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Identification of H-type BSE in Portugal Leonor Orge, Carla Guedes Machado, Luísa Ramalho, Renata Carvalho, João Silva, Paula Almeida, Paula Tavares, Cristina Ochoa, Carla Lima, Maria J Marques Pinto & J Pedro Simas To cite this article: Leonor Orge, Carla Guedes Machado, Luísa Ramalho, Renata Carvalho, João Silva, Paula Almeida, Paula Tavares, Cristina Ochoa, Carla Lima, Maria J Marques Pinto & J Pedro Simas (2015) Identification of H-type BSE in Portugal, Prion, 9:1, 22-28, DOI: 10.1080/19336896.2014.997615 To link to this article:

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Prion, 9:22–28, 2015 Ó 2015 Taylor & Francis Group, LLC ISSN: 1933-6896 print / 1933-690X online DOI: 10.1080/19336896.2014.997615


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Identification of H-type BSE in Portugal Leonor Orge1,2, Carla Guedes Machado1, Luısa Ramalho3, Renata Carvalho1, Jo~ao Silva1, Paula Almeida1, Paula Tavares1, Cristina Ochoa1, Carla Lima1, Maria J Marques Pinto3, and J Pedro Simas4,* Laborat orio de Patologia polos Benfica e Vair~ao; Instituto Nacional de Investiga¸c~ao Agraria e Veterinaria, IP; Lisboa e Vair~ao, Portugal; 2 CECAV; Centro de Ci^encia Animal e Veterinaria; Universidade de Tras-os-Montes e Alto Douro; Vila Real, Portugal; 3 Dire¸c~ao Geral de Alimenta¸c~ao e Veterinaria; Lisboa, Portugal; 4 Instituto de Medicina Molecular; Faculdade de Medicina; Lisboa, Portugal 1

ABSTRACT. During the bovine spongiform encephalopathy (BSE) epidemic, Portugal was the third most affected country. As a result of a successful national eradication plan, the number of BSE affected animals has been progressively declining in Portugal with no cases identified in 2013. However, within the scope of this active surveillance scheme, we have identified the first H-type BSE case born after the introduction of the reinforced ban in fallen stock. Here, we report the phenotypic features of this case and the analysis of the protein coding sequence of prnp as well as the prnp promoter and intron 1 insertion-deletions. KEYWORDS. H-BSE, Portugal, prnp, PrPres

ABBREVIATIONS. BSE, bovine spongiform encephalopathy; prnp, prion protein gene; PrP, prion protein; PrPres, protease resistant prion protein; EU, European Union; OIE, Office International of Epizooties; MBM, meat and bone meal; BARB, born after the reinforced ban of meat and bone meal; aa, amino acid

*Correspondence to: J Pedro Simas; Email:[email protected] Received October 1, 2014; Revised October 30, 2014; Accepted December 7, 2014. Color versions of one or more figures in this article can be found online at 22

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Classical bovine spongiform encephalopathy (C-BSE) is an acquired prion disease of cattle disseminated by the ingestion of infected tissues in meat-and-bone meal (MBM) used as feed supplement.1 It emerged in the United Kingdom in 19862 and then spread to continental Europe, Japan and North America. Its link to variant Creutzfeldt-Jakob disease in humans3 triggered an intensive active surveillance in cattle and reinforced food safety measures. Under the extended screening for BSE in cattle population, 2 other forms of BSE were identified almost 20 y later. Both forms are referred to as atypical BSE and are rare. While they do present different pathological features, their main diagnostic and characteristic features compared to that in C-type BSE are the molecular features of their PrPres in Western Blots, which are the unglycosylated PrPres band in L-type BSE and H-type BSE migrating respectively at lower and higher position in the gel, differences in glycoprofile, protease susceptibility and coexistence of 2 triple band PrPres populations.46 L-type BSE is also known as bovine amyloidotic spongiform encephalopathy (BASE). It is difficult to assess the precise number of atypical BSE affected animals detected worldwide due to the lack of reporting. Over 80 atypical BSE cases,7 including 35 cases of H-type BSE,7 were detected in the European Union (EU) but only 13 are referred worldwide by the Office International of Epizooties (OIE).8 Although the risk of developing H-type BSE does not increase with the age of the animals, as observed for the L-Type,9 most cases occur in older animals of the age of 8 y and older although in one instance a weak and incompletely proven H-type case has been reported in Switzerland in a 6.5 y old animal.10 As all the cases have been detected under the active surveillance program, clinical description is scarce and the available material for pathological characterization is mostly in very poor conditions. In a few H-type BSE natural cases, minimal spongiform lesions (solitary tract nucleus and spinal tract nucleus of trigeminal nerve) and mild PrPres deposits (neuropil punctate and coarse particulate, intraneuronal and intraglial types) were reported in brainstem (both at


the obex-dorsal motor nucleus of the vagus nerve, caudal olivarius nucleus, cunneate nucleus, hypoglossal nucleus, spinal tract nucleus of trigeminal nerve, solitary tract nucleus, reticular formation- and at the midbrain).10–12 In H-type BSE experimentally challenged cattle, animals developed either dull or a nervous clinical phenotype. PrPres immunolabelling showed PrPres accumulation predominantly at the obex, as intraglial type in the white matter tracts including the cerebellum.13 H-type BSE discriminatory diagnosis has to rely on the PrPres electrophoretic profiles obtained with 2 different antibodies. According to the AHVLA Bio-Rad TeSeE-based Hybrid Method that uses the antibodies Sha31 (C-terminal PrP region antibody) and P4 (N-terminal PrP region antibody), there is a visible signal and high molecular mass migration (compared to Classical BSE) with Sha31, an equivalent signal with P4 and a predominance of the diglycosylated band.14 Additionally, H-type BSE presents a lower extra band at 10–12 kDa revealed by antibodies (e.g., SAF84) binding PrP C-terminally of the Sha31 epitope confirmed as unglycosylated fraction.6,15 Despite several transmission studies revealing that H-type BSE is caused by a distinctive prion strain,13,16-18 some isolates acquired Ctype BSE properties after serial passages in rodent models, suggesting a putative role of this atypical form in the origin of BSE epidemics.19-21 Nevertheless, its etiology and pathogenesis are still poorly understood. Thus, studies have been carried out in order to gain insight about this atypical BSE form. One such study demonstrated that PrPres is not confined to central nervous system as it was detected in peripheral nervous system of intracerebrally challenged cattle.22 It has been pointed out that H-type BSE could be a spontaneous prion disease due to its low and stable incidence as well as the older age of the affected animals (revised by Sala et al.9). This hypothesis is also supported by data showing that atypical BSE is not influenced by the prion gene (prnp) promoter (23-bp) and intron 1 (12-bp) deletion variations22 that are related to an increased susceptibility to classical BSE.24-26 However, an

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Orge et al.

American H-type BSE was associated to a new prnp allele K21127 and the referred prnp deletion variants were overrepresented in French and Polish atypical BSE cases.28 These particular characteristics of some atypical BSE affected animals reinforce the need to study each case detected in order to further understand its pathogenesis. After the emergence of BSE, Portugal became one of the EU Member states with higher incidence of this disease. Between 1990 and 2013, a total of 1088 BSE cases were confirmed, representing a 3.5 prevalence expressed in number of cases per million of adult cattle. The first strain characterization study concerning Portuguese BSE cases from 1996 to 2004 confirmed their similarity to that described as classical BSE regarding lesion profile, patterns and distribution of PrPres as well as PrPres electrophoretic profile.29 The occurrence of putative atypical BSE cases will be investigated under a retrospective study included in the EU recent request, thus contributing to the assessment of the BSE forms in Portugal. Here we report the phenotypic features of this first case of H-type BSE in Portugal and the analysis of the protein coding sequence of prnp as well as the prnp promoter and intron 1 insertion-deletions. Since 2001, BSE active surveillance is based on PrPres screening in the brainstem by the Bio-Rad TeSeETM SAP ELISA rapid test done following manufacturer’s instructions. In 2011, a 13 y old cow, cross breed for meat production, under the fallen stock stream, tested positive by the rapid test and was further analyzed for the presence of vacuolation by histopathology, for the deposition of PrPres by immunohistochemistry as well as for the analysis of the PrPres electrophoretic profile by Western immunoblotting. The prnp analysis including the open reading frame and insertion-deletion variants in the promoter region and in intron 1 was also performed. Brainstem was divided sagittally with one half frozen for rapid test analysis, Western immunoblotting and the other half fixed in 10% saline formalin and processed for routine paraffin histology. For immunohistochemistry, the anti-PrP monoclonal antibody- 12F10 (aa 142– 160, SPI-bio) diluted 1/100 was used after

pretreatment of sections with formic acid followed by autoclaving and visualization by Vectastain- peroxidase and diaminobenzidine (Vector). For Western immunoblotting analysis of PrPres, frozen brainstem tissue (500ml) was processed according to the protocol of TeSeE Western blot kit (Bio-Rad with the kit monoclonal anti-PrP antibody Sha31, aa 156–163 bovine PrP protein15 diluted 1/10). The confirmation of the H-type BSE phenotype was carried out using the discriminatory molecular method with the C-terminal anti-PrP antibody Sha31 and the N- terminal anti-PrP monoclonal antibody P4 (aa 101–107 bovine PrP protein,15 R-Biopharm, 0.1mg/ml) diluted 1/500 as recommended by the European Union Reference Laboratory (AHVLA Bio-Rad TeSeE- based Hybrid Western Blot method).14 The specificity of PrPres immunolabelling was controlled using tissues from known C-type BSE-positive case and H-type BSE control (sourced from the AHVLA proficiency testing) and BSE-negative case. For prnp genotyping, genomic DNA extracted from ear tissue (WizardÒ Genomic DNA purification kit, Promega) was subjected to PCR with prnp specific primers to analyze the protein coding region according to Choi et al.30 and to determine the insertion-deletion variants in the promoter region (the 23 bp) and in intron 1 (12bp) as reported by Sander et al.24. The purified PCR product of the prnp protein coding region was then submitted to automated cycle sequencing in both orientations. Sequence data was aligned using the BLAST (Basic Local Alignment Search Tool) with the Genbank accession numbers: EU809428, AJ298878, AY367642 and AY367643. Based on the size of the PCR product, the indel variants were directly identified by electrophoresis on a 4% NuSieve agarose gel accordingly to Sander et al.24 The atypical BSE case detected in Portugal was born and raised in the classical BSE second most affected region. It did not receive milk replacers or starters and the diet was based on commercial feed for cattle since 1999, hence considered a BARB (Born After Reinforced Ban of meat and bone meal) case. Most H-type BSE natural cases reported did not show clinical signs10 or showed essentially difficulty in



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FIGURE 1. Phenotypical features of the first Portuguese H-BSE. (a) PrPres immunolabelling in the reticular formation of the brainstem: linear pattern (arrow) and the predominant intraglial pattern as single granules in the white matter (arrowheads) (b). The box in picture (a) outlines the area shown in picture (b) at higher magnification. Specific PrPres signal was visualized with Vectastain–peroxidase, DAB (Vector). All sections were counterstained with Mayer’s haematoxylin. The magnifications used are indicated. (c) Western blot analysis of PrPres after proteinase K treatment using Sha31 and P4 immunolabelling: Sha31 shows a higher molecular mass migration (orange arrow) by comparison to C-BSE; with P4 there is immunolabelling in the Portuguese H-BSE (PT H-BSE) similar to that presented by the H-BSE control. Absence of signal in C-BSE and L-BSE. H-BSE, CBSE and L-BSE controls from AHVLA proficiency testing. Scrapie represents classical scrapie control. M, molecular mass marker (kDa).

rising and/or lateral recumbency.11,27 The present case showed fear, a behavioral change related to the nervous clinical phenotype described in the experimental transmissions.13 The brainstem presented a positive result (1.827; 2.199; 2.262) by the rapid test. The histopathological exam resulted unsuitable due to autolysis changes. By immunohistochemistry, intraneuronal and linear PrPres deposition types were detected as usually observed in Ctype BSE. However, the main feature of this case was the intraglial type seen in the white matter tracts of the brainstem (Fig. 1(a) and (b)) indicative of an atypical form as reported in natural and experimental H-type BSE cases.10,12,13 Interestingly not all H-type BSE

cases show this feature as with other natural cases a fine punctate deposition in the neuropil was the predominant feature reported.11 This suggests the existence of some variability in PrPres distribution; however more data of natural cases is needed. The cerebellum was unavailable for analysis. This is unfortunate as this neuronanatomical region is an important target area to discriminate atypical BSE forms when using immunohistochemistry13 and thus recommended to be included in bovine sample collection procedure in active surveillance. The Western immunoblotting was consistent with that described for H-type BSE showing a higher molecular weight of the unglycosylated fraction of PrPres when compared to C-type BSE


Orge et al.

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FIGURE 2. PCR analysis of the 12-bp and 23-bp indel polymorphisms on the PT H-BSE case. Electrophoresis on a 4% NuSieve agarose gel showing a single fragment for both the 12-bp and the 23-bp indicating homozygous for insertion in both loci, according to Sander et al., 200424 and Choi et al., 2012.30

(Fig. 1c). The immunolabelling with N-terminal PrP antibody P4 confirmed the H-type BSE form. In this animal only a synonymous polymorphism at codon 78 (Q78Q, CAG/CAA) was detected, as previously reported in French, American and Swedish H-type BSE cases.11,22,26 Neither other silent mutations in codons 23, 113, 185 and 192 observed by the same authors, nor the E211K mutation associated to an American H-type BSE case27 were present. Both alleles contained the 6-copy octapeptide repeat region, considered as the wild type cattle genotype. Thus, there was no increase in the number of octapeptide repeats that had enhanced susceptibility to BSE in transgenic mice31 even though in BSE cattle cases this was never observed.24,32 Regarding polymorphisms in the regulatory regions of the prnp- 23-bp deletion (promoter region)24 and 12-bp deletion (intron 1),25 associated to increased susceptibility to classical BSE, our H-type BSE was homozygous for insertion in both loci (Fig. 2), the genotype with a presumed link to BSE resistance, as the insertion variants have the potential to lower the host

prion protein expression levels (revised by Brunelle et al.23). The effect of these polymorphisms in atypical BSE has not been clarified yet due to the reduced number of cases analyzed, the limitation of genetic analysis to the prnp open reading frame thus excluding other gene regions10,11,33 and even to the report of contradictory results. Brunelle and collaborators23 concluded that these polymorphisms do not influence susceptibility to atypical BSE, but, on the other hand, an association of bovine prion gene haplotype with atypical BSE was reported.34 Moreover, in a few H-type BSE cases the expression of prnp was increased35 and the deletion gene variants 12bp and 23bp were overrepresented in both H-type and Ltype BSE.28 Therefore, genetic characterization of further atypical BSE cases is required to elucidate the genetic background of these prion diseases, if there is any. In this report, we highlight the importance of BSE type identification. Given the potential link of atypical cases of BSE with the aetiology of the BSE epidemic, it is of paramount importance to understand their epidemiology and

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pathogenesis since several BSE control measures have now been lifted. Hence, it is important to assure an efficient detection of any atypical BSE shift to classical BSE. Accurate reporting of each type of BSE by all countries is also crucial for determining the real magnitude of this prion disease. In order to achieve this goal, all EU member states have performed a discriminatory test to all the BSE cases confirmed after 1st of July 201336 and they will also have to carry out in 2015 a retrospective classification of all positive BSE cases diagnosed from 2003 to 2009, according to a request from the European Commission.7

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.

ACKNOWLEDGMENTS We would like to thank all the people involved in BSE prevention, control and eradication plan in Portugal. The authors would also like to thank Joana Castro from the rendering plant for supplying the ear tissue needed for DNA extraction. REFERENCES 1. Wilesmith JW, Wells GA, Cranwell MP, Ryan JB. Bovine spongiform encephalopathy: epidemiological studies. Vet Rec 1988; 123:638-44; PMID:3218047 2. Wells GA, Scott AC, Johnson CT, Gunning RF, Hancock RD, Dawson M, Bradley R. A novel spongiform encephalopathy in cattle. Vet Rec 1987; 121:419-20; PMID:3424605 3. Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, Collinge J, Doey LJ, Lantos P. The same prion strain causes vCJD and BSE. Nature 1997; 389:44850; PMID:9333232 4. Casalone C, Zanusso G, Acutis PL, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jacob disease. Proc Natl Acad Sci USA 2004; 101:3065-70; PMID:14970340


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Identification of H-type BSE in Portugal.

During the bovine spongiform encephalopathy (BSE) epidemic, Portugal was the third most affected country. As a result of a successful national eradica...
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