Shigeo Fukuda

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrPSc, a protease-resistant misfolded isoform of the cellular prion protein PrPC. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrPSc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrPSc in BSE-affected cattle therefore remains unknown. Methodology/Principal Findings We report here that PrPSc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrPSc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrPSc in blood was not substantiated in the BSE-affected cattle examined. Conclusions/Significance The distribution of PrPSc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrPSc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.

Intraspecies transmission of L-type-like bovine spongiform encephalopathy detected in Japan

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.

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues.

Detection of disease-associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures.

A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrPSc) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrPSc was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrPSc was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrPSc immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrPSc might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrPSc in the extracerebral tissues of BSE-affected cattle.

Properties of L-type bovine spongiform encephalopathy in intraspecies passages.

The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrPSc) throughout the brain. Although immunolabeled PrPSc obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrPSc molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.

Brainstem auditory evoked potentials in experimentally-induced bovine spongiform encephalopathy

This study was carried out to evaluate the features of neurological dysfunction in experimentally-induced bovine spongiform encephalopathy (BSE)-infected cattle using brainstem auditory evoked potentials (BAEP). The progressive prolongation of peak latency of waves III and V was observed right-and-left bilaterally at the onset of neurological symptoms. The peak latency of wave V and the I-V interpeak latency (IPL) in BSE cattle 22 and 24 months after intracerebral inoculation were significantly (P<0.05) prolonged compared with the control cattle. In addition, the amplitude of the BAEP waves of the BSE cattle were low compared with the control cattle. Hearing loss occurred in the BSE cattle that showed advanced neurological symptoms such as tremor. It is thought that this BAEP data reflects a functional disorder in the central auditory nerve pathways characteristic of experimentally-induced BSE.

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle

To the Editor: A definitive diagnosis of bovine spongiform encephalopathy (BSE) in cattle usually relies on Western blot and immunohistochemical testing of samples from the obex region of the brainstem. These conventional diagnostic tests can detect the presence of the abnormal (disease-associated) form of the prion protein (PrPSc) in brain samples several months before the onset of clinical signs; however, there is no appropriate, universal tool for early preclinical and antemortem diagnosis of BSE. Furthermore, confirmation of the disease is currently only possible by postmortem examination of brain tissues. In this study, we used the serial protein misfolding cyclic amplification (sPMCA) technique to determine the presence of PrPSc in saliva samples collected from BSE-infected cows before and after the onset of disease (1). In a previous study (2), we analyzed the tissue distribution of PrPSc in cattle up to 66 months after they were orally inoculated with a relatively low dose (5 g) of homogenized brainstem from animals with naturally occurring BSE in England. In 2011, after publication of that study and 83.3 months after the cows were inoculated, clinical signs of BSE developed in 1 cow (no. 5444); necropsy was performed 84.7 months after inoculation. In addition, we used saliva samples from 2 BSE-affected cows (nos. 5413 and 5437) (2) to determine the presence of PrPSc. We collected saliva samples from animals at 4 monthly intervals, beginning in 2009, 56 months after inoculation. Samples were stored at −80°C until analysis. Using the sodium phosphotungstic acid precipitation method, we concentrated (100-fold) individual 1-mL saliva samples from each time point. We then diluted the concentrated samples 1:10 with the normal isoform of prion protein substrate containing 0.5% potassium dextran sulfate. Using the sPMCA technique as described (1), we amplified the samples in 3–8 tubes, and we used Western blot to analyze the proteinase K–treated sPMCA products (2). Using Western blot and immunohistochemical tests, we detected the accumulation of PrPSc in brains collected at necropsy from the 3 cows examined. In addition, using the sPMCA technique, we detected PrPSc signal in 1) saliva samples that were concentrated from samples collected from the same 3 cows at necropsy and in 2) concentrated saliva samples that were collected from 2 of the cows (nos. 5413 and 5444) at the early clinical stages of disease. After saliva samples underwent 3 rounds of amplification, we detected PrPSc in a saliva sample that was collected from cow number 5437 two months before the clinical onset of clinical symptoms (Figure). For 2 of the cows (nos. 5413 and 5437), the positive ratio of salivary PrPSc at round 4 of amplification increased as the disease progressed (Figure). Because PrPSc signal could be detected in BSE-infected brain homogenates diluted up to 10−10 after 2 rounds of amplification (1), we estimated PrPSc levels in the nonconcentrated original saliva samples to be lower than those in BSE-infected brain homogenate diluted to 10−12. No PrPSc signal was detected in samples collected from the 3 cows 3–5 months before the onset of clinical symptoms or from age-matched noninfected controls, even after 4 rounds of amplification. Figure Western blot detection, using the serial protein misfolding cyclic amplification technique, of the abnormal (disease-associated) form of the prion protein (PrPSc) in concentrated saliva samples from 3 cows experimentally infected by inoculation with the ... We demonstrated the presence of PrPSc in saliva of BSE-affected cows during the clinical stage of the disease, and in 1 case, at the preclinical or asymptomatic stage. Our findings suggest that PrPSc is likely to be detected in the saliva of BSE-affected cattle during the clinical stage of disease, after accumulation of PrPSc in the brain. PrPSc was found in the salivary glands of BSE-affected cattle at the terminal stage of infection (1). Therefore, once the infectious agent reaches the central nervous system, it may spread centrifugally from the brain to the salivary glands through the autonomic nervous system. Infectivity of saliva and the presence of PrPSc in saliva have been reported in other ruminants affected with transmissible spongiform encephalopathy. Infectivity of saliva was demonstrated in deer with chronic wasting disease (3) and in scrapie-affected sheep (4); the immunolabeled PrPSc accumulated in the salivary glands of scrapie-affected sheep (5). A low level of PrPSc was detected in concentrated buccal swab samples of preclinical scrapie-infected sheep by using sPMCA (6,7). These results suggest that small amounts of PrPSc may accumulate in the salivary glands and are then secreted into saliva. The presence of infectious prions in saliva may explain the facile horizontal transmission of scrapie in sheep (4–6) and chronic wasting disease in deer (4,8). There has been no epidemiologic evidence, however, that saliva, milk, blood, and cerebrospinal fluid from BSE-infected cattle are infectious (9). Nonetheless, the potential risk for BSE transmission by body fluids or excretions from BSE-infected cattle is cannot be ruled out by the current data.

The Presence of Disease-Associated Prion Protein in Skeletal Muscle of Cattle Infected with Classical Bovine Spongiform Encephalopathy

ABSTRACT The aim of this study was to investigate the presence of disease-associated prion protein (PrPSc) in the skeletal muscle of cattle infected with classical bovine spongiform encephalopathy (C-BSE). The study was carried out systematically in 12 different muscle samples from 43 (3 field and 40 experimental) cases of C-BSE; however, muscle spindles were not available in many of these cases. Therefore, analysis became restricted to a total of 31 muscles in 23 cattle. Even after this restriction, low levels of PrPSc were detected in the muscle spindles of the masseter, intercostal, triceps brachii, psoas major, quadriceps femoris and semitendinosus muscles from 3 field and 6 experimental clinical-stage cases. The present data indicate that small amounts of PrPSc are detectable by immunohistochemistry in the skeletal muscles of animals terminally affected with C-BSE.

Localization of TGF-β and TGF-β receptor in bovine term placentome and expression differences between spontaneous and induced parturition

INTRODUCTION Mechanisms of detachment of fetal membrane after parturition in cattle are poorly understood. Glucocorticoids trigger the initiation of parturition and may facilitate the placental maturation. We compared the disappearance of trophoblast binucleate cells (BNCs) and expression of transforming growth factor-β (TGFB) in term placentomes between spontaneous and induced parturition to investigate the influences of glucocorticoids on the placental maturity. METHODS Cows were delivered spontaneously (SP group) or after the administration of prostaglandin (PG) F(2)α (PG group); dexamethasone, PGF(2)α, and estriol (DEX group); and triamcinolone acetonide, PGF(2)α, and betamethasone (BET group) and placentomes were collected immediately after parturition. The number of BNCs in hematoxylin and eosin stained section was examined. Protein localization and mRNA levels of TGFB and its receptor (TGFBR) were analyzed using immunohistochemistry and qRT-PCR, respectively. RESULTS TGFB1 is characteristically localized in the maternal septum in caruncle in contrast to TGFB2 and TGFB3, which are mainly found in cotyledonary villi and maternal epithelial cells. TGFBR1 and TGFBR2 colocalized in BNCs. The number of BNCs was lower in the SP group than in PG and DEX groups. mRNA levels of TGFB1, TGFBR1 and TGFBR2 in the SP group differed from PG and DEX groups. There was no difference between SP and BET groups in all analyses. DISCUSSION These results indicate that parturition inductions using PGF(2)α or dexamethasone were not able to induce disappearance of BNCs and change of TGFB signaling. Results in the BET group suggest that investigation into types, dose, and dosage schedule of glucocorticoids may facilitate placental maturation.

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in cattle. During passaging of H-BSE in transgenic bovinized (TgBoPrP) mice, a novel phenotype of BSE, termed BSE-SW emerged and was characterized by a short incubation time and host weight loss. To investigate the biological and biochemical properties of the BSE-SW prion, a transmission study was conducted in cattle, which were inoculated intracerebrally with brain homogenate from BSE-SW–infected TgBoPrP mice. The disease incubation period was approximately 15 months. The animals showed characteristic neurological signs of dullness, and severe spongiform changes and a widespread, uniform distribution of disease-associated prion protein (PrPSc) were observed throughout the brain of infected cattle. Immunohistochemical PrPSc staining of the brain revealed the presence of intraglial accumulations and plaque-like deposits. No remarkable differences were identified in vacuolar lesion scores, topographical distribution patterns, and staining types of PrPSc in the brains of BSE-SW– vs H-BSE–infected cattle. PrPSc deposition was detected in the ganglia, vagus nerve, spinal nerve, cauda equina, adrenal medulla, and ocular muscle. Western blot analysis revealed that the specific biochemical properties of the BSE-SW prion, with an additional 10- to 12-kDa fragment, were well maintained after transmission. These findings indicated that the BSE-SW prion has biochemical properties distinct from those of H-BSE in cattle, although clinical and pathologic features of BSW-SW in cattle are indistinguishable from those of H-BSE. The results suggest that the 2 infectious agents, BSE-SW and H-BSE, are closely related strains.