected in the infectivity levels were not due to differences between individual cattle. A low level of infectivity was also detected in the sciatic nerve from animals sampled at 30 and 33 months after challenge. All other tissues or fluids investigated, including skeletal muscle, blood and urine, revealed no detectable infectivity throughout the time course studied (Table 1). Tissues with no detectable infectivity in the highly sensitive bioassay based on transgenic mice (without a transmission barrier for BSE prions) may be considered as being of very low risk for oral infection in humans, where a strong transmission barrier is present (Lasmezas et al., 2005).

In conclusion, our results confirm that BSE infectivity in asymptomatic cattle is essentially restricted to the nervous system, as reported previously for terminally BSE-diseased cattle (Buschmann & Groschup, 2005), and is consistent with the idea that BSE infectivity, after oral uptake, propagates only poorly in some intestinal lymphatic tissues (mainly Peyer’s patches) and from there spreads centripetally to the CNS, probably by intraneural spread via the peripheral nervous system.

	ACKNOWLEDGEMENTS

 
We thank the VLA (New Haw, Addlestone, Surrey, UK) for kindly providing tissues of experimentally infected cows at different time points. The authors wish to thank Dr J. Grassi from CEA (Commissariat à l’Energie Atomique), France, for providing the Sha31 antibody. Thanks are also due to Bio-Rad for supplying the ELISA TeSeE kits. This work was supported by grants INIA-CAL01-018, UE-FAIR-CT97-3306 and INIA-RTA-2006-0091.

REFERENCES

Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., 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.[Abstract/Free Full Text]

Andreoletti, O., Morel, N., Lacroux, C., Rouillon, V., Barc, C., Tabouret, G., Sarradin, P., Berthon, P., Bernardet, P. & other authors (2006). Bovine spongiform encephalopathy agent in spleen from an ARR/ARR orally exposed sheep. J Gen Virol 87, 1043–1046.[Abstract/Free Full Text]

Aucouturier, P., Carp, R. I., Carnaud, C. & Wisniewski, T. (2000). Prion diseases and the immune system. Clin Immunol 96, 79–85.[CrossRef][Medline]

Bons, N., Mestre-Frances, N., Belli, P., Cathala, F., Gajdusek, D. C. & Brown, P. (1999). Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc Natl Acad Sci U S A 96, 4046–4051.[Abstract/Free Full Text]

Borchelt, D. R., Davis, J., Fischer, M., Lee, M. K., Slunt, H. H., Ratovitsky, T., Regard, J., Copeland, N. G., Jenkins, N. A. & other authors (1996). A vector for expressing foreign genes in the brains and hearts of transgenic mice. Genet Anal 13, 159–163.[Medline]

Bosque, P. J., Ryou, C., Telling, G., Peretz, D., Legname, G., DeArmond, S. J. & Prusiner, S. B. (2002). Prions in skeletal muscle. Proc Natl Acad Sci U S A 99, 3812–3817.[Abstract/Free Full Text]

Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J. & other authors (1997). Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389, 498–501.[CrossRef][Medline]

Brun, A., Castilla, J., Ramirez, M. A., Prager, K., Parra, B., Salguero, F. J., Shiveral, D., Sanchez, C., Sanchez-Vizcaino, J. M. & other authors (2004). Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11. Neurosci Res 48, 75–83.[CrossRef][Medline]

Buschmann, A. & Groschup, M. H. (2005). Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J Infect Dis 192, 934–942.[CrossRef][Medline]

Castilla, J., Gutierrez Adan, A., Brun, A., Pintado, B., Ramirez, M. A., Parra, B., Doyle, D., Rogers, M., Salguero, F. J. & other authors (2003). Early detection of PrP(res) in BSE-infected bovine PrP transgenic mice. Arch Virol 148, 677–691.[CrossRef][Medline]

Castilla, J., Gutierrez-Adan, A., Brun, A., Pintado, B., Parra, B., Ramirez, M. A., Salguero, F. J., Diaz San Segundo, F., Rabano, A. & other authors (2004). Different behavior toward bovine spongiform encephalopathy infection of bovine prion protein transgenic mice with one extra repeat octapeptide insert mutation. J Neurosci 24, 2156–2164.[Abstract/Free Full Text]

Feraudet, C., Morel, N., Simon, S., Volland, H., Frobert, Y., Creminon, C., Vilette, D., Lehmann, S. & Grassi, J. (2005). Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J Biol Chem 280, 11247–11258.[Abstract/Free Full Text]

Grassi, J., Comoy, E., Simon, S., Creminon, C., Frobert, Y., Trapmann, S., Schimmel, H., Hawkins, S. A., Moynagh, J. & other authors (2001). Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue. Vet Rec 149, 577–582.[Abstract/Free Full Text]

Hathaway, L. J. & Kraehenbuhl, J. P. (2000). The role of M cells in mucosal immunity. Cell Mol Life Sci 57, 323–332.[CrossRef][Medline]

Heggebo, R., Press, C. M., Gunnes, G., Ulvund, M. J., Tranulis, M. A. & Lsverk, T. (2003). Detection of PrPSc in lymphoid tissues of lambs experimentally exposed to the scrapie agent. J Comp Pathol 128, 172–181.[CrossRef][Medline]

Heppner, F. L., Christ, A. D., Klein, M. A., Prinz, M., Fried, M., Kraehenbuhl, J. P. & Aguzzi, A. (2001). Transepithelial prion transport by M cells. Nat Med 7, 976–977.[CrossRef][Medline]

Herzog, C., Sales, N., Etchegaray, N., Charbonnier, A., Freire, S., Dormont, D., Deslys, J. P. & Lasmezas, C. I. (2004). Tissue distribution of bovine spongiform encephalopathy agent in primates after intravenous or oral infection. Lancet 363, 422–428.[CrossRef][Medline]

Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C., Gowland, I., Collinge, J., Doey, L. J. & Lantos, P. (1997). The same prion strain causes vCJD and BSE. Nature 389, 448–450, 526.[CrossRef][Medline]

Jeffrey, M., Ryder, S., Martin, S., Hawkins, S. A., Terry, L., Berthelin-Baker, C. & Bellworthy, S. J. (2001). Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). I. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J Comp Pathol 124, 280–289.[CrossRef][Medline]

Lasmezas, C. I., Comoy, E., Hawkins, S., Herzog, C., Mouthon, F., Konold, T., Auvre, F., Correia, E., Lescoutra-Etchegaray, N. & other authors (2005). Risk of oral infection with bovine spongiform encephalopathy agent in primates. Lancet 365, 781–783.[Medline]

Madec, J. Y., Groschup, M. H., Calavas, D., Junghans, F. & Baron, T. (2000). Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock. Microb Pathog 28, 353–362.[CrossRef][Medline]

Prusiner, S. B. (2004). Early evidence that a protease-resistant protein is an active component of the infectious prion. Cell 116, S109

Race, R., Jenny, A. & Sutton, D. (1998). Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis. J Infect Dis 178, 949–953.[Medline]

Scott, M., Foster, D., Mirenda, C., Serban, D., Coufal, F., Walchli, M., Torchia, M., Groth, D., Carlson, G. & other authors (1989). Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847–857.[CrossRef][Medline]

Scott, M., Groth, D., Foster, D., Torchia, M., Yang, S. L., DeArmond, S. J. & Prusiner, S. B. (1993). Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73, 979–988.[CrossRef][Medline]

Thomzig, A., Kratzel, C., Lenz, G., Kruger, D. & Beekes, M. (2003). Widespread PrP(Sc) accumulation in muscles of hamsters orally infected with scrapie. EMBO Rep 4, 530–533.[CrossRef][Medline]

Thomzig, A., Schulz-Schaeffer, W., Kratzel, C., Mai, J. & Beekes, M. (2004). Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie. J Clin Invest 113, 1465–1472.[CrossRef][Medline]

van Keulen, L. J., Schreuder, B. E., Meloen, R. H., Mooij-Harkes, G., Vromans, M. E. & Langeveld, J. P. (1996). Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol 34, 1228–1231.[Abstract]

Wells, G. A., Dawson, M., Hawkins, S. A., Green, R. B., Dexter, I., Francis, M. E., Simmons, M. M., Austin, A. R. & Horigan, M. W. (1994). Infectivity in the ileum of cattle challenged orally with bovine spongiform encephalopathy. Vet Rec 135, 40–41.[Medline]

Wells, G. A., Hawkins, S. A., Green, R. B., Austin, A. R., Dexter, I., Spencer, Y. I., Chaplin, M. J., Stack, M. J. & Dawson, M. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 142, 103–106.[Abstract/Free Full Text]

Wells, G. A., Spiropoulos, J., Hawkins, S. A. & Ryder, S. J. (2005). Pathogenesis of experimental bovine spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Vet Rec 156, 401–407.[Abstract/Free Full Text]

Wilesmith, J. W., Wells, G. A., Cranwell, M. P. & Ryan, J. B. (1988). Bovine spongiform encephalopathy: epidemiological studies. Vet Rec 123, 638–644.[Abstract]

Received 16 October 2006; accepted 22 December 2006.