These authors contributed equally to this work.
Intraspecies transmission of L-type-like bovine spongiform encephalopathy detected in Japan
Article first published online: 21 AUG 2009
© 2009 The Societies and Blackwell Publishing Asia Pty Ltd
Microbiology and Immunology
Volume 53, Issue 12, pages 704–707, December 2009
How to Cite
Fukuda, S., Iwamaru, Y., Imamura, M., Masujin, K., Shimizu, Y., Matsuura, Y., Shu, Y., Kurachi, M., Kasai, K., Murayama, Y., Onoe, S., Hagiwara, K., Sata, T., Mohri, S., Yokoyama, T. and Okada, H. (2009), Intraspecies transmission of L-type-like bovine spongiform encephalopathy detected in Japan. Microbiology and Immunology, 53: 704–707. doi: 10.1111/j.1348-0421.2009.00169.x
- Issue published online: 30 NOV 2009
- Article first published online: 21 AUG 2009
- Received 7 May 2009; revised 2 August 2009; accepted 4 August 2009.
- atypical bovine spongiform encephalopathy;
It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L-type-like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.
bovine amyloidotic spongiform encephalopathy
bovine spongiform encephalopathy
transmissible spongiform encephalopathy
TSE, including BSE and Creutzfeldt–Jakob disease, are neurodegenerative and fatal disorders in humans and animals. The key event in the pathogenesis of TSE is the conformational change from the normal host prion protein (PrPC) to the abnormal, disease-associated form (PrPSc), which is thought to be the main, if not the only, constituent of the TSE agents. Classical BSE was first recognized in the United Kingdom (UK) in 1986 (1), and has subsequently spread to other European countries, Japan and North America. Until recently, it was believed that the BSE agent was a single strain based on biological, neuropathological and biochemical characteristics in field BSE cases (2–4). However, since 2003, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been reported (5). At present, atypical BSE are classified as the H-type and L-type according to the higher and lower molecular masses of the unglycosylated form of proteinase K (PK)-treated PrPSc in western blot analysis, respectively, compared with those from classical BSE isolates (6). Among the L-type BSE cases reported from various countries (5), the Italian L-type BSE cases were further characterized by the presence of PrP-positive amyloid plaques in the brain (7) and is termed as BASE. BASE prion was experimentally transmitted to cattle, and the phenotypes of BASE prion have been characterized in detail (8). However, it remains to be determined whether the L-type BSE prions detected in other countries are identical to BASE prion and are classified into a single prion strain. Resolving this issue is crucial for future research aimed at exploring the origin of atypical BSE, assessing the risk of atypical BSE and reviewing of the current administrative measures for BSE controls.
In Japan, two atypical BSE cases have been identified to date. The first case showed an L-type-like electrophoretic mobility of the unglycosylated PrPSc on western blot analysis (9). The second case was identified in an aged beef cattle, Japanese Black (BSE/JP24), and showed PrP-positive amyloid plaques in histopathological examination of the brain and a distinct glycoform profile (10). Although such properties seem to be similar to those reported in a BASE case (7), unlike with the BASE prion, shortening of the incubation periods was observed in bovinized mice serially passaged with the BSE/JP24 prion (11). Thus, it remains controversial whether the BSE/JP24 prion is identical to the BASE prion. These observations prompted us to characterize the phenotypes of the BSE/JP24 prion propagated in its natural host by comparison with those of the classical BSE prion. Hence, we have inoculated with brain homogenates from classical BSE and BSE/JP24 isolates into Holstein cattle and assessed their risk against cattle species.
This study was approved by the Animal Ethical Committee and the Animal Care and Use Committee of National Institute of Animal Health, and Hokkaido Animal Research Center.
Six Holstein calves aged 2–3 months were intracerebrally inoculated with 1 ml of 10% (w/v) brain homogenates prepared from the medulla oblongata of classical BSE prion-affected cattle from the UK (n= 3) or BSE/JP24 (n= 3). The cattle were clinically monitored for signs of disease. The BSE/JP24 prion-affected cattle appeared to display the clinical signs indicative of BSE, such as mild anxiety and/or hyperesthesia evoked by sudden loud noises or waving of a clipboard at 344 ± 14 (mean ± SD) days post inoculation (dpi). The time of onset of clinical signs in BSE/JP24 prion-affected cattle was substantially earlier than that in cattle inoculated with classical BSE prion in our experiments (at 548 ± 25 dpi) or than that in a previous study (12). With disease progression, both experimentally transmitted cattle showed an ataxic gait, which appeared to be due to uncoordinated hind limbs and had difficulty rising in the terminal stage of the disease. In accordance with a recent report describing the differences in clinical signs between BASE prion-affected and classical BSE prion-affected cattle (8), the BSE/JP24 prion-affected cattle were inactive and displayed little aggression.
We killed the classical BSE and BSE/JP24 prion-affected animals at the terminal stage of disease with incubation periods of 675 ± 57 and 486 ± 11 dpi, respectively (Table 1). A shorter incubation period was observed previously in transgenic mice and cattle challenged with the BASE and German L-type BSE prion (8, 13), and in transgenic mice challenged with Japanese L-type-like BSE prion (11). Thus, the short incubation period in experimental transmission might be a common feature of BASE, L-type and L-type-like BSE prion. We then examined for the accumulation of PrPSc in brain tissues by western blot and immunohistochemical analyses. Western blot analysis for PrPSc from PK-treated brain homogenates was carried out as described previously (14). To detect PK-treated PrPSc, anti-PrP mAbs 6H4 (Roche Diagnostics, Basel, Switzerland) and T2 (15) were used (Fig. 1a and b). The unglycosylated fragments of PK-treated PrPSc derived from BSE/JP24 prion-affected cattle migrated slightly faster than those from the classical BSE prion-affected cattle (Fig. 1b). The signal intensities in di- mono-, and non-glycosylated fragments of PK-treated PrPSc were measured and semiquantified by densitometric analysis (Fig. 1a and c). The relative amounts of these fragments from BSE/JP24 prion-affected cattle resembled those from the original BSE/JP24 isolate. The glycoform ratios were distinguishable from those observed in classical BSE prion-affected cattle. These data suggest that the molecular properties of PrPSc from BSE/JP24 prion were sustained in the transmitted cattle.
|Code||Breed||Inoculum||Incubation time (days)||Clinical duration (days)|
|Mean: 675 ± 57||Mean: 128 ± 46|
|Mean: 486 ± 11||Mean: 141 ± 25|
Vacuolar lesion profile was determined in hematoxylin–eosin-stained sections as described previously (3). For the immunohistochemical detection of PrPSc, dewaxed sections were pretreated with the chemical solutions as described previously (16) and immunolabeled with mAbs F99/97.6.1 (VMRD, Pullman, WA, USA) and T1 (17) using the tyramide signal amplification system (NEN Life Science Products, Boston, MA, USA). Vacuolation was more severe in the midbrain, thalamus, hypothalamus and frontal cortex (Fig. 2a) of BSE/JP24 prion-affected cattle compared to that of classical BSE prion-affected cattle. Immunohistochemical analysis showed that the pattern of PrPSc deposition in BSE/JP24 prion-affected cattle was characterized by diffuse synaptic-punctuate staining, low-grade stellate-type PrPSc deposits, and amyloid PrP plaques (Fig. 2b). However, no striking differences were identified in the topography of PrPSc deposition between the classical BSE and BSE/JP24 prion-affected cattle. PrPSc deposits were pronounced in the neuropil of the thalamus and midbrain, particularly in the periaqueductal gray matter of the brains from both experimentally BSE prion-affected cattle (data not shown). Consistent with this, western blot analysis also showed that there were no marked differences in the topography of PrPSc deposition, except for high PrPSc levels in the frontal cortex of BSE/JP24 prion-affected cattle (Fig. 2c). Interestingly, these immunohistochemical and neuropathological properties closely resembled those of the BASE-affected cattle (8).
In summary, we demonstrated the successful transmission of the BSE/JP24 prion to cattle. The BSE/JP24 prion-affected cattle sustained the molecular properties of PK-treated PrPSc as those of the original BSE/JP24 isolate. Although most brain regions except for the medulla oblongata of the original BSE/JP24 isolate were unable to be investigated due to inadequate specimen collection, in comparison to experimentally BSE/JP24 prion-affected cattle, both neuropathological features, such as severe vacuolation in the medulla oblongata at the obex level and the presence of PrPSc plaques, closely resembled each other. Based on molecular properties of PK-treated PrPSc and a detailed comparison of the immunohistochemical and neuropathological properties, the BSE/JP24 prion was distinguishable from those in the classical BSE prion, and appear to be rather similar to the BASE prion (8).
Of interest, experimental transmission of the BSE/JP24 prion to cattle induced a shorter incubation period and more severe neuropathological changes compared to the classical BSE prion, suggesting that the BASE and BSE/JP24 prion might be more virulent in cattle species. However, such speculation conflicts with reports that atypical BSE field cases have been mainly found in adult and aged cattle (5). The reason for this discrepancy in incubation periods between experimentally and naturally affected cattle is unknown. These observations may imply that atypical BSE are sporadic forms of BSE. Alternatively, the route of infection and/or prion titer may be attributed to the relatively long incubation period in natural atypical cases. Further studies using orally BSE/JP24 prion-affected cattle will be needed to address this issue.
We thank Dr Y. Tagawa (National Institute of Animal Health) for providing the mAb T2 used in this study. We also thank Ms M. Kakisaki, Ms M. Sakurai, Ms Y. Miyama and Ms N. Tabeta for their technical assistance. This work was supported in part by a Grant-in-Aid of the BSE and the Prion Disease Control Project from the Ministry of Agriculture, Forestry, and Fisheries of Japan; a grant for BSE research from the Ministry of Health, Labor, and Welfare of Japan.
- 11987) A novel progressive spongiform encephalopathy in cattle. Vet Rec 121: 419–20., , , , , , , (
- 21996) Transmission and strain typing studies of scrapie and bovine spongiform encephalopathy. In: CourtL., DodetB. eds. Transmissible Subacute Spongiform Encephalopathies: Prion Diseases. Paris : Elsevier, pp. 259–62., , , , (
- 31996) BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases. Vet Rec 138: 175–7., , , , , (
- 41998) Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation. J Infect Dis 178: 693–9., , (
- 52008) Review on the epidemiology and dynamics of BSE epidemics. Vet Res 39: 15., , , , (
- 62007) Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe. J Clin Microbiol 45: 1821–9., , , , , , , , , , , , , , (
- 72004) Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA 101: 3065–70., , , , , , , (
- 82008) Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes. PLoS Pathog 4: e1000075., , , , , , , , , , , , , , , , , (
- 9Expert Committee for BSE Diagnosis, Ministry of Health, Labour and Welfare of Japan. 2003. Atypical proteinase K-resistant prion protein (PrPres) observed in an apparently healthy 23-month-old Holstein steer. Jpn J Infect Dis 56: 221–2., , , , , , , ;
- 102007. Accumulation of mono-glycosylated form-rich, plaque-forming PrPSc in the second atypical bovine spongiform encephalopathy case in Japan. Jpn J Infect Dis 60: 305–8., , , , , ,
- 112008) Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle. Prion 2: 123–8., , , , , , , , , , (
- 122005) Pathogenesis of experimental bovine spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Vet Rec 156: 401–7., , , (
- 132006) Atypical BSE in Germany–proof of transmissibility and biochemical characterization. Vet Microbiol 117: 103–16., , , , , , , , , (
- 142006) Distribution of PrP(Sc) in cattle with bovine spongiform encephalopathy slaughtered at abattoirs in Japan. Jpn J Infect Dis 59: 100–7., , , , , , , , , , , , (
- 152004) Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein. J Vet Med Sci 66: 515–20., , , , , , , (
- 162005) An alternative pretreatment procedure in animal transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry. J Histochem Cytochem 53: 1199–202., , (
- 172007) Species-specificity of a panel of prion protein antibodies for the immunohistochemical study of animal and human prion diseases. J Comp Pathol 136: 9–17., , , , , , , (