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.

The occurrence of avian diseases has been greatly reduced by improved sanitation on poultry farms and the development of prevention programs through vaccination and other measures. However, the poultry industry still faces several concerns. Marek’s disease (MD) has caused significant economic losses to the poultry industry in the past; however, the introduction of vaccines has successfully contributed to a reduction in MD occurrences. Nevertheless, sporadic MD occurrences are still observed, and increased virulence has been reported owing to the changes in viral characteristics. In addition to infectious diseases, infestation with poultry red mites (PRMs; Dermanyssus gallinae), which are small hematophagous ectoparasites of poultry, has caused severe declines in productivity of the poultry industry worldwide. Furthermore, in addition to animal hygiene, problems caused by PRMs are of concern in terms of public health and animal welfare. The emergence of acaricide-resistant PRMs has made their control difficult. Therefore, the establishment of novel control strategies is eagerly awaited. Among the issues of avian diseases facing the poultry industry, this review highlights research on the mechanisms of increased virulence of MD virus and the development of a novel approach for PRM control.

In 2015, the third novel virulence plasmid pVAPN of Rhodococcus equi was discovered in bovine isolate from Ireland. The first bovine case of R. equi infection in Japan was reported in 2018 at a meat inspection station in Yokkaichi, Mie Prefecture, involving a Japanese Black heifer. Subsequently, additional cases were reported through annual surveillance in Miyazaki’s prefectures, Oita, Shiga, Gifu, Aichi, and Kyoto City, bringing total number of confirmed cases to 12. This review provides an overview of host-associated virulence plasmids of R. equi, a brief history of bovine R. equi case reports in Japan, and data analysis on the occurrence, clinical signs, and pathological findings of these 12 cases, along with a discussion on infection mechanisms and pathogenesis.

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.

The most common bovine mastitis pathogen is the Staphylococcus species, consisting of Staphylococcus aureus and non-aureus staphylococci (NAS). Lactoferrin (Lf) is an iron-binding protein with antimicrobial and antibiofilm properties. Lf has a metal-free form (apo-Lf) and a natural form (native-Lf), and their differences were reported to affect their activity against bacteria. However, its effects on bovine mastitis-causing Staphylococcus spp. remain unclear. Fifteen S. aureus and 49 NAS strains were isolated from bovine mastitis cases, and their growth and biofilm-forming abilities were observed. Bacterial growth and biofilm formation were observed by culturing each strain with/without bovine milk apo-/native-lactoferrin (200 μg/mL). Without Lf treatment, the growth and biofilm formation abilities of S. aureus were significantly higher and lower, respectively, than those of NAS. The growth of S. aureus and NAS significantly decreased during apo-Lf treatment and significantly reduced the total amount of biofilm produced by S. aureus whereas native-LF treatment did not affect the growth and biofilm formation of Staphylococcus species. These results confirmed the ability of Lf to act as an antimicrobial and antibiofilm substance against mastitis-causing Staphylococcus spp., although various responses from each strain were observed. Additionally, the iron-binding state of Lf affected growth but did not affect the biofilm formation ability. Differences in the responses of Staphylococcus strains to Lf may help explain their pathogenicity, requiring further research.

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.

This study aimed to determine the effects of precentrifugation storage time and temperature on coagulation and fibrinolysis parameters in Japanese Black calves. Blood samples were collected from calves under 6 weeks of age into plastic tubes containing 3.2% sodium citrate. Whole blood was stored at either room temperature or under refrigeration prior to centrifugation. Prothrombin time, activated partial thromboplastin time, fibrinogen level, and antithrombin level were measured. The results indicated that plasma parameters in samples centrifuged within 9 hr of collection showed no more than ± 10% variation compared with those in samples centrifuged within 1 hr of collection.

C-reactive protein (CRP) is a key biomarker for diagnosis and prognosis in dogs. Accurate and consistent CRP measurements are essential for clinical decision-making. In Japan, different CRP measurement systems are commonly used, but their compatibility has not been validated. In this study, we evaluated the correlation and agreement among three CRP assay systems (immunonephelometry, immunoturbidimetry, and dry chemistry) using 138 dog plasma samples. Strong correlations (r >0.9) were observed, but regression analysis and Bland-Altman plots revealed systematic errors, indicating that these systems are not interchangeable. These findings highlight the need for system-specific reference ranges and clinical decision thresholds and suggest that a common calibration agent could enhance comparability and support the standardization of CRP measurements in veterinary medicine.

Exotic animal cafes are a growing phenomenon within Japanese cities. These cafes are registered under the same license as other animal exhibits such as zoos, often allowing visitors to directly touch the animals or interact with animals within close proximity. The aim of this research was to report on current standards of animal care found in Japanese exotic animal cafes according to animal class. We included 79 exotic animal cafes with data collected on observable husbandry and management conditions by four trained assessors. Qualifiable welfare standards under the quantifiable scores were used to support guidance in scoring methodology. Welfare categories were assessed to identify whether some animal classes received better care in exotic animal cafés. Kruskal–Wallis tests assessed differences in welfare scores across animal classes, with Bonferroni-adjusted pairwise comparisons identifying inter-class variation. Among 231 animals identified, 51% were mammals, 31% were birds and 18% were reptiles. Scores across all categories were low for all taxa assessed, with significant differences between animal classes for the environmental and nutritional provisions and the performance of natural behavior with birds scoring the lowest for all these categories. Results conclude a widespread lack of appropriate animal care provision across all facilities, resulting in poor welfare for a range of species. This study, the first comprehensive assessment of welfare standards in Japanese exotic animal cafes, concludes an urgent need for effectively enforced facility and national animal protection standards for captive exotic species in Japan.

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.

Supraventricular tachyarrhythmia (SVTA) in dogs can cause cardiac dysfunction and may progress to heart failure. Pimobendan, an inotropic agent with calcium-sensitizing and phosphodiesterase III inhibition, is widely used in veterinary medicine. However, its influence on myocardial injury under persistent SVTA remains incompletely understood. This study aimed to evaluate whether pimobendan improves systolic function without exacerbating myocardial injury, using a canine model of rapid atrial pacing (RAP). Fourteen healthy Beagle dogs were allocated to either a control or pimobendan group. RAP was performed for three weeks at 390 bpm. Cardiac function was assessed by echocardiography before (baseline) and after RAP (3W). Concurrently, cardiac troponin I (cTnI) concentrations were measured to assess myocardial injury. In the control group, a significant increase in cTnI was observed at 3W compared with baseline (0.023 ± 0.029 ng/mL vs. 0.176 ± 0.173 ng/mL). Additionally, in the control group, echocardiographic parameters worsened, including fractional shortening (FS) (37.8 ± 4.6% vs. 24.5 ± 6.8%) and stroke volume (SV) (15.8 ± 3.1 mL vs. 10.5 ± 2.0 mL) at 3W compared with baseline. In contrast, the pimobendan group showed no significant differences in these echocardiographic parameters. cTnI concentrations also remained within the normal range without significant differences. These results suggest that pimobendan preserved systolic function without aggravating myocardial injury during sustained SVTA. Pimobendan may therefore be safe and effective cardiac inotropic agent for the treatment of dogs with SVTA.

The Tokara horse is a breed of native Japanese horse, for which hematological and biochemical data are lacking. The aim of this study was to obtain current hematological and biochemical data on Tokara horses. We enrolled 90 Tokara horses kept at the Kaimon Foothills Nature Park (Kagoshima) that underwent physical examinations and blood sampling between 2022 and 2024. All horses in this study were regarded as clinically healthy, based on physical examinations. Hematological and biochemical parameters also showed no abnormal values. Based on the above results, we consider that the data from this study will serve as reference values for blood tests in Tokara horses. However, we consider further research is needed on horses kept at two other locations (Nakanoshima and Iriki farm, Kagoshima).

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.

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.

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.

Osteoarthritis (OA) or degenerative joint disease (DJD) is a degenerative joint disease that progressively causes loss of joint function and is the most common and costly form of arthritis. Non-steroidal anti-inflammatory drugs (NSAIDs) are currently one of therapeutic treatment options for OA. Because NSAIDs do not alter the underlying pathophysiological process on the structural degradation of joint tissue but merely control signs of pain and inflammation, recent developments have allowed the use of treatments termed disease modifying osteoarthritic drugs (DMOADs) aimed at targeting the pathophysiologic processes of OA with the view of preventing, retarding progression of, or reversing morphologic changes associated with OA. Several in vitro and experimental studies have reported potential efficacy of glucosamine (GlcN) and pentosan polysulfate (PPS) as novel therapeutic agents of OA. This review will evaluate the clinical efficacy and safety of DMOADs with particular focus on GlcN and PPS based on the prescribed outcome measures by European Medicines Agency.

Cancer in dogs remains a major challenge in modern veterinary medicine. Immunotherapy using immune checkpoint inhibitors (ICIs) is available for various human tumor types, and recent veterinary clinical studies have shown that ICIs are a promising approach for treating canine cancers. A canine chimeric anti-PD-L1 antibody, c4G12 (HFC-L1), has been investigated for canine cancer immunotherapy; however, its clinical benefits have not been well characterized in tumors other than pulmonary metastatic (stage IV) oral malignant melanoma (OMM). To explore the efficacy and safety of HFC-L1, we conducted a clinical study in dogs with stage I–III OMM or other tumor types (n=12). HFC-L1 treatment at a dose of 5 mg/kg every 2 weeks was well tolerated, and no grade 3 or higher treatment-related adverse events were reported. Among the dogs eligible for response evaluation (n=10), a partial response was observed in one dog with squamous cell carcinoma, resulting in an objective response rate of 10%. In addition, in a dog with ceruminous cell carcinoma, clinical evidence of a tumor response was observed in metastatic lung lesions. Together, these results suggest that the HFC-L1 therapy is applicable for the treatment of various tumor types, although its clinical benefits should be further evaluated in clinical studies involving a larger number of dogs with each tumor type.

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.

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.

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.

Inflammation is a fundamental defense mechanism in animals, and its accurate detection is essential for diagnosis and treatment in veterinary medicine. Serum amyloid A (SAA), an acute-phase positive protein (APP), is widely used to detect inflammation in domestic animals. However, its application in wildlife, particularly raccoon dogs (Nyctereutes procyonoides koreensis), remains underexplored. This study aimed to establish a reference interval (RI) for SAA in raccoon dogs and explore its applicability as a biomarker of inflammation. A total of 83 blood samples from 43 raccoon dogs collected from the Korean Wildlife Rescue Center were categorized into three clinical groups: clinically healthy (CH), mild injury (M), and severe injury (S). The SAA concentrations were significantly higher in the S group than in the CH and M groups, whereas no significant difference was observed between the CH and M groups. The RI for SAA in CH raccoon dogs was determined to be 0.13–1.4 mg/L. Although SAA may have limited sensitivity for detecting mild or localized inflammation, the results support its use as a reliable marker of severe inflammation in raccoon dogs. Future studies should explore other APPs to improve the diagnostic accuracy of inflammation in wildlife health assessment.