Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Myogenesis, the formation of muscle fibers, is affected by certain glycoproteins, including chondroitin sulfate (CS), which are involved in various cellular processes. We aimed to investigate the mechanism underlying CS-E-induced suppression of myotube formation using the myoblast cell line C2C12. Differentiated cells treated with 0.1 mg/ml CS-E for nine days showed multinucleated and rounded myotubes with myosin heavy chain positivity. No difference was found between the CS-E-treated group with rounded myotubes and CS (−) controls with elongated myotubes in the levels of phospho-cofilin, a protein involved in the dynamics of actin cytoskeleton. Interestingly, N-cadherin, which is involved in the gene expression of myoblast fusion factors (myomaker and myomixer), was significantly downregulated at both the mRNA and protein levels following CS-E treatment. These results suggest that N-cadherin downregulation is one of the mechanisms underlying the CS-E-induced suppression of myotube formation.

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method, the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.

Understanding the immune dynamics in the respiratory mucosa of calves is necessary for a good management of bovine respiratory disease. Immune dynamics in the respiratory mucosa in humans and experimental animals has been assessed by flow cytometric analysis of bronchoalveolar lavage fluid (BALF); however, few reports have addressed this subject in calves. The aim of this study was to establish a universal method to analyze bronchoalveolar lavage fluid (BALF) by flow cytometry and to obtain basic knowledge of bovine respiratory mucosal immune dynamics. We investigated the immune cell populations in BALF and evaluated the surface antigen expression of alveolar macrophages in calves using flow cytometer. To further analyze the surface antigen variation observed in alveolar macrophages in detail, stimulation assays were performed in vitro. BALF cells were separated into three distinct populations based on their light scatter plot, which were considered to be macrophages, lymphocytes, and neutrophils. In most individuals, most of the BALF immune cells were alveolar macrophages, but an increased proportion of lymphocytes and neutrophils was observed in some individuals. Analysis of each surface antigen expression in alveolar macrophages showed that CD21 and MHC class II expression changed in response to changes in the leukocyte population. Moreover, when alveolar macrophages were stimulated with interferon-γ in vitro, the expression of CD21 was drastically reduced and MHC class II was increased, suggesting that functional changes in alveolar macrophages themselves are involved in the immune dynamics.

High-intensity exercise and competition are associated with depressed immune function. Young horses, which participate in high-intensity exercise and competitions, are at increased risk for the development of infectious disease due to depression of immune function. The effects of branched-chain amino acid (BCAA) supplementation on the immune status of young racing horses were evaluated, determining whether BCAA might help to avoid or reduce immune suppression during exercise and competitions. Twenty horses (10 male and 10 female) were treated with BCAA supplementation; another twenty untreated horses (10 male and 10 female) constituted control group. Peripheral blood was collected from each animal and evaluated for lymphocyte subsets, phagocytosis analysis of monocytes and granulocytes, lymphocyte proliferative response, and expression of cytokine-encoding messenger ribonucleic acids (mRNAs). The numbers of CD4⁺, CD8⁺, and major histocompatibility complex (MHC) class II⁺ cells in females of the treated group were significantly higher than those in females of the control group. The lymphocyte proliferative response in female of the treated group also was significantly higher than that in females of the control group. In addition, expression of mRNAs encoding interleukin-1β (IL-1β) and interferon-γ (IFN-γ) in females of the treated group was significantly higher than that in females of the control group. There were no significant differences between males of the treated and control groups. The results of this study indicated the positive effects of BCAA supplementation in counteracting immunosuppression in young female racing horses during and following high-intensity exercise.

Intestinal lymphangiectasia (IL) is often observed in dogs with chronic small intestinal diseases. Hypoplasia of the lymphatic vessel due to decreased lymphangiogenesis, which has been suggested in human idiopathic IL, may contribute to the pathogenesis of canine IL. This study aimed to evaluate the diameter and number of lymphatic vessels in full-thickness small intestinal specimens of dogs with IL. Immunohistochemical labeling of lymphatic endothelial cell markers was performed on retrospectively retrieved full-thickness small intestinal specimens. Sixteen dogs with histologically confirmed IL were included, of which 10 had lymphoplasmacytic enteritis (LPE), and six had granulomatous lymphangitis (GL). Nine dogs that died from non-gastrointestinal disorders and with little or no abnormalities in the small intestine were used as controls. Lymphatic vessel diameters in dogs with IL were significantly increased in all layers of the small intestine, including the villus lacteal, lamina propria, submucosa, muscularis, and mesentery, compared with controls (all P<0.01). There was no significant difference in the lymphatic vessel diameters between dogs with LPE and GL (all P>0.05). There was no significant difference in the number of lymphatic vessels between dogs with IL and the controls in all layers of the small intestine (all P>0.05). This study demonstrated that IL was observed in all layers of the small intestine, including the submucosa, muscularis, and mesentery, independent of the underlying disease. Factors other than reduced lymphatic vessels would contribute to the pathogenesis of IL in dogs.

A 10-month-old, intact male Toy Poodle was referred for a postural abnormality. Blood biochemical tests revealed a marked increase in plasma creatine phosphokinase (CPK) concentration. The isoenzyme test showed that 99% of serum CPK consisted of CPK-MM. Histopathological evaluation of muscle biopsy samples confirmed scattered degeneration and necrosis of myofibers. Immunohistochemistry for dystrophin showed an absence of staining in muscle cells. Based on these findings, the dog was diagnosed with dystrophin-deficient muscular dystrophy. Whole genome sequencing using genomic DNA extracted from blood revealed a single base pair insertion in exon 45 of the Duchenne muscular dystrophy (DMD) gene. This is the first report on muscular dystrophy in Toy Poodles and identified a novel mutation in the DMD gene.

Bordetella bronchiseptica (B. bronchiseptica) is associated with respiratory tract infections in laboratory animals. In our laboratory animal facility, B. bronchiseptica was isolated from 21 of 27 apparently healthy rabbits obtained from a breeding farm contaminated with B. bronchiseptica. Restriction fragment length polymorphism (RFLP) analysis showed that the flagellin genotype of isolates from the laboratory animal facility and breeding farm was type A, which is seen relatively frequently in rabbits in Europe. To examine its pathogenicity, guinea pigs, rats, and mice were inoculated intranasally with a representative strain isolated in the laboratory animal facility. Following inoculation of 10⁷ colony forming unit (cfu), severe inflammation was observed in the lungs of guinea pig and mice, although the inflammation was less severe in rats. The strain was recovered from the trachea and lungs of these species after inoculation with lower dose such as 10³ or 10⁴ cfu. These results suggest that the isolated strain causes respiratory tract infection in guinea pigs, rats, and mice, and that its pathogenicity higher in mice than in rats. This study extends our knowledge of interpreting the microbiologic status of laboratory animals, which will contribute to the development of reliable and reproducible animal experiments.

Acetaminophen-induced liver injury in mice is a model system of human acetaminophen overdose and oxidative stress in vivo. The system is technically established, and we usually obtain severe liver damage in the treated mice; however, it is possible that the degree of liver damage is affected by the type of chow fed to mice. Thus, in this experiment, we investigated the effect of different chows on mice by comparing acetaminophen-induced liver damage, liver antioxidant level, and serum amino-acid concentrations. The results showed that differences in chows, even standard ones, affected mouse physiological conditions, with the response to oxidative stress greatly affected.

The Cre-loxP system is widely used to investigate the cell-type specific roles of genes of interest. Cre-driver mice are required for cell-type specific knockout during the Cre-loxP reaction. To maintain Cre-driver mouse strains, Polymerase chain reaction (PCR)-oriented genotyping targeting the Cre gene cassette is usually conducted. In this study, we instead applied a colorimetric loop-mediated isothermal amplification (LAMP) method for Cre-genotyping. Among four sets of primers designed by the in silico program, one set effectively amplified the Cre cassette of three Cre-driver strains, but not of C57BL/6 mice. This LAMP-oriented method reduces assay time by less than half compared to the PCR-based method, and can be carried out using a conventional isothermal incubator. Applying this LAMP method may accelerate genotyping of Cre-driver mice.

Porcine circovirus type 2 (PCV2) exists widely in swine populations worldwide, and healthy PCV2 virus carriers have enhanced the severity of the infection, which is becoming more difficult to control. This study investigated the regulatory effect of Panax notoginseng saponins (PNS) on the oxidative stress and histone acetylation modification induced by PCV2 in vitro and in mice. In vitro, PNS significantly increased the scavenging capacities of superoxide anion radicals (O₂^•−) and hydroxyl radicals (^•OH) and reduced the content of hydrogen peroxide (H₂O₂) induced by PCV2 in porcine alveolar macrophages (3D4/2). In addition, PNS decreased the protein expression level of histone H4 acetylation (Ac-H4) by increasing the activity of histone deacetylase (HDAC) in PCV2-infected 3D4/2 cells. In vivo, PNS enhanced the scavenging capacities of ^•OH and O₂^•− and reduced the content of H₂O₂ in the spleens of PCV2-infected mice. PNS also reduced the protein expression level of histone H3 acetylation (Ac-H3) by reducing the activity of histone acetylase (HAT) and increasing the activity of HDAC in the spleens of PCV2-infected mice. PCV2 infection activated oxidative stress and histone acetylation in vitro and in mice, but PNS ameliorated this oxidative stress. The research can provide experimental basis for exploring the antioxidant effect and the regulation of histone acetylation of PNS on PCV2-infected 3D4/2 cells and mice in vitro and in vivo, and provide new ideas for the treatment of PCV2 infection.

Postoperative ileus (POI) is a postsurgical gastrointestinal motility dysfunction caused by mechanical stress to the intestine during abdominal surgery. POI leads to nausea and vomiting reduced patient quality of life, as well as high medical costs and extended hospitalization. Intestinal inflammation caused by macrophages and neutrophils is thought to be important in the mechanism of POI. Surgery-associated tissue injury and inflammation induce the release of adenosine triphosphate (ATP) from injured cells. Released ATP binds the purinergic P2X7 receptor (P2X7R) expressed on inflammatory cells, inducing the secretion of inflammatory mediators. P2X7R antagonists are thought to be important mediators of the first step in the inflammation process, and studies in chemically induced colitis models confirmed that P2X7R antagonists exhibit anti-inflammatory effects. Therefore, we hypothesized that P2X7R plays an important role in POI. POI models were generated from C57BL/6J mice. Mice were treated with P2X7R antagonist A438079 (34 mg/kg) 30 min before and 2 hr after intestinal manipulation (IM). Inflammatory cell infiltration and gastrointestinal transit were measured. A438079 ameliorated macrophage and neutrophil infiltration in the POI model. Impaired intestinal transit improved following A438079 treatment. P2X7R was expressed on both infiltrating and resident macrophages in the inflamed ileal muscle layer. The P2X7R antagonist A438079 exhibits anti-inflammatory effects via P2X7R expressed on macrophages and therefore could be a target in the treatment of POI.

In modern society, stress caused by relationships and emotions is one of the greatest social problems. Similar to humans, domestic and captive animals live under various stresses. Several stresses have been associated with skin disorders, such as atopic dermatitis, but there is a lack of reliable and objective indicators for the characterization of this association. This study aimed to define the changes in fatty acid composition and amino acid concentration in the skin following repeated restraint stress in ICR mice. Mice subjected to 30 min of daily restraint stress for 8 days showed changes in the composition of saturated fatty acids, such as an increase in palmitic acid content, which are the substrates of Δ-9 desaturase. Conversely, unsaturated fatty acids decreased with stress treatment, which appeared to be a result of these fatty acids being the substrate of Δ-6 desaturase. Changes in fatty acid composition after stress treatment may be one of the factors that cause skin inflammation. The water-retention capacity may have been lowered by stress treatment because histidine and leucine, which are natural moisturizing factors, were significantly decreased. The collagen content in the skin gradually decreased after repeated stress treatment. Our results indicate that repeated restraint stress may impact skin health through changes in both the fatty acid composition and amino acid concentration in mice.

Mesotocin (MT) is an avian homologue of oxytocin (OT). Behavioral pharmacological studies in birds have suggested the involvement of MT in socially affiliative behavior. However, investigations of peripheral MT levels associated with social behavior are lacking because non-invasive methods to measure surrogate plasma MT have yet to be established. This study aimed to measure urinary MT in crows using a commercially available OT enzyme-linked immunosorbent assay kit. Urine samples were collected after intravenous injection of MT and centrifuged to separate urine and fecal components. We found that urinary MT was significantly elevated 15–30 min after MT injection. These results validate our method for the use of urine samples for the measurement of peripheral MT levels in crows.

The purpose of this observational study was to determine the characteristic computed tomographic (CT) myelography findings of cervical vertebral stenotic myelopathy (CVSM) lesions in Thoroughbred horses. A total of 23 Thoroughbred horses (age range, 155–717 days on CT examination; mean, 410.9 days) were analyzed. All 23 Thoroughbred horses underwent unenhanced radiography, radiographic myelography, and CT myelography. Unenhanced radiographs were observed the presence of cervical vertebral malalignment and osseous lesions. Radiographic myelograms were observed for signs of cervical spinal cord compression; additionally, CT myelograms were used to detect cervical vertebral osseous lesions. Ventral compressions were frequently observed in the cranial cervical vertebrae (C2–C4), whereas dorsal compressions were frequently observed in the caudal cervical vertebrae (C5–C7). Furthermore, osseous lesions of the caudal articular process developed more frequently than those of the cranial articular process. CT myelography in Thoroughbred horses is a useful method for detecting CVSM changes.

This study aimed to evaluate the clinical utility of ultrasonography in the diagnosis of a newborn calf presenting with extended swelling within its right flank, in addition to its therapeutic planning. Ultrasonograms of the bilateral flanks identified thinning of the external and internal oblique abdominal muscles in whole areas of the abdominal walls. A right lateral abdominal hernia associated with thin abdominal muscular structures was diagnosed ultrasonographically. The right flank abdominal hernia was successfully reconstructed through a modified Mayo mattress suture. This allowed the overlapping of the two very thin structures of the abdominal walls, resulting in the creation of a thicker structure of the right lateral abdominal walls. Reconstruction of the abdominal walls using this method could prevent re-protrusion of the viscera during calf growth.

This paper reports on two cases of laparoscopic nephrotomy employed in the treatment of canine dioctophymosis, which is considered a unusual procedure and a new treatment proposal heretofore not performed in veterinary medicine. Two patients were treated, one with a history of hematuria and the other with incidental finding of the parasite in the abdominal cavity during elective ovariohysterectomy. Both dogs were subjected to abdominal ultrasound, which produced images indicating the presence of the parasite in the right side kidney, but with partial parenchymal preservation. The patients were therefore subjected to laparoscopic nephrotomy. The surgical procedure was effective in treating dioctophymosis and enabled minimum tissue invasion during surgery, in addition to preservation of the kidney.

Neonicotinoid pesticides (NNs) cause behavioral abnormalities in mammals, raising concerns about their effects on neural circuit activity. We herein examined the neurological effects of the NN clothianidin (CLO) by in vivo Ca²⁺ imaging using two-photon microscopy. Mice were fed the no-observed-adverse-effect-level (NOAEL) dose of CLO for 2 weeks and their neuronal activity in the primary somatosensory cortex (S1) was observed weekly for 2 weeks. CLO exposure caused a sustained influx of Ca²⁺ in neurons in the S1 2/3 layers, indicating hyperactivation of neurons. In addition, microarray gene expression analysis suggested the induction of neuroinflammation and changes in synaptic activity. These results demonstrate that exposure to the NOAEL dose of CLO can overactivate neurons and disrupt neuronal homeostasis.

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1–97 and 189–397 (Gc_1–97 and Gc_189–397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.

The enzyme-linked immunosorbent assay (ELISA) was applied to detect antibodies against Brucella abortus in serum samples from four seal species at nine coastal locations of Hokkaido, Japan. These antibodies were detected in 27% (32/118) of Western Pacific harbor seals (Phoca vitulina stejnegeri) at Cape Erimo. The antibodies were observed in spotted seals (P. largha) in one out of six at Nemuro, in two out of three at Rebun Island, in one out of two at Bakkai, and in examined one at Soya. They were also found in respective examined one ribbon seal (Histriophoca fasciata) and one ringed seal (Pusa hispida) at Akkeshi. Harbor seals that tested positive were mostly yearlings (35%, 20/57) and juveniles (45%, 10/22), while only one pup (1/13) and one subadult (1/5) tested positive with low titers of the antibody; no antibodies were observed in adults (n=21). These results suggest that Brucella mainly infected harbor seals from the environment while weaning, and the bacteria were cleared during the early life stage of the seals. In spotted seals, however, antibodies were also detected in adults, suggesting that spotted seals could become infected with Brucella even as adults. It is also possible that a different, more persistent strain of Brucella may have infected the spotted seals.