Atypical H-Type Bovine Spongiform Encephalopathy in a Cow Bornafter the Reinforced Feed Ban on Meat-and-Bone Meal in Europe
Claudia Guldimann,a Michaela Gsponer,a Cord Drögemüller,b Anna Oevermann,a and Torsten Seuberlicha
NeuroCentre, National and OIE Reference Laboratory for BSE and Scrapie,a and Institute of Genetics,b Vetsuisse Faculty, University of Bern, Bern, Switzerland
The significance of atypical bovine spongiform encephalopathies (BSE) in cattle for controlling the BSE epidemic is poorly un-derstood. Here we report a case of atypical H-type BSE in a cow born after the implementation of the reinforced feed ban in Eu-rope. This supports an etiology of H-type BSE unrelated to that of classical BSE.
CASE REPORT
A6.5-year-old Red Angus cow presented downer cow syn-drome after the birth of a dead calf in Switzerland in February
2012. The animal was born in Germany in 2005 and imported intoSwitzerland at the age of 17 months. According to the owner, therewere no signs of illness of the cow prior to giving birth. Afteremergency slaughter, medulla oblongata samples were taken incompliance with the Swiss statutory bovine spongiform encepha-lopathy (BSE) surveillance. The initial BSE rapid test (CheckWestern; Prionics) (21) performed by a regional laboratory waspositive. Consequently, the medulla oblongata sample was sent,together with the remaining brain, which was still available at theslaughterhouse, to the Swiss BSE Reference Laboratory. There, theanimal was confirmed BSE positive with the TeSeE Western blot(Bio-Rad) (2), using limited proteinase K digestion and immuno-detection with two prion protein-specific monoclonal antibodies(MAbs), Sha31 (11) and 12B2 (16). Molecular masses of protei-nase K-resistant prion protein peptides (PrPres) in the Westernblot were determined with Quantity One software version 4.6.2(Bio-Rad). In comparison to a classical (C-type) BSE control sam-ple, the PrPres bands seen in this case showed �1.3- to 1.4-kDahigher molecular masses as well as an additional band at �7.2kDa. Also, the sample reacted with MAb 12B2 (Fig. 1). This isconsistent with the molecular phenotype of H-type BSE (14). Thedistribution of the disease-associated prion protein (PrPd)throughout the brain was determined by enzyme-linked immu-nosorbent assay (ELISA) (BSE-scrapie antigen test kit; Idexx).PrPd was detected mainly in the thalamus and the obex and, to alesser extent, in the cerebellar cortex, hippocampus, lobus pyrifor-mis, and basal nuclei (Fig. 2). Histopathological analysis was per-formed on hematoxylin-and-eosin (H&E)-stained paraffin sec-tions of the same brain regions as those analyzed in the ELISA.Minimal spongiform lesions were present in the obex region (Fig.3a) and in the midbrain, but not in other brain structures. Byimmunohistochemistry (using MAb F99) (17), mild PrPd depositswere observed in the dorsal motor nucleus of the vagus nerve, thecaudal olivary nucleus (Fig. 3b), the cuneate nucleus (Fig. 3c), thehypoglossal nucleus, the spinal tract nucleus of the trigeminalnerve, and the solitary tract nucleus (Fig. 3d), as well as in themidbrain and thalamus. These deposits were of the coarse par-ticulate, intraneuronal, and intraglial type. There was no PrPd
labeling in the cerebellum, hippocampus, basal nuclei, andcerebral cortex. The entire open reading frame of the bovineprion protein was sequenced and revealed no DNA variant in
comparison to the reference sequence (GenBank accession no.AJ298878.1). In particular the E211K mutation thought tocause a genetic variant of H-type BSE (19) was not present.After laboratory confirmation of the disease, the carcass of theanimal, including all by-products, was destroyed, and no ma-terial entered the food chain.
BSE is a transmissible and neurodegenerative disease thatemerged in the United Kingdom in the mid-1980s and later incontinental Europe, Japan, and North America (26). It is causedby prions, which are misfolded cellular prion proteins (PrPd) thataccumulate in the brain of affected cattle. Prion diseases may ei-ther be acquired, (i.e., transmitted by infection), have a geneticbasis, or develop spontaneously as sporadic cases (9). Three typesof BSE are currently differentiated: the C-, L-, and H-types. WhileC-type BSE has been by far the most frequent form of the disease,L- and H-type BSEs, also referred to as atypical BSEs, are rareconditions that present biochemically and biologically distinctcharacteristics from C-type BSE (6, 8). C-type BSE is acquired andprion transmission occurs by the ingestion of infected tissues—inruminants notably of meat-and-bone meal (MBM) being used asa feed supplement (27). Due to an incubation period of severalyears, the average age of BSE-affected cattle was 5 to 6 years duringthe epidemic, but cases have also been identified in much youngerand older animals. H-type BSE has only been diagnosed in about30 cattle worldwide, all over 8 years of age (24). Detection wasmostly by active surveillance schemes (i.e., by laboratory testing oflarge numbers of adult cattle not suspected of having clinical BSE).Biological strain typing by experimental transmission to rodentmodels and cattle indicated that H-type BSE is caused by a prionagent distinct from those of C- and L-type BSEs (4, 5, 15, 18).Down to the present day, the pathology and etiology of H-typeBSE remain poorly understood (24). Especially the question ofwhether the disease is acquired, genetic, or sporadic leaves a major
Received 15 August 2012 Returned for modification 19 September 2012Accepted 25 September 2012
gap in our knowledge. The case presented here extends the under-standing of H-type BSE in several aspects.
Whole brain pathology of H-type BSE has only been reportedin a bull of the miniature zebu breed (23, 25) and in experimen-tally intracerebrally inoculated cattle (15, 18). For all other naturalH-type BSE cases, only brain stem samples were available, and it isnot known which brain structures are the best suited diagnostictarget. The zebu was 19 years old and showed prominent neuro-logical signs, and spongiform lesions as well as PrPd deposits weresevere and widespread in the brain. In contrast, the animal de-scribed here was 6.5 years old, central nervous system (CNS)-specific neurological signs were not observed, and spongiformlesions as well as PrPd deposits in the brain were minimal. All ofthese findings support that the cow was in an early, preclinicalstage of the disease. In this regard, it is important to point out thatthese minimal lesions and PrPd deposits were found in the graymatter structures of the obex region of the medulla oblongata, themidbrain, and the thalamus. These findings are essentially similarto those in preclinical C-type BSE (1, 12, 13, 22) and support thatsampling of the obex region in surveillance schemes implementedfor C-type BSE might be similarly suitable for detection of natu-rally occurring H-type BSE.
The main disease control measure of C-type BSE is the ban onmammalian MBM in ruminant feed. This feed ban was enforced
in Switzerland and the European Union in the early 1990s andconsiderably reduced the number of newly infected cattle. How-ever, the recycling of the C-type BSE agent in the cattle populationwas not blocked until the MBM feed ban was reinforced in 2001,now excluding the use of animal proteins in feed of all farmedanimals (10). Whether H-type BSE similarly is transmitted orallyin the cattle population with MBM as a vehicle remains unknown.If oral transmission occurs and is the sole etiology, the reinforcedMBM feed ban should be an appropriate measure to prevent thespread of H-type BSE as well, and H-type BSE should not be de-tected in animals born after its implementation, i.e., after 2001 inSwitzerland and Germany. To our knowledge, this is the first re-port of an H-type BSE-affected animal being born after the rein-forced MBM feed ban in the respective country. Therefore, thiscase provides further evidence that the etiology of H-type BSE maybe unrelated to the ingestion of prion-contaminated meat-and-bone meal. Taken together, this supports the widely expressedpostulate that H-type BSE originates from a spontaneous misfold-ing of cellular PrP with a pathophysiology similar to that of spo-radic Creutzfeld-Jakob disease in humans (7, 20). Alternatively,other yet unknown routes of transmission or genetic determi-nants must be considered. This said, H-type BSE might persistafter eradication of C-type BSE. What are the implications of thisscenario? Studies with mice provided experimental evidence thatH-type BSE may shift its disease phenotype to that of C-type BSE(3) upon transmission. It has therefore been hypothesized that theC-type BSE epidemic originated from spontaneously occurringH-type BSE cases. If this was the case, there would be a constantrisk that C-type BSE would reemerge in the cattle population oncethe feed ban is discontinued. Consequently, some measures ofdisease control would need to be maintained indefinitely. Sincethe standards for the determination of a country’s BSE risk statuscurrently do not differentiate between BSE subtypes (28), BSE riskassessments will certainly need to take such considerations intoaccount. This highlights the need for continuing research into therelationship between classical and atypical BSE variants to providethe scientific basis for future disease surveillance and control pol-icies.
FIG 1 Bio-Rad TeSeE hybrid Western blot using MAb Sha31 and MAb 12B2.Molecular masses of individual proteinase K-resistant prion protein peptides(PrPres) are indicated below the brackets. Note the differences in the molecularmasses and 12B2 reactivities between the H-type BSE case and the C-type BSEcontrol as well as the additional PrPres peptide at �7 kDa.
FIG 2 Neuroanatomical pathological prion protein (PrPd) distribution. PrPd
distribution in the brain of the H-type BSE case was determined by the Idexxtest. The test cutoff is indicated by the dashed line.
This work was funded by the Swiss Federal Veterinary Office.We are indebted to Jan Langeveld for kindly providing monoclonal
antibody 12B2, Dagmar Heim for scrutinizing the manuscript, and An-dreas Zurbriggen for continuous support.
REFERENCES1. Arnold ME, et al. 2007. Estimating the temporal relationship between
PrPSc detection and incubation period in experimental bovine spongi-form encephalopathy of cattle. J. Gen. Virol. 88:3198 –3208.
2. Arsac JN, Biacabe AG, Nicollo J, Bencsik A, Baron T. 2007. Biochemicalidentification of bovine spongiform encephalopathies in cattle. Acta Neu-ropathol. 114:509 –516.
3. Baron T, et al. 2011. Emergence of classical BSE strain properties duringserial passages of H-BSE in wild-type mice. PLoS One 6:e15839. doi:10.1371/journal.pone.0015839.
4. Baron TG, Biacabe AG, Bencsik A, Langeveld JP. 2006. Transmission ofnew bovine prion to mice. Emerg. Infect. Dis. 12:1125–1128.
5. Beringue V, et al. 2006. Isolation from cattle of a prion strain distinctfrom that causing bovine spongiform encephalopathy. PLoS Pathog.2:e112. doi:10.1371/journal.ppat.0020112.
6. Biacabe AG, Laplanche JL, Ryder S, Baron T. 2004. Distinct molecularphenotypes in bovine prion diseases. EMBO Rep. 5:110 –115.
8. Casalone C, et al. 2004. Identification of a second bovine amyloidotic
spongiform encephalopathy: molecular similarities with sporadicCreutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. U. S. A. 101:3065–3070.
9. Colby DW, Prusiner SB. 2011. Prions. Cold Spring Harbor Perspect.Biol. 3:a006833. doi:10.1101/cshperspect.a006833.
10. Ducrot C, Arnold M, de Koeijer A, Heim D, Calavas D. 2008. Review onthe epidemiology and dynamics of BSE epidemics. Vet. Res. 39:15. doi:10.1051/vetres:2007053.
11. Feraudet C, et al. 2005. Screening of 145 anti-PrP monoclonal antibodiesfor their capacity to inhibit PrPSc replication in infected cells. J. Biol.Chem. 280:11247–11258.
12. Hoffmann C, et al. 2007. Prions spread via the autonomic nervous systemfrom the gut to the central nervous system in cattle incubating bovinespongiform encephalopathy. J. Gen. Virol. 88:1048 –1055.
13. Iwata N, et al. 2006. Distribution of PrP(Sc) in cattle with bovine spon-giform encephalopathy slaughtered at abattoirs in Japan. Jpn. J. Infect.Dis. 59:100 –107.
14. Jacobs JG, et al. 2007. Molecular discrimination of atypical bovine spon-giform encephalopathy strains from a geographical region spanning awide area in Europe. J. Clin. Microbiol. 45:1821–1829.
15. Konold T, et al. 2012. Experimental H-type and L-type bovine spongi-form encephalopathy in cattle: observation of two clinical syndromes anddiagnostic challenges. BMC Vet. Res. 8:22. doi:10.1186/1746-6148-8-22.
16. Langeveld JP, et al. 2006. Rapid and discriminatory diagnosis of scrapieand BSE in retro-pharyngeal lymph nodes of sheep. BMC Vet. Res. 2:19.doi:10.1186/1746-6148-2-19.
17. Nentwig A, et al. 2007. Diversity in neuroanatomical distribution ofabnormal prion protein in atypical scrapie. PLoS Pathog. 3:e82. doi:10.1371/journal.ppat.0030082.
FIG 3 Histopathology and immunohistochemistry. (a) Dorsal motor nucleus of the vagus nerve (H&E stain). A vacuole is indicated by the arrow. (b) Caudalolivary nucleus with predominant intraneuronal PrPd labeling. (c) Cuneate nucleus, intraneural (arrow), and intraglial PrPd labeling and mild staining of theneuropil. A similar staining pattern was observed in the spinal tract nucleus of the trigeminus. (d) Solitary tract nucleus with sparse glial PrPd labeling.
Case Report
December 2012 Volume 50 Number 12 jcm.asm.org 4173
18. Okada H, et al. 2011. Experimental H-type bovine spongiform enceph-alopathy characterized by plaques and glial- and stellate-type prion pro-tein deposits. Vet. Res. 42:79. doi:10.1186/1297-9716-42-79.
19. Richt JA, Hall SM. 2008. BSE case associated with prion protein genemutation. PLoS Pathog. 4:e1000156. doi:10.1371/journal.ppat.1000156.
20. Sala C, et al. 2012. Individual factors associated with L- and H-typebovine spongiform encephalopathy in France. BMC Vet. Res. 8:74. doi:10.1186/1746-6148-8-74.
21. Schaller O, et al. 1999. Validation of a Western immunoblotting proce-dure for bovine PrP(Sc) detection and its use as a rapid surveillancemethod for the diagnosis of bovine spongiform encephalopathy (BSE).Acta Neuropathol. 98:437– 443.
22. Schulz-Schaeffer WJ, Fatzer R, Vandevelde M, Kretzschmar HA. 2000.Detection of PrP(Sc) in subclinical BSE with the paraffin-embedded tissue(PET) blot. Arch. Virol. Suppl. 2000:173–180.
23. Seuberlich T, et al. 2006. Spongiform encephalopathy in a miniaturezebu. Emerg. Infect. Dis. 12:1950 –1953.
24. Seuberlich T, Heim D, Zurbriggen A. 2010. Atypical transmissible spon-giform encephalopathies in ruminants: a challenge for disease surveillanceand control. J. Vet. Diagn. Invest. 22:823– 842.
25. Tester S, et al. 2009. Biochemical typing of pathological prion protein inaging cattle with BSE. Virol. J. 6:64. doi:10.1186/1743-422X-6-64.
26. Wells GA, et al. 1987. A novel progressive spongiform encephalopathy incattle. Vet. Rec. 121:419 – 420.
