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A feline spinal dermoid cyst treated with surgical intervention
Get PDFA 5-year-old neutered female mixed cat presented with reduced activity and ataxia of the hind limbs. Computed tomography and magnetic resonance imaging revealed an extradural mass compressing the spinal cord on the dorsal aspects from the 7th to 8th thoracic vertebra. Dorsal laminectomy was performed on the 7-8th thoracic vertebra and the cyst was totally removed, giving full resolution of the clinical signs. The cyst was diagnosed as a dermoid cyst. To our knowledge, this is the first report of feline dermoid cyst compressing the spinal cord that was diagnosed antemortem. The prognosis is favorable when the cyst is completely resected.journal articl
Usefulness of combining computed tomography and air sac fluid examination to rule out aspergillosis: case study in two gentoo penguins (Pygoscelis papua) with respiratory clinical signs
Get PDFPulmonary aspergillosis occurring in captive penguins living in zoos and aquariums is a fatal disease owing to its high mortality rate. On conducting computed tomography, two gentoo penguins (Pygoscelis papua) with respiratory clinical signs that were housed at an aquarium in Hokkaido, Japan were found to have a certain amount of fluid in their air sacs. This fluid was collected and cultured for bacteria and fungi, the results of which both came back negative. These results enabled us to rule out bacterial infection or aspergillosis and supported our decision not to administer antibacterial and antifungal drugs. Overall, the combination of computed tomography and air sac fluid examination was useful for ruling out bacterial infection or aspergillosis in penguins with respiratory clinical signs.journal articl
Molecular form identification of anterior pituitary gland-secreted prolactin in chicken
No full textEndocrine changes during bird reproduction are well documented. Prolactin (PRL) exhibits a strong relationship between incubation and broody behavior. The molecular forms of PRL in the anterior pituitary gland during the reproductive cycle have already been previously identified but not those in the secreted form. To identify the molecular forms of secreted PRL during the reproductive cycle, we thus monitored the physiological status and incubation behavior of 10 Silkie hens by a video recording system over 1–2 years. Nine out of ten mature hens exhibited incubation behavior multiple times during the experiment. Ten hens demonstrated two interesting features. In a typical clutch, hens spent 10–15 min in the nest to lay an egg. Once they spent over 1 h in the nest, the nest occupancy increased incrementally. This shift in the nest occupancy occurred 7–10 days before the incubation onset and was highly repeatable. Based on the behavior of the hens, we cultured the anterior pituitary gland during four stages (premature non-laying, laying, trans, and incubation) with physiological PRL-releasing factor, vasoactive intestinal peptide (VIP). Based on our two-dimensional protein analysis, glycosylated PRL (G-PRL) displayed several isoforms with varying isoelectric points (pI), whereas we could detect one primary signal for non-glycosylated PRL (NG-PRL). However, 3–4 NG-PRL isoforms were detected in the anterior pituitary gland. These results suggested that secreted PRL, especially from the trans and incubation stages, contains various isoforms and it is post-translationally glycosylated and phosphorylated.journal articl
Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma
Get PDFOral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.journal articl
Molecular Mechanism of Oocyte Activation in Mammals: Past, Present, and Future Directions
Get PDFDuring mammalian fertilization, repetitive intracellular Ca2+ increases known as Ca2+ oscillations occur. These oscillations are considered crucial for successful fertilization and subsequent embryonic development. Numerous researchers have endeavored to elucidate the factors responsible for inducing Ca2+ oscillations across various mammalian species. Notably, sperm-specific phospholipase C zeta (PLCζ) emerged as a prominent candidate capable of initiating Ca2+ oscillations, particularly in mammals. Genetic mutation of PLCζ in humans results in the absence of Ca2+ oscillations in mouse oocytes. Recent studies further underscored PLCζ’s significance, revealing that sperm from PLCζ-deficient (Plcz1−/−) mice fail to induce Ca2+ oscillations upon intracytoplasmic sperm injection (ICSI). Despite these findings, observations from in vitro fertilization (IVF) experiments using Plcz1−/− sperm revealed some residual intracellular Ca2+ increases and successful oocyte activation, hinting at potential alternative mechanisms. In this review, we introduced the current hypothesis surrounding oocyte activation in mammals, informed by contemporary literature, and probed into the enigmatic mechanisms underlying mammalian fertilization-induced oocyte activation.journal articl
Developmen Validation of a Novel Four Gene-Pairs Signature for Predicting Prognosis in DLBCL Patients
Get PDFDiffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma. Because individual clinical outcomes of DLBCL in response to standard therapy differ widely, new treatment strategies are being investigated to improve therapeutic efficacy. In this study, we identified a novel signature for stratification of DLBCL useful for prognosis prediction and treatment selection. First, 408 prognostic gene sets were selected from approximately 2500 DLBCL samples in public databases, from which four gene-pair signatures consisting of seven prognostic genes were identified by Cox regression analysis. Then, the risk score was calculated based on these gene-pairs and we validated the risk score as a prognostic predictor for DLBCL patient outcomes. This risk score demonstrated independent predictive performance even when combined with other clinical parameters and molecular subtypes. Evaluating external DLBCL cohorts, we demonstrated that the risk-scoring model based the four gene-pair signatures leads to stable predictive performance, compared with nine existing predictive models. Finally, high-risk DLBCL showed high resistance to DNA damage caused by anticancer drugs, suggesting that this characteristic is responsible for the unfavorable prognosis of high-risk DLBCL patients. These results provide a novel index for classifying the biological characteristics of DLBCL and clearly indicate the importance of genetic analyses in the treatment of DLBCLjournal articl
Rapid formation of picture-word association in cats
Get PDFIt is well known that dogs are capable of following human verbal instructions. However, very little is known about the equivalent ability in cats. In this study, we used a switched stimuli task to examine whether cats rapidly form picture-word association, which is a fundamental ability for word learning. We presented cats with two meaningless picture-word combinations, in the habituation phase. Then, on half of the trials we switched the combination (switched condition), but the other half of the trials remained as before (non-switched condition). If cats rapidly form picture-word association, they were expected to look at the monitor for longer in the switched condition, reflecting detection of the change. We used human speech as stimuli in Exp.1, and mechanical sounds (electronic sounds) in Exp.2. Cats expressed detection of the switched combination in Exp.1, where human speech and objects were paired. However, in Exp.2 where non-social sounds and objects were paired, there was no statistical difference between switched and non-switched conditions, although there was a main effect of condition when the data from the two experiments were pooled. These results demonstrate that cats can rapidly form picture-word association. Further research should investigate whether domestication has played a role in this ability.journal articl
ブタおよびイノシシからの哺乳類オルソレオウイルスの浸潤状況および遺伝子多様性に関する研究
Get PDF麻布大学博士(獣医学)Mammalian orthoreoviruses (MRVs) are non-enveloped double-stranded RNA viruses with a broad host range. MRVs are prevalent worldwide, and in Japan, they have been isolated from various hosts, including humans, dogs, cats, wild boars, and pigs, and they have also been found in sewage. However, information on the status of MRV prevalence and genomic characterization in pigs and wild boars in Japan is lacking. Therefore, in this study, prevalence and genetic characterization of MRVs were investigated using fecal samples of porcine and wild boar collected for a surveillance of enteric viruses in Japan. Virus isolation and next generation sequencing were carried out on 421 fecal samples from pigs and 216 rectal contents from wild boars collected in 2017-2022 in Japan. MRVs were isolated from five (1.2%) of the samples from pigs and one wild boar sample found to be positive for MRV. Genetic analysis of the S1 gene of these strains revealed that the Japanese porcine MRV isolates could be classified as MRV-2 and MRV-3. Whole genome analysis showed that Japanese porcine MRVs exhibited genetic diversity, although they shared sequence similarity with porcine MRV and MRV prototype human strains isolated in the 1950s sequences in the DDBJ/EMBL/GenBank database. Several potential intragenetic reassortment events were detected among MRV strains from pigs, sewage, and humans in Japan, suggesting zoonotic transmission. Furthermore, homologous recombination events were identified in the M1 and S1 genes of Japanese porcine MRV. Structural modeling of the S1 protein showed structural differences of S1 between the strains, suggesting diversity in antigenic properties of MRVs. These findings imply that different strains of Japanese porcine MRV share a porcine MRV genomic backbone and have evolved through intragenetic reassortment and homologous recombination events. The results of this study shed light on the genetic diversity, plasticity and evolution of porcine MRV and prevention of MRV infection not only in porcine but also in human.哺乳類オルソレオウイルス（Mammalian orthoreoviruses： MRVs）は世界中に分布し、ヒトやブタおよびイノシシを含め、コウモリ、ウシ、ウマ、イヌ、ネコといったあらゆる種類の哺乳類に感染することが知られている。ヒトにおいてはMRVに感染しても一般的に症状は認められないと考えられていたが、呼吸器疾患や消化器疾患を伴う症例が散発的に報告され、さらに神経疾患や急性呼吸器疾患など、より重篤な症状にMRVが関与することが報告されるようになった。ブタにおいてもMRV感染はほとんどの場合、症状を示さないと考えられていたが、中国、韓国、米国およびイタリアにおいて、MRVの単独感染または他の病原体との同時感染による重篤な下痢症例が報告されている。
MRVはレオウイルス目スピナレオウイルス科オルソレオウイルス属の中の種として分類され、エンベロープを有さない2層構造を持つカプシドから構成される正20面体の構造を示し、10本の分節型2本鎖RNAをゲノムとして保有する。10本の遺伝子分節は、8つの構造タンパク質であるλ1（L3遺伝子）、λ2（L2遺伝子）、λ3（L1遺伝子）、μ1（M2遺伝子）、μ2（M1遺伝子）、σ1（S1遺伝子）、σ2（S2遺伝子）およびσ3（S4遺伝子）、および4つの非構造タンパク質であるμNS（M3遺伝子）、μNSC（M3遺伝子）、σNS（S3遺伝子）およびσ1s（S1遺伝子）をコードしている。μ1、σ3およびσ1は外殻カプシドタンパク質を構成し、このうちσ1は中和抗体によって認識されるオルソレオウイルスの血清型特異的抗原である。σ1タンパク質の抗原性状とS1遺伝子の遺伝的近縁性に基づいて、MRVはLang（T1L）、Jones（T2J）、Dearing（T3D）、Abney（T3A）およびNdelle（T4N）をプロトタイプウイルス株とするMRV-1（T1）、MRV-2（T2）、MRV-3（T3）およびMRV-4（T4）の血清型に分類されている。MRVは分節型の遺伝子を保有しているため、異なる血清型間であっても1個体の宿主に2株以上のMRVが同時に感染する、重複感染を起こしたに容易に遺伝子再集合が発生し、遺伝子再集合体が生成される。配列の多様性と有利な遺伝的進化を得た遺伝子再集合体は、病原性を増大し、宿主域を拡大すると考えられている。
MRVは多くの宿主に感染して人獣共通感染症を引き起こし、我が国においてもヒトにおける症例が報告されているものの、我が国のブタにおけるMRV感染は呼吸器疾患のある個体の呼吸器からMRV-1が、下痢症状のある個体とない個体の糞便からMRV-2が分離された報告がそれぞれ2件あるのみで、我が国のブタやイノシシにおけるMRVの分布状況は不明である。また、上記の報告にはMRV遺伝子の情報はなく、我が国のブタおよびイノシシにおけるMRVの分布実態およびMRVの遺伝子情報の収集は急務であった。そこで本研究では我が国に飼養されているブタおよび分布しているイノシシの糞便からMRVを検出し、詳細な遺伝子解析を実施した。
【第１章】ブタおよびイノシシの糞便からの哺乳類オルソレオウイルスの検出
腸管系ウイルス調査のため2020～2022年に採取された、北海道、本州および九州で飼養されているブタ349頭および北陸地方に生息するイノシシ126頭の糞便乳剤の遠心上清から株化細胞であるMA104細胞を用いてウイルス分離を行ったところ、ブタの糞便材料を接種したMA104細胞の349検体中47検体に細胞変性効果（CPE）が観察された。この47検体の培養上清からRNAを抽出し、ライブラリーを作製して次世代シークエンス（NGS）を実施したところ、20検体からA群ロタウイルス、13検体からエンテロウイルスGおよび10検体からサペロウイルスの遺伝子が検出され、残りの4検体からはMRVのほぼ完全長の配列が得られた。同じ糞便遠心上清から直接RNAを抽出し、同様な方法でライブラリーを作製してNGSを実施したところ、MRVが分離された4検体を含めていずれの検体からもMRVが検出されなかった。MRVが検出された4検体については、サペロウイルスの全遺伝子配列およびC群ロタウイルス、アストロウイルス、エンテロウイルスG、テシオウイルスの部分配列が確認されたが、MRV遺伝子は見つからなかった。
腸管系ウイルスの場合、糞便材料から直接RNAを抽出したメタゲノムアプローチによるNGSが行われるが、腸内に存在する細菌のリボゾーマルRNAが障害となりウイルス遺伝子が検出されにくいことがある。今回、糞便から直接抽出したRNAからのNGSではMRVは検出されなかったことから、MRVの検査においてはMA104細胞を用いたウイルス分離の方がNGSよりも感度が高いことが示された。今回のMRV検出率はブタが1.1% （4頭/349頭）であり、イノシシの126検体からは検出されなかった。MRVが検出されたブタはすべて健康個体であったが、下痢症状を呈する検体は15検体と少なかったことから、下痢症との関連を調べるためには下痢検体をさらに増やして検討する必要があると思われた。
【第２章】分子系統樹解析および遺伝子相同性解析による哺乳類オルソレオウイルスの疫学的分析
第1章で得られた4株に、2017年から2019年にブタおよびイノシシの糞便についてNGSを実施し、MRVが陽性となったブタおよびイノシシそれぞれ1検体から分離した2株のMRVを加え、DDBJ/EMBL/GenBankデータベースに登録されている各種動物由来MRVとともに全遺伝子分節の系統樹解析および塩基配列の相同性解析を実施した。その結果、S1遺伝子塩基配列を用いた系統解析で我が国のブタおよびイノシシ由来MRVはMRV-2（豚由来2株）およびMRV-3（豚由来2株およびイノシシ由来1株）に分類された。MRV-1およびMRV-2は我が国のブタからの分離がすでに報告されているが、MRV-3は我が国では犬から報告があるのみで、我が国のブタおよびイノシシにMRV-3が感染していることは本研究で初めて明らかになった。その他の分節の系統樹解析から、我が国のブタおよびイノシシ由来のMRVは国外のブタ由来MRVおよびヒト由来のMRVプロトタイプ株と相同性の高い分節を共有していることが明らかとなった。配列の比較からも、我が国のブタおよびイノシシ由来MRVは世界的に分布しているブタ由来MRVの遺伝子分節をバックボーンに持つことが明らかとなり、これを基本にヒト由来のプロトタイプMRVから同義置換により派生した可能性が考えられた。さらに我が国のブタおよびイノシシ由来MRVのいくつかの遺伝子分節は、我が国のヒトあるいは下水由来MRV遺伝子分節と相同性が確認されたことから、ヒト由来MRVとの遺伝子再集合の結果作出された可能性が示唆された。
【第3章】遺伝子再集合、遺伝子相同組換えおよびS1蛋白の構造予測による哺乳類オルソレオウイルス遺伝子の進化の分析
RNAウイルスは遺伝子再集合および遺伝子相同組換えにより変異するため、我が国のブタおよびイノシシ由来のMRVの起源や進化を考察する上で、これらを解析することは重要と考えられる。我が国のイノシシ由来MRV株WB/To14株は、L2、L3およびM3遺伝子は我が国の動物園で飼育されていたライオン由来のMRVと高い相同性を示した。動物園のライオンとイノシシの直接の接点がないことから、ライオンは餌を介してイノシシ由来MRVと類似性を有するMRVに感染した可能性が考えられる。S2遺伝子に関しては大阪で2014年にヒトから分離されたMRV株との相関性が高く、S1遺伝子は免疫誘導を起こすことから変異の激しい遺伝子分節であるが、WB/To14株は2021年に関東地方でブタから分離されたMRVに高い相同性を保有していた。これらのことはブタとイノシシ間にMRVのやり取りがあることを示しており、イノシシはヒトやブタのMRV感染の感染源になる可能性があることが示された。また、遺伝子相同組換え解析において、DDBJ/EMBL/GenBankデータベースから得た配列データを用いた組換え解析では、ブタ由来のMRVを含むL3遺伝子およびM2遺伝子に確からしさが強く支持される遺伝子組換えが存在することが示された。以上のことから我が国のブタおよびイノシシ由来MRVは、我が国に分布する他の宿主由来のMRVと遺伝子再集合および遺伝子相同組換えを起こし、進化を続けていることが示された。
またAlphaFold2を利用したMRVのS1蛋白質の立体構造予測の結果から、遺伝子型間にアミノ酸配列の相同性は50%未満と低く、C端側においては構造の相違が認められたが、N端側の棒状の構造は遺伝子型に関わらず共通して確認されたことから、MRVは抗原性状に関わると考えられるC端側は変異するがC端側の構造を保ちつつ進化しているものと推察された。
【総括】
MRVは我が国のブタおよびイノシシに感染しており、国内の他の宿主由来のMRVと遺伝子分節をやり取りし、さらには遺伝子組換えを起こしてブタおよびイノシシ群中で進化を続けていることが明らかとなった。イノシシにおいては、上記のメカニズムにより進化したMRVをヒトおよびブタをはじめとした家畜に伝播する感染源になりうることが示唆された。本研究で得られた知見がMRVの疫学解明の一助となり、MRV感染症の診断や予防の一助となることを期待したい。doctoral thesi
Effect of feeding spent coffee grounds on the methane production in bovine rumen
Get PDFThe environmental impact of methane, a greenhouse gas emitted from ruminants, is a pressing issue and methods to control methane emissions from ruminants are being investigated worldwide. In this study, we investigated the effects of the administration of spent coffee grounds
(SCG) on methane production in the rumen in two cows. In the control condition (days 1 and 2), the cows were fed a basic diet twice daily (roughage and concentrate), and in the SCG condition (days 1 and 2) sequentially, the cows were fed the same basic diet and administered SCG into the rumen twice daily. The methane and carbon dioxide concentrations in rumen gas were measured via a fistula after feeding on days 2 in both cases of the study. The measurements were made using a newly developed gas measurement system with a portable gas monitor, and data were obtained for the control condition and SCG condition at each measurement time. The methane ratio at each measurement time was calculated from the methane and carbon dioxide concentrations, and compared between the two conditions. Statistical analysis showed no significant difference between the two conditions in the methane ratios after the morning (P=0.108) and afternoon feedings (P=0.345). However, the methane ratios before the morning (P=0.043) and afternoon feedings (P=0.008) were significantly lower in the SCG condition than in the control condition, suggesting that the administration of SCG may suppress methane production in the rumen.journal articl
Availability and issues of 3D-printed skull models for veterinary anatomy laboratories from students’ perspective before and during the COVID-19 pandemic
Get PDFThree-dimensional (3D)-printed models of bones are a convenient and durable alternative to real bone specimens, and they have been used in anatomy laboratories. It is necessary to identify the precise advantages of 3D-printed models from all perspectives; not only the improvement in students' knowledge of anatomy but also the students' assessment of such models. Here, students of veterinary medicine and animal science evaluated the reproducibility and effectiveness of 3D-printed models as a learning tool by completing our questionnaires, with a focus on their understanding of the skull-morphological differences among dog breeds. With the COVID-19 pandemic having obliged veterinary universities to provide courses online, we also investigated how the pandemic affected the students' evaluation of the 3D-printed models. The questionnaire results revealed that the animal science students were satisfied with the reproducibility of the 3D-printed models, but the veterinary students were not (they preferred to use real specimens). The skull differences were well understood by both types of students, indicating that 3D-printed models are effective for learning about rare skeletal specimens. The veterinary students who experienced the COVID-19 pandemic tended to choose real specimens more often than those who did not have this experience. Our results suggest that the use of 3D-printed models as an introduction and the use of real specimens in anatomy laboratory courses can be adequate for veterinary students. Together our findings suggest ways to improve the educational performance of 3D-printed models for veterinary students who need to understand the anatomy of many species.journal articl