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Virulence factor genes analysis of extended spectrum β-Lactamase producing Escherichia coli isolated from broiler chickens in Indonesia. 印尼肉鸡产β-内酰胺酶大肠杆菌扩展谱毒力因子基因分析。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-03 Epub Date: 2025-10-28 DOI: 10.1292/jvms.25-0330
Nofita Nurbiyanti, Chaerul Basri, Puji Rahayu, Imron Suandy, Hadri Latif

Extended spectrum β-lactamase producing Escherichia coli (ESBL-Ec) is recognized as an opportunistic pathogen with zoonotic potential, posing a threat to public health. This study aimed to identify virulence factor genes (VFGs), determine ESBL-Ec pathotypes and pathovars, and analyze their distribution in isolates from broiler cecum across eight Disease Investigation Center (DIC) areas in Indonesia. A total of 120 ESBL-Ec isolates were proportionally selected based on regional prevalence. The VFGs were detected using Oxford Nanopore Technologies (ONT) MinION sequencing and analyzed with bioinformatics pipelines. Of the total isolates, 113 met the quality threshold. A total of 4,269 non-redundant orthologs and alleles related to VFGs were detected, representing 99 VFG types grouped into 25 clusters, indicating substantial genetic diversity. The fim gene cluster was the most frequently detected (20.1%), whereas the esp cluster exhibited the highest allele diversity (18.2%). The VFGs encoding the ExPEC were predominant (66.7%), while the remaining VFGs encoded the InPEC (33.3%). Each isolate potentially encodes at least two pathovars, with nearly 80% encoding three or more. The diversity of VFGs and pathovar combinations highlights the disease potential and zoonotic risk of ESBL-Ec.

产广谱β-内酰胺酶大肠杆菌(esblc - ec)被认为是一种具有人畜共患潜力的机会性病原体,对公众健康构成威胁。本研究旨在鉴定毒力因子基因(vfg),确定ESBL-Ec的致病型和致病型,并分析其在印度尼西亚8个疾病调查中心(DIC)地区肉鸡盲肠分离株中的分布。根据地区流行情况按比例选择120株ESBL-Ec分离株。利用Oxford Nanopore Technologies (ONT) MinION测序技术检测vfg,并通过生物信息学管道进行分析。其中113株符合质量标准。共检测到4269个与VFG相关的非冗余同源物和等位基因,代表99种VFG类型,分为25个簇,表明具有丰富的遗传多样性。其中,film基因簇的等位基因多样性最高(20.1%),esp基因簇的等位基因多样性最高(18.2%)。编码expc的vfg占多数(66.7%),其余编码InPEC的vfg占33.3%。每个分离株可能编码至少两种病原体,其中近80%编码三种或更多。vfg和病原体组合的多样性突出了ESBL-Ec的疾病潜力和人畜共患风险。
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引用次数: 0
Forensic detection and quantification of methomyl in animal stomach contents using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) and liquid Chromatography tandem mass spectrometry (LC-MS/MS) in Japan. 日本采用液相色谱四极杆飞行时间质谱法(LC-Q-TOF/MS)和液相色谱串联质谱法(LC-MS/MS)对动物胃内容物中的灭多威进行法医检测和定量。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-03 Epub Date: 2025-10-29 DOI: 10.1292/jvms.25-0287
Yared Beyene Yohannes, Aki Tanaka, Yuko Kihara, Tsuyoshi Tajima, Shouta Nakayama, Collins Nimako, Yoshinori Ikenaka, Mayumi Ishizuka

Pesticide poisoning in animals is a rising forensic issue due to widespread chemical use. This study analyzed stomach contents from 44 deceased animals (domestic and wild) in Japan using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) for non-targeted screening and liquid Chromatography tandem mass spectrometry (LC-MS/MS) for quantification after Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction. Non-target screening detected multiple pesticide classes, with carbamates and herbicides being the most prevalent. Methomyl was identified in 29 cases (65.9%), with concentrations up to 157 mg/kg in cats, 75 mg/kg in a civet, and 30 mg/kg in a dog, exceeding the oral LD50 of methomyl in dogs (10 mg/kg) and cats (estimated to be similar). These findings suggest acute poisoning and possible intentional misuse. The toxicological significance of methomyl highlights the need for stricter regulations, better monitoring, and increased public awareness to prevent further animal poisonings.

由于化学品的广泛使用,动物农药中毒是一个日益严重的法医问题。本研究采用液相色谱四极杆飞行时间质谱法(LC-Q-TOF/MS)进行非靶向筛选,液相色谱串联质谱法(LC-MS/MS)进行快速、简单、廉价、有效、坚固、安全(QuEChERS)提取,分析了日本44只死亡动物的胃内容物。非靶标筛选检测到多种农药,其中氨基甲酸酯类和除草剂最为普遍。在29例(65.9%)病例中鉴定出灭多威,猫的浓度高达157 mg/kg,果子狸的浓度高达75 mg/kg,狗的浓度高达30 mg/kg,超过了灭多威在狗(10 mg/kg)和猫(估计相似)中的口服LD₅0。这些发现提示急性中毒和可能的故意误用。灭多威的毒理学意义突出表明,需要更严格的法规、更好的监测和提高公众意识,以防止进一步的动物中毒。
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引用次数: 0
Timing of human chorionic gonadotropin administration affects conception rates after embryo transfer in crossbred beef heifers. 人绒毛膜促性腺激素给药时间影响杂交肉牛胚胎移植后受孕率。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-09 DOI: 10.1292/jvms.25-0224
Ken Hazano, Akira Goto, Shingo Haneda, Motozumi Matsui

This study aimed to determine the effect of the timing of human chorionic gonadotropin (hCG) administration to embryo transfer (ET) recipients on the reproductive outcome of ET in crossbred beef heifers. Heifers (n=1,887) were randomly assigned to the non-treatment group (n=1,160), and hCG was administered on day 5 (n=201), 6 (n=210), 7 (n=213), or 8 (n=103) after estrus. The χ2 test showed that the conception rates in the hCG administration on day 5 (79.6%, P=0.0001) and day 6 (75.2%, P=0.0047) were higher than those in the non-treatment group (65.2%). However, the conception rates with hCG administration on day 7 (70.9%, P=0.1103) and day 8 (70.9%, P=0.2499) were comparable to the non-treatment. A multiple logistic regression analysis, furthermore, showed that early treatment (hCG on day 5 or 6 after estrus) positively affected the conception rates compared to non-treatment (odds ratios (OR)=1.723 [95% confidence intervals (CI)=1.316-2.257]), but not late treatment (hCG on day 7 or 8 after estrus) (OR=1.299 [95% CI=0.981-1.719]). None of the confounding variables, including years, season at the time of ET, age in months, and number of ET, significantly affected the conception rate. In conclusion, hCG administration on day 5 or 6 after estrus for ET recipients improved the conception rate, but not on day 7 or 8, in beef heifers.

本研究旨在确定人绒毛膜促性腺激素(hCG)给药时间对杂交肉牛胚胎移植(ET)生殖结果的影响。将1887头母牛随机分为未处理组(1160头),分别于发情后第5天(201头)、第6天(210头)、第7天(213头)、第8天(103头)给予hCG。卡方检验显示,hCG组第5天受孕率(79.6%,P=0.0001)和第6天受孕率(75.2%,P=0.0047)均高于未治疗组(65.2%)。然而,在第7天和第8天给予hCG的受孕率(70.9%,P=0.1103)与未给予治疗的受孕率(70.9%,P=0.2499)相当。此外,多元logistic回归分析显示,与未治疗相比,早期治疗(发情后第5天或第6天的hCG)对受孕率有积极影响(比值比(or) = 1.723[95%可信区间(CI) = 1.315 -2.257]),但对晚期治疗(发情后第7天或第8天的hCG)没有积极影响(or = 1.299 [95% CI = 0.981-1.719])。包括年、ET时的季节、月龄和ET次数在内的混杂变量均未显著影响受孕率。综上所述,雌激素受体在发情后第5天或第6天给予hCG可提高肉牛受孕率,但在第7天或第8天则没有。
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引用次数: 0
Evaluation of two human diagnostic agarose gel electrophoresis kits for bovine serum protein fractionation. 两种用于牛血清蛋白分离的琼脂糖凝胶电泳试剂盒的评价。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-09 DOI: 10.1292/jvms.25-0272
Norio Yamagishi, Khaing Shwe Sin Soe, Hijiri Suyama, Akari Kawamura, Shinobu Tsuchiaka, Kouki Itagaki, Sueun Kim, Shingo Ishikawa

We evaluated two human diagnostic agarose gel electrophoresis (AGE) kits (J711 and J718) for bovine serum protein fractionation using 79 bovine samples, comparing the results with a biochemistry analyzer (BA). While six protein fractions were commonly observed, β2- and γ-globulin (Glb) fusion (β2γ-Glb) occurred in some inflammatory and neonatal cases. The results using both AGE kits correlated with those of the BA for total albumin and Glb, with J711 showing superior agreement. However, inter-kit variability was observed for individual protein fractions, particularly α1-, α2-, and β1-Glb, which showed proportional or constant errors and moderate-to-poor reliability, despite good agreement for β2γ-Glb. These findings necessitate caution in interpreting fraction-specific results and underscore the importance of stating the AGE kit in veterinary diagnostics.

我们对两种人类诊断琼脂糖凝胶电泳(AGE)试剂盒(J711和J718)对79份牛血清蛋白的分离进行了评估,并将结果与生物化学分析仪(BA)进行了比较。虽然通常观察到6个蛋白部分,但在一些炎症和新生儿病例中出现β2-和γ-球蛋白(Glb)融合(β2γ-Glb)。两种AGE试剂盒的检测结果均与BA对总白蛋白和Glb的检测结果相关,其中J711表现出较好的一致性。然而,对于单个蛋白质组分,特别是α1-, α2-和β1-Glb,观察到试剂盒间的差异,尽管β2γ-Glb的一致性很好,但它们显示出比例或恒定的误差和中等到较差的可靠性。这些发现在解释部分特异性结果时需要谨慎,并强调了在兽医诊断中说明AGE试剂盒的重要性。
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引用次数: 0
Impact of different culture media and drying treatment on the isolation of Melissococcus plutonius, the causative agent of European foulbrood, from honey bee larval specimens. 不同培养基和干燥处理对蜜蜂幼虫标本中欧洲臭蝇病原菌——深蜂美利索球菌分离的影响。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-02 DOI: 10.1292/jvms.25-0347
Yuko Kitamura, Yusuke Kusajima, Yuka Sekiya, Reiko Kawaguchi, Yuka Hayashi, Hisako Kakinuma, Mariko Okamoto, Daisuke Takamatsu

Melissococcus plutonius, the bacterium causing European foulbrood in honey bees, includes two distinct types: typical strains, which are fastidious and not easy to culture, and atypical strains, which grow more robustly and tolerate higher sodium levels. Several culture media have been used to isolate this pathogen, and growth dynamics and survival vary according to strain type and medium. The most commonly used media differs across countries, as does the ratio of typical to atypical strains isolated, implying that the choice of medium influences the type of M. plutonius that is more likely to be recovered. This study examined how two commonly used media, Basal and KSBHI, affect the isolation of typical and atypical strains from larval samples co-infected with both types. Both media supported the growth of both strains, and no isolation preference was observed, although KSBHI tended to yield more non-M. plutonius bacteria. Applying a drying treatment to larval smears reduced these secondary invaders but it shifted the isolation balance toward atypical strains. Drying resistance tests revealed that atypical strains survived longer under dry conditions than typical strains, which likely explains their increased recovery after drying. These results indicate that while both media can isolate either strain type, using both media in parallel is better for ensuring isolation of both types. Additionally, although drying reduces secondary invaders, it may bias the results toward atypical strains. These insights contribute to a more accurate diagnosis and epidemiological understanding of European foulbrood.

在蜜蜂中引起欧洲恶臭的美利索球菌(Melissococcus plutonius)包括两种截然不同的类型:典型菌株,它们挑剔且不易培养;非典型菌株,它们生长得更强壮,能耐受更高的钠含量。已有几种培养基用于分离该病原体,根据菌株类型和培养基的不同,其生长动态和存活率各不相同。各国最常用的培养基各不相同,分离出的典型菌株与非典型菌株的比例也各不相同,这意味着培养基的选择会影响更有可能被恢复的普鲁托尼分枝杆菌的类型。本研究考察了两种常用培养基,基底和KSBHI,如何影响从两种类型共感染的幼虫样本中分离典型和非典型菌株。两种培养基都支持两种菌株的生长,并且没有观察到分离偏好,尽管KSBHI倾向于产生更多的非m。plutonius细菌。对幼虫涂片进行干燥处理减少了这些次生入侵者,但使分离平衡向非典型菌株转移。抗干燥性试验表明,非典型菌株在干燥条件下比典型菌株存活的时间更长,这可能解释了它们在干燥后的恢复速度增加。这些结果表明,虽然两种介质可以分离任何一种菌株,但同时使用两种介质可以更好地确保两种菌株的分离。此外,尽管干燥减少了次生入侵者,但它可能使结果偏向非典型菌株。这些见解有助于更准确的诊断和流行病学的理解欧洲污秽。
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引用次数: 0
Evaluation of oxygen reserve index as an early warning indicator of hypoxemia in anesthetized dogs. 氧储备指数作为麻醉犬低氧血症预警指标的评价。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-24 DOI: 10.1292/jvms.25-0197
Taku Hirokawa, Takaharu Itami, Keiko Kato, Yujiro Minamoto, I-Ying Chen, Chihiro Sugita, Yusuke Endo, Taku Miyasho, Kazuto Yamashita

The oxygen reserve index (ORi) is a non-invasive parameter recently introduced in human medicine that detects impending hypoxemia earlier than peripheral oxygen saturation (SpO2). This study evaluated the utility of ORi as an early warning indicator of hypoxemia in anesthetized dogs. Six beagle dogs were subjected to planned apnea under two oxygen conditions (FiO2 1.0 and 0.4). Changes in ORi, SpO2, and arterial oxygen partial pressure (PaO2) were monitored. Warning times (WT) were calculated as the time difference between each timepoint and SpO2 reaching 90%. We defined Added warning times (AWT) as the time between an ORi warning point and the moment SpO2 fell to 96%. When ORi decreased to half of its baseline value or reached 0.4, it provided early warnings of 43.2 ± 24.6 sec and 44.7 ± 37.0 sec, under FiO2 1.0, and 29.5 ± 21.9 sec and 37.0 ± 22.5 sec under FiO2 0.4, before SpO2 began to decrease. A significant positive correlation was observed between ORi and PaO2 when SpO2 was ≥97% (r=0.74, P<0.0001). Receiver operating characteristic (ROC) analysis showed high diagnostic accuracy of ORi for predicting PaO2 ≥150 mmHg (area under the curve [AUC]=0.92), with an optimal cutoff value of 0.48 (sensitivity 92%, specificity 78.8%). Unlike in humans, ORi continued to change at PaO2 levels above 200 mmHg in dogs, suggesting species-specific differences. In conclusion, ORi can detect deterioration in oxygenation earlier than SpO2 in anesthetized dogs and provides valuable non-invasive real-time monitoring of oxygen reserves during controlled apnea under anesthesia.

氧储备指数(ORi)是最近在人体医学中引入的一种非侵入性参数,它可以比周围氧饱和度(SpO₂)更早地检测出即将发生的低氧血症。本研究评估了ORi作为麻醉犬低氧血症早期预警指标的效用。6只beagle犬在两种氧气条件下(FiO₂1.0和0.4)进行计划呼吸暂停。监测ORi、SpO₂、动脉氧分压(PaO₂)的变化。预警次数(WT)是根据每个时间点与SpO₂达到90%的时间差来计算的。我们将添加警告时间(AWT)定义为从ORi警戒点到SpO₂下降到96%的时刻之间的时间。当ORi下降到基准值的一半或达到0.4时,在FiO₂1.0下,预警时间分别为43.2±24.6秒和44.7±37.0秒,FiO₂0.4下,预警时间分别为29.5±21.9秒和37.0±22.5秒,然后SpO₂开始下降。当SpO 2≥97%时,ORi与PaO 2呈显著正相关(r=0.74, P
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引用次数: 0
Systemic amyloid A amyloidosis in a captive Cape hyrax (Procavia capensis). 系统性淀粉样蛋白圈养海岬兔(Procavia capensis)的一种淀粉样变性。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-15 DOI: 10.1292/jvms.25-0316
Aya Terao, Susumu Iwaide, Natsumi Kobayashi, Tomoaki Murakami, Yoshiyuki Itoh, Masakazu Nakada, Tomoyuki Shibahara

Amyloidosis is a disease caused by amyloid deposition and has been reported in various animal species; however, reports on Afrotherian species, including Cape hyraxes, are extremely limited. In this report, we describe the case of a 4-year-old captive Cape hyrax affected by amyloidosis. Histological examination revealed amyloid deposits in the liver, spleen, kidneys, and heart, which stained orange-red with Congo red and exhibited typical birefringence under polarized light. Mass spectrometry-based proteomic analysis and immunohistochemistry identified serum amyloid A in the amyloid deposits. Based on these findings, the case was diagnosed as amyloid A amyloidosis. This is a first case of amyloid A amyloidosis in Cape hyrax and, to our knowledge, the first documented case in any Afrotherian species.

淀粉样变性是一种由淀粉样蛋白沉积引起的疾病,在各种动物中都有报道;然而,关于非洲动物物种,包括海角狸的报道非常有限。在本报告中,我们描述了一例4岁圈养角兔淀粉样变的病例。组织学检查示肝、脾、肾、心淀粉样蛋白沉积,呈橙红色刚果红染色,偏振光下呈典型双折射。基于质谱的蛋白质组学分析和免疫组织化学鉴定了淀粉样蛋白沉积物中的血清淀粉样蛋白A。基于这些发现,本病例被诊断为淀粉样蛋白A淀粉样变性。这是hyrax的第一例淀粉样蛋白a淀粉样变性,据我们所知,这是在任何非洲动物物种中记录的第一例。
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引用次数: 0
Intestinal expression of diamine oxidase (DAO) and the relationship between blood DAO activity and haptoglobin concentration in diarrheal calves. 腹泻犊牛肠道二胺氧化酶(DAO)表达及血DAO活性与触珠蛋白浓度的关系
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-03 DOI: 10.1292/jvms.25-0324
Akihiro Kozutsumi, Minoru Okamoto, Hana Takeuchi, Marina Otsuka, Kenji Tsukano, Nao Kondo, Kana Kamitani, Keiko Ikeda, Natsuki Takami, Haruyuki Hirata, Kazuyuki Suzuki

The objectives of this study were (1) to confirm the expression of diamine oxidase (DAO) in the intestines of healthy calves immune-histologically and to clarify whether the expression of DAO in the intestines of calves with diarrhea differs from that of healthy animals, and (2) to clarify the relationship between DAO and intestinal inflammation using haptoglobin (HPT), a biomarker commonly used to evaluate inflammation. The abomasum, duodenum, jejunum and ileum, cecum, colon, and rectum of each animal were sampled at necropsy for immunohistochemical staining targeting DAO. DAO was diffusely expressed in all areas from the abomasum to the rectum in healthy calves, whereas its expression was reduced in calves with diarrhea. Four Japanese black calves with mild dehydration caused by diarrhea were included in the study to investigate the potential relationship between DAO and HPT. DAO activity was extremely low in diarrhea cases, whereas HPT levels were high. When an oral rehydration solution (ORS) was administered to these cases for three consecutive days, HPT significantly decreased logarithmically and fecal properties returned to normal on the fourth day of treatment. In contrast, DAO activities were significantly increased by oral rehydration therapy (ORT), and DAO activities were significantly negatively correlated with HPT concentrations. The present results confirmed that DAO is expressed in the epithelial cells of the intestinal mucosa in cattle, and that both the small and large intestines significantly contribute to blood DAO levels. Furthermore, DAO expression was reduced by enteritis.

本研究的目的是:(1)从免疫组织学上证实健康犊牛肠道中二胺氧化酶(DAO)的表达,并阐明腹泻犊牛肠道中DAO的表达是否与健康犊牛不同;(2)利用常用于评估炎症的生物标志物触球蛋白(HPT)阐明DAO与肠道炎症之间的关系。每只动物尸检时取皱胃、十二指肠、空肠和回肠、盲肠、结肠和直肠进行针对DAO的免疫组化染色。DAO在健康犊牛从皱胃到直肠的所有部位均有弥漫性表达,而在腹泻犊牛中表达减少。以4头因腹泻引起轻度脱水的日本黑犊为研究对象,探讨DAO与HPT之间的潜在关系。腹泻病例中DAO活性极低,而HPT水平较高。当这些病例连续3天口服补液(ORS)后,HPT显著呈对数下降,粪便特性在治疗第4天恢复正常。相比之下,口服补液治疗(ORT)显著提高了DAO活性,并且DAO活性与HPT浓度呈显著负相关。本研究结果证实了DAO在牛肠粘膜上皮细胞中表达,并且小肠和大肠对血液DAO水平有显著影响。此外,肠炎导致DAO表达降低。
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引用次数: 0
Feline vitamin D-dependent rickets type 2 caused by a missense variant in the vitamin D receptor gene. 猫维生素D依赖性佝偻病2型由维生素D受体基因错义变异引起。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-12 DOI: 10.1292/jvms.25-0307
Sora Suzuki, Hitomi Masuyama, Jiro Miyamae, Hiroaki Hemmi, Naoaki Matsuki, Kohei Murakami

An 11-month-old female kitten was evaluated for lameness and an inability to close her mouth. The kitten had marked hypocalcemia with elevated intact parathyroid hormone and 1,25(OH)2D3 levels. Radiographic imaging indicated generalized osteopenia and dysplasia of temporomandibular joints. Since a nutritionally complete diet was being fed, dysfunction of vitamin D receptors (VDR) was suspected. Genetic tests revealed three variants (c.439A >G, c.509C >T, and c.529_530insGCA) in the VDR gene, and c.509C >T, replacing proline 170 with leucine (p.Pro170Leu), was predicted to be damaging by in silico analysis. VDR-deficient feline kidney epithelial cells were transfected with wild-type or p.Pro170Leu VDR expression vectors, revealing impaired responsiveness to 1,25(OH)2D3 in mutants. Based on these findings, the cat was diagnosed with vitamin D-dependent rickets type 2.

一只11个月大的母小猫被评估为跛行和无法闭上她的嘴。小猫有明显的低钙血症,甲状旁腺激素和1,25(OH)2D3水平升高。影像学表现为全身性骨质减少及颞下颌关节发育不良。由于营养完全的饮食,维生素D受体(VDR)功能障碍被怀疑。基因检测显示VDR基因有3个变异(c.439A>G、c.509C>T和c.529_530insGCA),其中c.509C>T用亮氨酸代替脯氨酸170 (p.Pro170Leu),通过硅分析预测其具有破坏性。用野生型或p.Pro170Leu VDR表达载体转染VDR缺陷猫肾上皮细胞,发现突变体对1,25(OH)2D3的反应性受损。根据这些发现,这只猫被诊断为维生素d依赖性2型佝偻病。
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引用次数: 0
Improvement and development of immersion fixatives for enhanced tissue preservation in the whole eyeball specimens. 提高全眼球组织保存的浸泡固定液的改进和发展。
IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES Pub Date : 2025-12-01 Epub Date: 2025-10-22 DOI: 10.1292/jvms.25-0411
Muneyoshi Hyoto, Hiroyuki Imai, Akari Nishida, Kaoru Fujino, Ryoji Aoyagi, Kiyoshi Kano, Takeshi Kusakabe

The wall of eyeball limits the penetration of the common fixatives, raising inadequate fixation or degeneration of internal tissue and posing challenges for preparing thin sections of the whole eyeball. In addition, many formaldehyde-containing fixatives exhibit the strong cross-linking effect that can excessively mask antigenic epitopes, thereby hampering immunological analyses. This study tried to develop an available fixative that exerts high permeability to mouse whole eyeballs and applicability to immunostaining. Glyoxal is known to have milder cross-linking capacity compared to formaldehyde, bringing better preservation of epitopes. We substituted the formaldehyde component in Davidson's solution with glyoxal and supplemented 1-butanol, methanol, 2-mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl). Using modified fixatives and the GBMT solution, which was named by the initial letters of each component listed above, mouse eyeballs were immersion-fixed with the modified fixatives, and both tissue morphology and immunostaining quality were evaluated. The results showed that the GBMT solution prepared with glyoxal and specific combinations of additives improved morphological preservation of the eyeball tissues and enhanced immunofluorescence signals for certain cytoplasmic antigens, compared to the standard Davidson's solution. Our findings demonstrated that the successful development of a novel fixative enables both high-quality whole eyeball sectioning and improved immunostaining performance through immersion fixation alone.

眼球壁限制了普通固定物的穿透,引起固定不充分或内部组织退化,并对制备整个眼球的薄切片提出了挑战。此外,许多含甲醛的固定剂表现出强烈的交联效应,可以过度掩盖抗原表位,从而阻碍免疫学分析。本研究试图开发一种对小鼠全眼球具有高通透性和适用于免疫染色的固定液。与甲醛相比,乙二醛具有更温和的交联能力,可以更好地保存表位。我们用乙二醛代替戴维森溶液中的甲醛成分,并补充1-丁醇、甲醇、2-巯基乙醇和三(2-羧乙基)磷酸盐酸盐(TCEP-HCl)。使用改良的固定剂和GBMT溶液(按上面列出的每种成分的首字母命名),用改良的固定剂浸泡固定小鼠眼球,评估组织形态学和免疫染色质量。结果表明,与标准的Davidson’s溶液相比,由乙二醛和特定添加剂组合制备的GBMT溶液改善了眼球组织的形态保存,增强了对某些细胞质抗原的免疫荧光信号。我们的研究结果表明,一种新型固定物的成功开发既可以通过浸泡固定实现高质量的全眼球切片,又可以提高免疫染色性能。
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引用次数: 0
期刊
Journal of Veterinary Medical Science
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