Role of Mel1/Prdm16 in bone differentiation and morphology.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16-/-) was embryonic lethal at E 11.5. Heterozygous mice showed abnormal cartilage and bone formation in the postnatal skull and long bones, suggesting that Mel1/Prdm16 expression plays an important role in bone development. In osteoblast and chondrocyte cell lines, Mel1/Prdm16 promotes the differentiation of chondrocytes and regulates the differentiation of osteoblasts. Transient repression of the master regulator Runx2 is required for chondrocyte differentiation at an early stage of differentiation. However, in Mel1/Prdm16-suppressed ATDC5 cells, the initial suppression of Runx2 was lacking and its expression was upregulated at the beginning of differentiation, suggesting that chondrogenic differentiation is suppressed in Mel1/Prdm16+/- mesenchymal progenitor cells because Runx2 expression is upregulated during the early stage of differentiation. Thus, the Mel1/Prdm16 gene may be involved in the early repression of Runx2 expression during osteochondral differentiation and promote chondrogenic differentiation.

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus has a negative economic impact on broiler farms in Japan.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, immunosuppressive and causes a negative economic impact on poultry industry. IBDV-vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio) reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in the first and second flocks, respectively, using immunohistochemical staining and RT-PCR. IBDV isolates from both flocks were closely related to the China KM523643 strain. Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV infection was appeared to be the main influencing factor on BF atrophy and BB ratio reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary infection. The disease caused by IBDV had a negative economic impact in the farm. RESEARCH HIGHLIGHTS New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers. After vIBDV had infected broilers, at 21 days old they became immunosuppressed. High mortality at 30 days old in broilers was due to secondary infection. New vIBDV has a negative economic impact on broiler farms in Japan.

A persistent epidemic of porcine epidemic diarrhoea virus infection by serological survey of commercial pig farms in northern Vietnam.

BACKGROUND: Porcine epidemic diarrhoea (PED) is a highly contagious infectious disease with negative economic impacts on the swine industry. PED outbreaks were reported from 2009 to 2015, but sporadic infection has been observed until now in Vietnam. However, the seroprevalence of PEDV infection has not yet been reported for commercial pig farms in Vietnam. The aim of this study was to assess the seroprevalence of PEDV infection in Vietnamese pig farms to reveal the endemic status of PEDV in northern Vietnam. RESULTS: A serological survey of PEDV infection was carried out using indirect ELISA in commercial pig farms in Hai Duong, Hung Yen and Thai Binh provinces in northern Vietnam in 2019. Twenty sera were randomly collected from each of 10 commercial pig farms, from each province; none of the farms had vaccinated for PEDV. Serological evidence of natural PEDV infection, expressed as a high antibody titre, was observed in the pig farms in all 3 provinces. The OD values were significantly higher (p < 0.001) for pig sera from Thai Binh than from Hai Duong and Hung Yen. No significant differences (p > 0.05) were detected for seropositivity to PEDV based on locality, age, pig breed and farm size. CONCLUSIONS: This study indicates serological evidence of natural PEDV infection with high antibody titre in commercial pig farms. PEDV infection was widespread among the pig population in these 3 provinces and that good management and strict biosecurity are needed at these pig farms.

Severe Degenerative Changes in Cryptorchid Testes in Japanese Black Cattle.

This is a histopathologic and endocrinologic study of 6 calves diagnosed with cryptorchidism. Cases 1-3 were diagnosed as resembling testicular regression syndrome. In cases 1 and 2, the extracted tissue was a small, firm, gray-white mass, and there was lack of obvious testicular tissue in case 3. Histopathologically, the excised tissue in cases 1-3 was a fibrotic testicular remnant with inflammation, mineralization, hemosiderin-laden macrophages or lipofuscin-laden macrophages, and lack of germ cells and interstitial endocrine cells. These findings were compared with cases 4-6, which were diagnosed as testicular hypoplasia due to cryptorchidism. These cases had small but otherwise grossly unremarkable intra-abdominal testicular tissue and histologically had a few germ cells and sustentacular cells with arrested spermatogenesis and an increase in interstitial endocrine cells. Cases 1-3 had more severe degenerative changes compared with cases 4-6. In case 2, the average diameter of the seminiferous tubules was much smaller than in cases 4-6, and there were few tubule cross sections. Anti-Müllerian hormone (214 pg/ml) was detected in the plasma of case 2. Based on the macroscopic and histopathologic findings as well as endocrinologic profiles, the testicular degeneration in cases 1-3 was considered similar to that of testicular regression syndrome. In this condition, it is thought that a normally developing intra-abdominal testis undergoes degeneration due to heat or a vascular disorder such as torsion.

Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. RESULTS: One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80-100%, respectively. CONCLUSIONS: The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

Development of a Japanese encephalitis virus genotype V virus-like particle vaccine in silkworms.

To counter the spread of multiple Japanese encephalitis virus (JEV) variants harboured in alternative host species and highly neurotoxic variants with new antigenicity, such as genotype V (Muar), methods for developing more effective and low-cost vaccines against a variety of epidemic JEV strains are required. Here, we successfully synthesized large amounts of a Muar virus-like particle (MVLP) vaccine for JEV in silkworm pupae by using a Bombyx mori nuclear polyhedrosis virus recombinant consisting of JEV codon-optimized envelope (E) DNA. In particular, histopathological examination suggested that MVLP was efficiently synthesized in body fat tissues as well as epithelial cells. Quantitative analysis indicated that one silkworm pupa produced 724.8 µg of E protein in the MVLP vaccine. Electron microscopic examination of purified MVLP vaccine defined a typical MVLP morphological structure. Detailed MVLP antigen assessment by immune-electron microscopy revealed that the majority of MVLPs were covered with approximately 10 nm projections. Boosted immunization with MVLP antigens in mice and rabbits tended to show improved plaque inhibition potency against homologous Muar and heterologous Nakayama, but less potency to Beijing-1 strains. Notably, mixed immune rabbit antisera against Nakayama and Muar VLP antigens led to an increase in the low antibody reaction to Beijing-1. Additionally, a stopgap divalent JEV vaccine consisting of MVLP and Nakayama VLP and its immune mouse serum significantly increased plaque inhibition titre against Muar, Nakayama and Beijing-1 strains. These findings suggested that low-cost MVLP vaccines prepared in silkworm pupae are suitable for providing simultaneous protection of individuals in developing countries against various JEV strains.

Overexpression of a virus-like particle influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion.

Ecological investigations of silkworms have revealed that Eri silkworms (Samia cynthia ricini) possess useful morphological and ecological characteristics for virus-like particle (VLP) production, namely non-seasonal breeding, longer lengths, and heavier weights than Bombyx mori silkworms. Furthermore, when vector DNA from Bombyx mori nuclear polyhedrosis virus (BmNPV), which is unable to replicate in Sf9 cells from Eri silkworms, was replaced with the Autographa californica nuclear polyhedrosis virus (AcNPV) vector, three improved AcNPV influenza virus recombinants capable of replication in Sf9 cells were obtained. Although VLP antigens produced previously in silkworms were not evaluated individually, the present recombinant Fukushima (FkH5) and Anhui (AnH7) VLP antigens were detected in tissue fluids and fat bodies of Eri silkworms. Here, we aimed to determine the function of the AcNPV vector and P143 gene by expressing recombinants in Sf9 cells and eri silkworm pupae. The FkH5 recombinant produced high yields of haemagglutinin (HA)-positive VLPs, showing a mean HA titre of 1.2 million. Similarly, high production of H7 HA VLPs was observed in the fat bodies of eri silkworm pupae. Antigenic analysis and electron microscopy examination of Eri-silkworm-produced H5 HA VLPs showed characteristic antigenicity and morphology similar to those of the influenza virus. Although FkH5 recombinants possessing the AcNPV vector did not replicate in Bm-N cells, the introduction of the helicase p143 gene from BmNPV resulted in their production in Bm-N and Sf9 cells.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks.

BACKGROUND: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013-2016, were sequenced and analyzed. RESULTS: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics. CONCLUSIONS: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd. RESULTS: In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed. CONCLUSIONS: The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

Experimental induction of chicken amyloid A amyloidosis in white layer chickens by inoculation with inactivated vaccines.

We investigated the amyloidogenic potential of inactivated vaccines and the localized production of serum amyloid A (SAA) at the injection site in white layer chickens. Hens in the treated group were injected intramuscularly three times with high doses of inactivated oil-emulsion Salmonella Enteritidis vaccine and multivalent viral and bacterial inactivated oil-emulsion vaccines at two-week intervals. Chickens in the control group did not receive any inoculum. In the treated group, emaciation and granulomas were present, while several chickens died between 4 and 6 weeks after the first injection. Hepatomegaly was seen at necropsy, and the liver parenchyma showed inconsistent discolouration with patchy green to yellowish-brown areas, or sometimes red-brown areas with haemorrhage. Amyloid deposition in the liver, spleen, duodenum, and at injection sites was demonstrated using haematoxylin and eosin staining, Congo red, and immunohistochemistry. The incidence of chicken amyloid A (AA) amyloidosis was 47% (28 of 60) in the treated group. In addition, RT-PCR was used to identify chicken SAA mRNA expression in the liver and at the injection sites. Furthermore, SAA mRNA was detected by in situ hybridization in fibroblasts at the injection sites, and also in hepatocytes. We believe that this is the first report of the experimental induction of systemic AA amyloidosis in white layer chickens following repeated inoculation with inactivated vaccines without the administration of amyloid fibrils or other amyloid-enhancing factors.

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α2u-globulin accumulation in the rat kidney.

Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as "hyaline droplets," whereas a specific type of deposits is referred to as "eosinophilic bodies" by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for α2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with α2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of α2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in α2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of α2u-globulin.

Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008.

Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position.

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aß production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood-brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.

Hepatocellular carcinoma with lung metastasis showing hemochromatosis in an Egyptian fruit bat (Rousettus aegyptiacus).

After an Egyptian fruit bat (Rousettus aegyptiacus) in a zoo became emaciated and died, a necropsy revealed multiple nodules on the liver and lung surfaces. Microscopy revealed that the liver nodules consisted of neoplastic hepatocytes and showed metastasis in the lung lobes. Most of the neoplastic cells in the liver and lung showed positive labeling for HepPar-1, cytokeratin 19, glypican-3, and Ki-67. Hepatocellular degeneration and necrosis were diffuse in the liver parenchyma. Berlin blue staining revealed large amounts of iron in normal and neoplastic cells. Based on these pieces of evidence, this case was diagnosed as hepatocellular carcinoma with hemochromatosis. This is believed to be the first report of hepatocellular carcinoma in an Egyptian fruit bat that has been immunophenotypically examined in detail by pathological examination.

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence.

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.

Novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P2 position.

Recently, we reported substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. We also designed and synthesized non-peptidic and small-sized inhibitors possessing a heterocyclic scaffold at the P(2) position. By studying the structure-activity relationship of these inhibitors, we found that the σ-π interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition mechanism. Hence, we optimized the inhibitors with a focus on their P(2) regions. In this Letter, a series of novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P(2) position are described along with the results of the related structure-activity relationship study. These small-sized inhibitors are expected improved membrane permeability and bioavailability.

Antimicrobial resistance in Salmonella serovars isolated from meat shops at the markets in North Vietnam.

A total of 97 out of 245 carcass, sewage effluent, and table surface samples in meat shops at the retail markets in North Vietnam showed Salmonella positive. Eleven Salmonella serovars, including Infantis, Anatum, Rissen, Reading, London, Typhimurium, Enteritidis, Agona, Newport, Emek, and Derby, were identified. The Salmonella isolates were tested for antimicrobial susceptibility and further investigated for antimicrobial resistance genes. Resistance to kanamycin, gentamicin, neomycin, nalidixic acid, chloramphenicol, trimethoprim, streptomycin, tetracycline, ampicillin, and sulphonamides was found in 28.9-56.7%. The isolates were neither resistant to ceftazidime nor norfloxacin. Sixty-four (66.0%) out of 97 isolates were resistant to at least one of 14 antimicrobials, and 55 (85.9%) out of the 64 isolates showed multidrug resistance. Thirteen resistance genes (bla(TEM), bla(OXA-1), aadA1, sul1, tetA, tetB, tetG, cmlA1, floR, dfrA1, dfrA12, aac(3)-IV, and aphA1-1AB) were detected in the resistant isolates. This study indicates that Salmonella isolated from meat shops were resistant to multiple antimicrobials, and the resistance genes were widespread among the serovars isolated.

Adenomyomatous uterine polyp in a Miniature Pinscher: histologic, immunohistochemical, and clinical aspects.

A 14-y-old Miniature Pinscher bitch was admitted to a veterinary clinic because of inappetence and a distended abdomen; ultrasound examination revealed a fluid-filled uterus with a single 1-cm hyperechoic nodule in its lumen. Ovariohysterectomy was performed. Grossly, the uterine horns were distended irregularly and asymmetrically, and the uterine lumen contained 200-300 mL of brown watery fluid. A single white polypoid 0.9-cm diameter nodule was present at the site of the distended uterine horn and arose from the endometrium with a narrow stalk. Histologically, the polyp consisted of dense, smooth muscle fascicles admixed with glandular components; its surface was covered by simple cuboidal epithelium with areas of squamous metaplasia. The myomatous cells expressed the myogenic markers smooth muscle actin and desmin. We interpreted the mass as an adenomyomatous uterine polyp, which is a rare variant of an endometrial polyp.

Dural Changes Induced by an Ultrasonic Bone Curette in an Excised Porcine Spinal Cord.

In spinal surgery, ultrasonic bone curettes are considered unlikely to cause mechanical injury to the dura; however, there is little evidence to support this claim. We investigated the effect of direct contact with an ultrasonic bone curette on the dura and the protective effect of covering the dura with a cotton pattie using an excised porcine spinal cord. The ultrasonic bone curette was pressed against the porcine spinal cord with constant force and activated for 1 s, with or without covering the dura with a cotton pattie. The dural surface and cross-section were observed using electron and light microscopy. When the ultrasonic bone curette was applied directly against the dura, most specimens showed non-perforating dural injuries. However, none of the specimens showed dural perforation. Histological changes were also observed. The use of a cotton pattie reduced the occurrence of these changes, although it did not prevent them when ultrasonic vibration was applied with a large force. We considered ultrasonic bone curettes to have a low risk of dural perforation and, thus, to be a safe surgical device as long as they did not accidentally make strong contact with the dura.