Dissemination of extended-spectrum cephalosporin (ESC)-resistant Salmonella is a public health concern in the egg production industry. ESC-resistant Salmonella often acquires the bla gene via insertion sequences (ISs). Therefore, this study aimed to assess antimicrobial resistance in Salmonella from Japanese layer breeding chains and egg processing chains, and determine the genetic profiles of IS-like elements in ESC-resistant Salmonella. Antimicrobial susceptibility testing was performed on 224 isolates from 49 facilities involving layer breeder farms, hatcheries, pullet-rearing farms, and layer farms in breeding chains along with egg processing chains. ESC-resistant Salmonella strains were whole-genome sequenced. Among them, 40 (17.9%) were resistant to at least streptomycin, tetracycline, ampicillin, chloramphenicol, cefpodoxime, nalidixic acid, ciprofloxacin, and/or kanamycin despite lacking resistance to azithromycin and meropenem. Moreover, 15 were ESC-resistant Salmonella harboring bla_CMY-2 (Salmonella enterica serovar Ohio, n=12; S. Braenderup, n=1; untypeable with O7:b:-, n=1) and bla_CTX-M-14 (S. Cerro, n=1). IncA/C₂ plasmids containing ISEcp1, IS26, and multiple antimicrobial resistance genes (including bla_CMY-2) were identified in S. Ohio isolates from pullet-rearing and layer farms belonging to the same company. Chromosomal integration of partial or whole IncA/C₂ plasmids was seen with two S. Ohio isolates via ISEcp1 or IS26, respectively. Antimicrobial resistance genes such as bla_CMY-2 might be transmitted among the upper and the lower levels of layer breeding chains via the replicon type IncA/C₂ plasmids containing ISEcp1 and IS26.

We have previously generated Large White pigs with high immune competence using a selection strategy based on phagocytic activity (PA), capacity of alternative complement pathway, and antibody response after vaccination against swine erysipelas. In this study, to identify the genetic changes caused by the immune selection pressure, we compared gene expression and polymorphisms in the promoter region between pigs subjected to the immune selection (immune-selected pigs) and those that were not (non-selected pigs). After lipid A stimulation, using a microarray analysis, 37 genes related to immune function and transcription factor activity showed a greater than three-fold difference in expression between macrophages derived from immune-selected and non-selected pigs. We further performed a polymorphic analysis of the promoter region of the differentially expressed genes, and elucidated the predominant promoter-types in the immune-selected and non-selected pigs, respectively, in the genes encoding ribonuclease L (RNASEL), sterile α motif and histidine-aspartate domain containing deoxynucleoside triphosphate triphosphohydrolase 1, signal transducer and activator of transcription 3, and tripartite motif containing 21. Analysis of the association between these promoter genotypes and the immune phenotypes revealed that the immune-selected promoter-type in RNASEL was associated with increased PA and was inherited recessively. Considering that RNASEL has been reported to be involved in antimicrobial immune response of mice, it may be possible to enhance the PA of macrophages and improve disease resistance in pig populations using RNASEL promoter-type as a DNA marker for selection.

Pharyngeal collapsibility has been used as diagnostic criteria in dogs, whereas the normal range and quantitative method have not been studied. Dynamic and static computed tomography (CT) was performed in 23 normal Beagle dogs to quantify the nasopharyngeal collapsibility at different locations. Using dynamic CT, maximum and minimum nasopharyngeal cross-sectional areas (CSAs) were measured at the level of the cranial end of the soft palate, pterygoid hamulus, foramen lacerum, bony labyrinth, and caudal end of the soft palate. The ratio of all maximum and minimum CSA to nasopharyngeal CSA at the level of the caudal hard palate (rCSAmax and rCSAmin) and the nasopharyngeal collapsibility were calculated. The differences of rCSAmax, rCSAmin, and nasopharyngeal collapsibility were analyzed at various locations. The nasopharyngeal collapsibility at the level of foramen lacerum, bony labyrinth, and caudal end of soft palate were higher than the others. At the level of the caudal end of the soft palate, rCSAmin was lower than that of the foramen lacerum and bony labyrinth, whereas rCSAmax at foramen lacerum was higher than that of the caudal end of the soft palate. These results indicated that the nasopharynx at the level of foramen lacerum and caudal end of the soft palate were considered notable locations for evaluating collapsibility. Dynamic CT could show the nasopharyngeal dynamic profile and will be an adequate modality for evaluating nasopharynx. Our results will be helpful for further comparative studies in dogs with and without nasopharyngeal collapse.

The size of canine focal liver lesions (FLLs) is known to be one of the predicting criteria for malignancy. However, there are discrepancies for the measurement of maximum lesion size, resulting in contradicting results among studies and incidences of false positive outcomes. Thus far, the morphometric changes of FLLs for distinguishing malignancy from benignancy remains undocumented. This study aimed to investigate morphometric characteristics of FLLs using computed tomography (CT). CT images of 40 dogs with histopathological confirmation of 49 liver lesions, including 39 hepatocellular carcinomas and 10 nodular hyperplasias were retrospectively reviewed. The morphometric parameters including size (long and short axis diameters measured on transverse image), shape (measured by long to short axis (L/S) ratio), volume, and surface appearance of a liver lesion were evaluated using univariate and stepwise multivariate analyses, respectively. The results of univariate analysis showed that long and short axis diameters, L/S ratio, volume, and surface appearance of a lesion were significantly different between hepatocellular carcinomas and nodular hyperplasias. Multivariate analysis revealed that short axis diameter (>3.30 cm; odds ratio (OR): 36.1, 95% confidence interval (CI): 3.36–387.05, P=0.0031) and L/S ratio (>1.23; OR: 18.1, 95% CI: 1.61–205.12, P=0.0191) were independent predictors of malignancy, with the area under the curve of 0.9154. These results suggest that the combination of short axis diameter and L/S ratio is a promising tool for predicting liver malignancy with outstanding discriminating ability.

Liver-type fatty acid-binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. L-FABP is a cytotoxic oxidation product secreted from the proximal tubules under ischemic and oxidative stress conditions. First, L-FABP gene expression in the kidney and liver was evaluated. Next, the urinary L-FABP concentrations in dogs with or without renal diseases were measured using a novel enzyme-linked immunosorbent assay kit. Urinary L-FABP was normalized relative to urinary creatinine (uCre) concentrations (µg/g uCre). Finally, the relationships between urinary L-FABP and renal biomarkers used in canine medicine or serum alanine transaminase (ALT) as an indicator of liver damage were examined. Serum and urine samples from 94 client-owned dogs including 23 dogs with renal diseases and 71 dogs without renal diseases were used for analysis. Relative L-FABP gene expression was confirmed both in the liver and kidney. Dogs with renal diseases had a significantly higher urinary L-FABP than those without, and its predictive cutoff value was 26 µg/g uCre. Urinary L-FABP was significantly correlated with serum creatinine (r=0.4674, P<0.01), urea nitrogen (r=0.4907, P<0.01), urine specific gravity (r=−0.5100, P<0.01), and urine protein/creatinine ratio (r=0.7216, P<0.01), but not with serum ALT. Hence, dogs with a high urinary L-FABP value were more likely to have renal diseases.

Epithelial–mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescence-immunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n=22) and cats (n=9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti‐vimentin antibodies. Enzymatic immunohistochemistry (IHC) revealed that 51.6% (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5% (5/8) of those cases. In cats, FICC could detect EMT in 100% (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.

Gel pads are commonly used in skin ultrasonography; however, the effects of their thickness are unknown. This study investigated the effects of pad thickness on measurements of skin thickness in 10 beagle dogs. Sonograms to measure neck skin thickness were captured without pads and using pads with thicknesses of 3, 5, 10, and 20 mm. Without pads, acoustic shading was observed due to air bubbles in the coupling gel. With 20-mm pads, echogenic artifacts were observed on the skin surface. Entry echo with 20-mm pads was significantly higher than with 3-mm pads. This suggests that visibility of the skin structure could be affected when a gel pad is not used or when a thick gel pad is selected.

Rhabdomyosarcoma (RMS) is an aggressive type of soft tissue sarcoma, and pleomorphic RMS is a rare subtype of RMS found in adult. p16 is a tumor suppressor which inhibits cell cycle. In human RMS, p16 gene is frequently deleted, but p16-null mice do not develop RMS. We reported that genetic ablation of p16 by the crossbreeding of p16 knock-out rats (p16-KO rats) improved the dystrophic phenotype of a rat model of Duchenne muscular dystrophy (Dmd-KO rats). However, p16/Dmd double knock-out rats (dKO rats) unexpectedly developed sarcoma. In the present study, we raised p16-KO, Dmd-KO, and dKO rats until 11 months of age. Twelve out of 22 dKO rats developed pleomorphic RMS after 9 months of age, while none of p16-KO rats and Dmd-KO rats developed tumor. The neoplasms were connected to skeletal muscle tissue with indistinct borders and characterized by diffuse proliferation of pleomorphic cells which had eosinophilic cytoplasm and atypical nuclei with anisokaryosis. For almost all cases, the tumor cells immunohistochemically expressed myogenic markers including desmin, MyoD, and myogenin. The single cell cloning from tumor primary cells gained 20 individual Pax7-negative MyoD-positive RMS cell clones. Our results demonstrated that double knock-out of p16 and dystrophin in rats leads to the development of pleomorphic RMS, providing an animal model that may be useful to study the developmental mechanism of pleomorphic RMS.

Diabetes mellitus (DM) is characterized by metabolic disorders and psychological deficits, including cognitive decline. Here, we investigated the effect of cordycepin on oxidative stress and protein expression in the brains of diabetic mice. Twenty-four mice were divided into four groups, one comprising untreated healthy mice (N); one comprising healthy mice treated with cordycepin (24 mg/kg body weight) (N+Cor); one comprising untreated DM mice; and one comprising DM mice treated with cordycepin (24 mg/kg body weight) (DM+Cor). After 14 days of treatment, cognitive behavior was assessed using the novel object recognition (NOR) test. The brain levels of oxidative stress markers (glutathione, catalase, and superoxide dismutase) were examined using the respective detection kits. Protein expression in brain tissues was assessed by liquid chromatography with tandem mass spectrometry (LC–MS/MS); the functions of the identified proteins were annotated by PANTHER, while major protein–protein interactions were assessed using STITCH. We found that cordycepin treatment significantly decreased body weight and food and water intake in the DM+Cor group compared with that in the DM group; however, no differences in blood glucose levels were found between the two groups. Cordycepin treatment significantly reversed cognitive decline in diabetic mice in the NOR test and ameliorated antioxidant defenses. Additionally, we identified ULK1 isoform 2, a protein associated with cognitive function via the activated AMPK and autophagic pathways, as being uniquely expressed in the DM+Cor group. Our findings provide novel insights into the cellular mechanisms underlying how cordycepin improves cognitive decline in diabetic mice.

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68⁺ M1-macrophages and major histocompatibility complex (MHC) class II⁺ macrophages remarkably increased persistently, whereas CD163⁺ M2-macrophages slightly increased. MHC class II⁺/CD68⁺ and MHC class II⁺/CD163⁺ macrophages were present, indicating that MHC class II⁺ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-β1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4⁺ and CD8⁺ T cells appeared in close relation with MHC class II⁺ macrophages, and mainly CD4⁺ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.

We report the pathologic features, local inflammatory response immunophenotype, and molecular identification results of cerebral nematodiasis in a young sitatunga (Tragelaphus spekii) from Texas. To the authors’ knowledge, this is the first report of cerebral nematodiasis by Parelaphostrongylus tenuis in a sitatunga, a bovid species introduced into the USA, and the first characterization of the local inflammatory response immunoprofile in this condition. A molecular identification method based on formalin-fixed and paraffin-embedded-polymerase chain reaction was described. These results contribute to knowledge on geographical distribution and host spectrum of P. tenuis, and highlight the relevance of this nematodiasis in naïve translocated or introduced bovid species into endemic areas.

Maternal obesity and diabetes are known to be involved in fetal myogenesis, but the later stages of myogenesis are not well understood. In this study, we investigated the influence of a hyperglycemic environment on L6 skeletal myoblast differentiation and the function of omega-7 palmitoleic acids. Exposure to a high concentration of glucose (25 mM) in high-glucose culture medium (HG) increased the expression of myogenic genes (MyoD, Myogenin, MRF4, Myhc2x, and Myhc2a) and the synthesis of myosin. HG also activated the PI3K/AKT pathway revealed muscle cell differentiation. Furthermore, the levels of reactive oxygen species (ROS) and an inflammatory cytokine (Tnfaip3; tumor necrosis factor alpha-induced protein 3), which are crucial for the growth and differentiation of skeletal muscle, were increased by HG. Palmitoleic acids suppressed the expression levels of myogenic regulatory genes and increased the expression level of a cell proliferation-related gene (Pax3). Trans-palmitoleic acid and eicosapentaenoic acid (TPA and EPA) increased the phosphorylation level of MAPK/ERK1/2 and downregulated ROS generation and Tnfaip3 expression. In contrast, cis-palmitoleic acid inactivated MAPK/ERK1/2, leading to increased ROS generation. In conclusion, a hyperglycemic environment mediated by HG induced excessive muscle differentiation. Palmitoleic acids inhibited myoblast differentiation by downregulating muscle-specific genes. Moreover, trans-palmitoleic acids may have beneficial antioxidant and/or anti-inflammatory effects in cells.

We determined a comprehensive immunohistochemistry of putative isoforms of enzymes for prostaglandin (PG) F₂α and PGE₂ biosynthesis and these PGs levels in placenta and fetal membrane of normal pregnant rats in vivo. Placenta and fetal membrane showed positive immunoreactions for phospholipase A₂ group 4A, but not group 2A, and cyclooxygenase (COX)-1 rather than COX-2. They showed positive immunoreactions for at least one isoform of each of PGF synthase and PGE synthase with tissue-dependent variations. PGF₂α and PGE₂ levels in both tissues were highest on day 12 and declined and remained low thereafter. Obtained data would be the basic information on the primary PGs synthesis in rat placenta and fetal membrane in normal pregnancy.

Few reports have investigated prognosis of canine gastrointestinal stromal tumor (GIST) cases treated by surgical resection alone. In the present study, we investigated the overall survival (OS) and prognostic factors for dogs with GIST treated by surgical complete resection alone. Fifty-three dogs were included, and the median OS was 18 months. Multivariate analysis showed that primary tumors in small intestine (P=0.04) is significantly associated with shorter OS, and median OS of the cases with cecum lesion and those with small intestine lesion was 22 and 6 months, respectively. The present study suggested primary tumor site was a novel prognostic factor for dogs with GIST treated by surgical complete resection alone.

Several studies have reported methods to estimate the parturition date of dogs using ultrasonographic measurements. However, these prediction models were mainly determined using ultrasonographic measurements of naturally pregnant small- and medium-sized dogs, and no such studies have been performed using dogs carrying cloned fetuses produced via somatic cell nuclear transfer. The present study evaluated the abilities of three reference formulas (Luvoni and Grioni, Milani et al., and Groppetti et al.), all of which were developed using data from naturally occurring pregnancies, to accurately predict the parturition date in surrogates carrying cloned German Shepherd (GS) fetuses. All three formulas were based on the use of inner chorionic cavity diameter (ICC) measurements, obtained via ultrasonography. For evaluation, a total of 54 ICC measurements were collected from 14 pregnant bitches carrying cloned GS fetuses. We found that the clinical accuracy of the breed-specific Groppetti et al. formula was highest among those of the three formulas tested, with 87% and 100% of the estimated parturition dates (calculated based on the ICC measurements) being within 1 and 2 days, respectively, of the actual delivery date. By contrast, the Luvoni and Grioni formula showed relatively low accuracy, and the Milani et al. formula showed higher accuracy than that reported previously for natural pregnancies.

Epizootic hemorrhagic disease (EHD) is an arthropod-borne disease of wild and domestic ruminants caused by the EHD virus (EHDV). To date, seven EHDV serotypes have been identified. In Japan, strain Ibaraki of EHDV serotype 2 has caused outbreaks of Ibaraki disease in cattle. In addition, EHDV serotype 7 (EHDV-7) has caused large-scale EHD epizootics. In mid-September 2016, eight cattle at a breeding farm in Fukuoka Prefecture, Japan developed fever. Since EHDV-7 was detected in sentinel cattle in western Japan in 2016, we suspected that the cause of this fever might be an EHDV-7 infection. In this study, we tested cattle for EHDV-7 and some other viruses. Consequently, EHDV was isolated from washed blood cells collected from three of the eight cattle, and genetic analysis of genome segment 2 revealed that this isolate was EHDV-7. Moreover, all affected cattle tested positive for anti-EHDV-7 neutralizing antibodies. Our results suggest that the fever was caused by EHDV-7 infection. In addition, we modified a conventional reverse transcription polymerase chain reaction assay for the specific detection of EHDV. This modified assay could detect various strains of EHDV isolated in Japan, Australia, and North America. Furthermore, the assay permitted the detection of EHDV-7 in blood cells collected from seven of the eight cattle. We believe that this modified assay will be a useful tool for the diagnosis of EHD.

I performed metaviromic analysis of publicly available RNA-seq data from reptiles to understand the diversity of filoviruses (family Filoviridae). I identified a coding-complete sequence of a filovirus from the common lancehead (Bothrops atrox (Linnaeus, 1758)), tentatively named Tapajós virus (TAPV). Although the genome organization of TAPV is similar to mammalian filoviruses, our phylogenetic analysis showed that TAPV forms a cluster with a fish filovirus. However, TAPV is still distantly related to all the known filoviruses, suggesting that TAPV can be assigned as a species of a novel genus in Filoviridae. To our knowledge, this is the first report identifying a filovirus in reptiles, and thus contributes to a deeper understanding of the diversity and evolution of filoviruses.

In this study, we performed a molecular phylogenetic analysis of six bovine papular stomatitis virus (BPSV) field strains detected from Japanese beef calves kept on a farm in Saga prefecture, a southwest part of Japan, from 2017 to 2020. The phylogenetic analysis based on a partial B2L gene (554-nt) showed that these field strains were divided into two lineages, a lineage (A-lineage) constructed by a Saga strain and strains obtained from various regions of Japan and the world, and other lineage (B-lineage) constructed by five Saga strains and strains obtained from France, USA and Iwate prefecture (a north part of Japan). Furthermore, a Saga field strain named BPSV_SAGAbv2 and strains obtained from USA and Iwate prefecture belonged to a sub-lineage blanched from B-lineage. This is the first report elucidating molecular epidemiological characters of field BPSVs obtained from Saga prefecture. The existence of the multiple lineages was thought to be related to a history of calf introduction from various regions of Japan into Saga prefecture.

The Shimokita Peninsula in Aomori Prefecture, Japan, which is inhabited by Japanese monkeys (Macaca fuscata), is the northernmost habitat for wild primates in the world. This study was the first to determine the conception dates of specific individuals and estimate the pregnancy rate of wild populations in this region. The pregnancy rate of animals aged 5 years or more at delivery was estimated to be 40.9% (27/66). Conception dates of each fetus were also estimated using a regression line of Pig-tail monkeys (Macaca nemestrina), which are taxonomically related to Japanese monkeys and have a similar physique. The conception dates were distributed across 90 days between September 24th and December 23rd, with a mean conception date of November 4th (SD=22.3 days, n=53). Using these findings, the mean birth date was estimated as April 25th, more than two weeks earlier than the mean birth date in previous research determined using direct observations carried out over the past 20 years ago. Global warming due to climate change is thought to be one of the main causes of this difference.

This study aimed to investigate the effect of mydriasis using topical rocuronium bromide on electroretinography (ERG) in domestic pigeons (Columba livia). Scotopic mixed rod and cone, photopic cone, and photopic flicker ERG were performed on nine eyes of nine healthy adult pigeons under sedation. Each pigeon underwent two sets of ERG recordings. First, without the induction of mydriasis (control) and the second time with the induction of mydriasis using topical rocuronium bromide (treatment). The results were compared using either the Student’s t-test or Wilcoxon rank-sum test, where a P-value of <0.05 was considered statistically significant. No significant differences were observed in the a- and b-wave implicit times and amplitudes during scotopic ERG between the two groups. The a- and b-wave amplitudes in the photopic cone were significantly higher in the treatment group (63.83 ± 32.33 and 191.75 ± 94.46 µV) compared to the control group (46.15 ± 27.60 and 116.76 ± 70.65 µV; P=0.045 and P=0.032, respectively). The photopic flicker amplitude was also significantly higher in the treatment group (76.23 ± 48.56 µV) than in the control group (42.18 ± 31.18 µV; P=0.044). No statistically significant differences were observed in the photopic cone and flicker implicit times between both groups. In conclusions, mydriasis induced by rocuronium bromide in pigeon resulting in higher amplitudes during the photopic ERG but not scotopic ERG.

Three eastern bongos (Tragelaphus eurycerus isaaci) presented acutely with hemorrhagic diarrhea at the Singapore Zoo, thought to be caused by a mouldy batch of hay. Repeated fecal tests were negative of parasites and common gastrointestinal bacteria including salmonella and campylobacter. The diarrhea resolved for all individuals after a week of leaf-only diet. However, 2 individuals developed signs of colic. Both animals were anesthetized for examination including blood tests and imaging studies. The findings were consistent of gastrointestinal ileus and a possible impaction. With intensive treatment involving repeated sedations for fluid therapy administration and treatments for gastrointestinal impaction, one individual eventually made a full recovery, but the other individual died due to septic peritonitis secondary to a rupture in the spiral colon. Persistent supportive therapy may be vital in treating severe gastrointestinal disease in this species.

Lions (Panthera leo) breed well under captivity, so contraception has been commonly conducted for population management, leading to a demand for a less invasive and reversible contraceptive approach in lions. In this study, we examined the efficacy of a commercial gonadotropin-releasing hormone vaccine as a method of suppressing reproductive activity in a sexually matured female lion. Under behavioral restraint, the vaccine was injected twice (days 0 and 109). After the initial vaccination, ovarian activity is still observed. After the second vaccination, contraceptive effect was confirmed for 246 days until restart of estrous cycles. We confirmed only a slight swelling around the injection site after the second vaccination. This study may suggest an alternative option for a contraceptive method in lions.