Many parasite species migrate to another site of infection after entering the host body. Such parasite dynamics are closely related to pathogenicity, but it is not easy to observe such parasite behavior deep within the organs. In recent years, technology that can make organs transparent has been developed that enables us to observe deep within organs ex vivo while maintaining their three-dimensional structure. This review describes a series of attempts to apply this technology to understand the behavior of Toxoplasma gondii in the host body. A series of studies has shown that T. gondii tachyzoites that infect leukocytes can reach target organs far from the site of invasion via the circulatory system. In addition, infected leukocytes in the bloodstream adhere more readily to vascular endothelial cells than uninfected leukocytes and are more likely to remain inside the target organs. When infected leukocytes adhere to the vascular endothelial cells of the target organ, the tachyzoites inside the cells immediately escape and infect the parenchyma of the organs. As described above, organ transparency technology is a powerful tool for understanding the internal dynamics of parasites.

To evaluate the immune enhancement effect of the extracellular polysaccharide of Lactobacillus plantarum on oprH recombinant subunit vaccine from Pseudomonas aeruginosa, a recombinant subunit vaccine of oprH (rOprH vaccine) was developed. The EP-rOprH vaccine was prepared with the extracellular polysaccharide of L. plantarum as an adjuvant. Mice were vaccinated with the rOprH and EP-rOprH vaccines, and the outer membrane protein (OMP) and inactivated vaccines were used as controls. The levels of serum antibody, interferon-γ (IFN-γ), interleukin (IL-2), and IL-4 were determined after vaccination. Finally, the protective efficacy of the vaccine was evaluated after challenge with virulent P. aeruginosa. Following vaccination, the serum antibody levels were significantly higher in mice vaccinated with the EP-rOprH vaccine than in those vaccinated with the rOprH vaccine (P<0.05). Moreover, the serum antibody levels detected in the EP-rOprH vaccine group were similar to those detected in the OMP vaccine group when P. aeruginosa suspension was used as the coating antigen. However, the levels in the EP-rOprH vaccine group were higher than those in the OMP vaccine and inactivated vaccine groups when the purified rOprH protein was used as the coating antigen (P<0.05). The level of IFN-γ, IL-2, and IL-4 in mice vaccinated with the EP-rOprH vaccine was significantly higher than that in mice vaccinated with the rOprH vaccine (P<0.05) and comparable to that in mice vaccinated with the OMP vaccine. The protective rates were 65%, 80%, 80%, and 95% with the rOprH, EP-rOprH, OMP, and inactivated vaccines, respectively. Thus, the extracellular polysaccharide of L. plantarum significantly enhanced the immune response and protection provided by the recombinant subunit vaccine of oprH.

Aims of our study were to evaluate the feasibility and diagnostic value of two-dimensional shear wave elastography in dogs with acute kidney injury, chronic kidney disease, and acute on chronic kidney disease, its correlation with renal functional (creatinine, urea), and prognostic parameters (serum calcium-phosphorus product, urinary output), and with contrast-enhanced ultrasound (qualitative and quantitative evaluation). The study was prospective. A group of healthy (Group A) and a group of nephropathic dogs (Group B) were included. Shear wave elastography was performed on the left kidney of the subjects of both groups; contrast-enhanced ultrasound was performed only in dogs with acute kidney injury and acute on chronic kidney disease. Sixty-four dogs were included (Group A, n=24; Group B, n=40). The renal stiffness values were significantly higher in Group B than Group A; optimal cut-off stiffness values for detection of renal pathology were: ≥1.51 m/sec (area under the curve, 0.84; 95% confidence interval 0.74–0.94) and ≥6.75 kPa (area under the curve, 0.84; 95% confidence interval 0.73–0.94). For contrast-enhanced ultrasound, a significant positive correlation was found between renal stiffness, area under the curve, and wash-out area under the curve values of cortex quantitative analysis. No correlations were found between renal stiffness and renal functional and prognostic parameters. Shear wave elastography showed diagnostic utility to detect renal abnormalities in dogs with acute kidney injury, chronic kidney disease and acute on chronic kidney disease, however, it could not differentiate between these different nephropathies.

The objective of this study was to investigate the thromboelastography (TEG) variables and platelet count after Asian pit viper (Gloydius) envenomation in dogs compared with healthy control dogs. Ten dogs with snake envenomation and twenty healthy dogs were included. There was a significant decrease in G (P=0.017), maximum amplitude (MA) (P=0.012), angle value (P=0.041), platelet count (P<0.001, as well as an increase in K value (P=0.034) and R value (P=0.007) in dogs with snake envenomation. The results of this study revealed that dogs with Asian pit viper (Gloydius) envenomation showed a hypocoagulable state compared with healthy dogs. Our findings may support the use of TEG for diagnosing and monitoring coagulopathy after Asian pit viper (Gloydius) envenomation in dogs.

Retroperitoneal hemangiosarcoma (RPHSA) is a rare tumor in dogs with a poorly understood prognosis after surgery. The objectives of this study were to investigate the clinical features and prognosis of canine RPHSA that had undergone surgical resection. In this single-center, retrospective cohort study, we reviewed the medical records of dogs that had undergone surgical resection for retroperitoneal tumors and received a histopathologic diagnosis of HSA between 2005 and 2021. The median progression-free survival (PFS) and overall survival (OS) were 77.5 days and 168 days, respectively. In the present study, canine RPHSA had an aggressive biological behavior similar to visceral HSA. Further studies in larger canine populations are needed to evaluate the efficacy of adjuvant chemotherapy.

Merkel cell carcinoma (MCC) is a rare skin tumor that shares a similar immunophenotype with Merkel cells, although its origin is debatable. More than 80% of human MCC cases are associated with Merkel cell polyomavirus infections and viral gene integration. Recent studies have shown that the clinical and pathological characteristics of feline MCC are comparable to those of human MCC, including its occurrence in aged individuals, aggressive behavior, histopathological findings, and the expression of Merkel cell markers. More than 90% of feline MCC are positive for the Felis catus papillomavirus type 2 (FcaPV2) gene. Molecular changes involved in papillomavirus-associated tumorigenesis, such as increased p16 and decreased retinoblastoma (Rb) and p53 protein levels, were observed in FcaPV2-positive MCC, but not in FcaPV2-negative MCC cases. These features were also confirmed in FcaPV2-positive and -negative MCC cell lines. The expression of papillomavirus E6 and E7 genes, responsible for p53 degradation and Rb inhibition, respectively, was detected in tumor cells by in situ hybridization. Whole genome sequencing revealed the integration of FcaPV2 DNA into the host feline genome. MCC cases often develop concurrent skin lesions, such as viral plaque and squamous cell carcinoma, which are also associated with papillomavirus infection. These findings suggest that FcaPV2 infection and integration of viral genes are involved in the development of MCC in cats. This review provides an overview of the comparative pathology of feline and human MCC caused by different viruses and discusses their cell of origin.

Postoperative ileus (POI) is a surgical complication that induces emesis and anorexia. Fuzapladib (FUZ), an inhibitor of leukocyte-function-associated antigen type 1 (LFA-1) activation, a leukocyte adhesion molecule, exerts anti-inflammatory effects by inhibiting leukocyte migration into the inflammatory site. In this study, we examined the prophylactic impact of FUZ on POI in a mouse model. POI model mice were generated by intestinal manipulation, and the effect of FUZ on intestinal transit and the infiltration of inflammatory cells into the ileal muscularis externa was assessed. The increased number of macrophages was significantly suppressed by FUZ, whereas the infiltration of neutrophils into the ileal muscularis externa was not sufficiently inhibited in the POI model mice. Additionally, FUZ did not ameliorate delayed gastrointestinal transit in POI model mice. In conclusion, our results suggest that FUZ does not improve delayed gastrointestinal transit but partially inhibits inflammation in the ileal muscularis externa in POI model mice. FUZ may be a potential anti-inflammatory agent for the management of post-surgical inflammation.

Cellular senescence is a highly stable state associated with cell cycle arrest, that is elicited in response to various stresses. The accumulation of senescent cells in tissues drives age-related diseases. Recent studies have shown that the cellular senescence enhances an extracellular vesicles (EV) secretion. EV are lipid-bilayer-capsuled particles released by various cells mediating cell-to-cell communication. It was recently reported that EV secreted by the senescent cells had several functions such as cancer cell proliferation and immune cell activation. In the present study, we investigated whether senescent cardiac fibroblasts-derived EV play an autocrine/paracrine role in the heart cells. Neonatal rat cardiac fibroblasts (NRCFs) were treated with doxorubicin (DOX) to induce cellular senescence. EV were isolated from NRCFs culture media. The vehicle-treated NRCFs-derived EV (D0-EV, 72 hr) increased a living cell number in NRCFs, which was attenuated by DOX (1,000 nM)-treated NRCFs-derived EV (D10³-EV, 72 hr). While D0-EV did not affect protein concentration in NRCFs, D10³-EV decreased it. Furthermore, D10³-EV significantly increased a ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I in NRCFs, indicating an induction of autophagy. In addition, D10³-EV increased phosphorylation of adenosine monophosphate-activated kinase (AMPK) α in NRCFs. In neonatal rat cardiomyocytes, however, NRCFs-derived EV (72 hr) had no effect on the living cell number, protein concentration, and ratio of LC3-II to LC3-I. In conclusion, we for the first time revealed that DOX-induced senescent NRCFs-derived EV induce autophagy in NRCFs perhaps partly through the activation of AMPKα.

Though lysophosphatidic acid (LPA) shows a variety of regulatory roles in reproduction, its action mechanisms in the gestational organs are still largely unknown. We here characterized cellular distribution of its six kinds of specific receptors (LPA1-6) in rat uteri by immunohistochemistry and quantitatively analyzed changes in Lpar1-6 mRNAs expression throughout pregnancy. Among LPA1-6, evident expression of LPA3, LPA4, and LPA6 was immunologically detected and less expression of immunoreactive LPA1 and LPA2 was also found. Luminal and glandular epithelial cells, stromal cells, and myometrial cells are sites of positive immunoreactions, and they are all likely to express three or more subtypes. All of Lpar1-6 mRNAs were expressed, and their alterations were variable depending on subtypes and gestational age. The present information suggests that diverse actions of LPA in the uterus involve varied expression of LPA receptors dependent on tissue/cell types, receptor subtype(s), and organ reproductive states and helps to understand uterine biology of LPA.

The aim of this study was to compare the intra and postoperative analgesic effects of sacrococcygeal epidural levobupivacaine with those of lumbosacral levobupivacaine in feline ovariohysterectomy. Thirty-six cats were premedicated with intramuscular acepromazine (0.05 mg/kg) and meperidine (6 mg/kg). Anesthesia was induced with intravenous propofol and maintained with isoflurane in oxygen. The cats were randomly assigned one of the three treatments receiving 0.33% levobupivacaine (0.3 mL/kg) into the sacrococcygeal (S-C group, n=12) or lumbosacral (L-S group, n=12) epidural space, or the same volume of 0.9% saline solution into one of the epidural approaches (Control group, n=12). Intraoperatively, cardiorespiratory variables, end-tidal isoflurane concentration (FE´ISO), and fentanyl requirements were recorded. Postoperative pain was assessed by the UNESP (Universidade Estadual Paulista)-Botucatu multidimensional composite pain scale and the Glasgow feline composite measure pain scale up to 8 hr post-extubation. Morphine was administered as rescue analgesia. Overall FE´ISO and fentanyl requirements were lower in the L-S and S-C compared to the Control (P=0.002–0.048, respectively). There was no significant difference in the cardiorespiratory variables during anesthesia, postoperative pain and rescue analgesia among groups. The time to standing after anesthesia was prolonged in the L-S and S-C groups than in the Control (P<0.001). Lumbosacral and sacrococcygeal epidural levobupivacaine resulted in similar decreases in isoflurane requirements and intraoperative fentanyl supplementation in the cats, with no postoperative benefits.

Cystine-glutamate transporter (xCT) is a plasma membrane transporter that imports cystine and indirectly contributes to the oxidative stress resistance associated with increased intracellular glutathione levels. Canine adipose-derived stem cells (CADSCs) include an xCT-positive subpopulation and show relatively low expression of osteogenic markers during in vitro osteogenic differentiation. Sulfasalazine (SSZ), a drug used to treat rheumatoid arthritis, suppresses xCT expression in cancer cells. In this study, we found that the SSZ treatment at 100 µM significantly suppressed xCT mRNA expression in CADSCs but did not significantly affect cell proliferation under the same conditions. Additionally, this treatment decreased the intracellular glutathione concentration. During in vitro osteogenic differentiation, the SSZ treatment at 50 µM and 100 µM significantly increased alizarin red staining and its quantification, as well as the concentration-dependent osteogenic differentiation markers (BMP1 and SPP) mRNA expression. Our results suggested that SSZ enhances the osteogenic differentiation potential of CADSCs and can potentially exhibit a superior therapeutic profile in canine bone regenerative medicine.

In the winter of 2021–2022, multiple subtypes (H5N8 and H5N1) of high pathogenicity avian influenza viruses (HPAIVs) were confirmed to be circulating simultaneously in Japan. Here, we phylogenetically and antigenically analyzed HPAIVs that were isolated from infected wild birds, an epidemiological investigation of affected poultry farms, and our own active surveillance study. H5 subtype hemagglutinin (HA) genes of 32 representative HPAIV isolates were classified into clade 2.3.4.4b lineage and subsequently divided into three groups (G2a, G2b, and G2d). All H5N8 HPAIVs were isolated in early winter and had HA genes belonging to the G2a group. H5N1 HPAIVs belong to the G2b and G2d groups. Although G2b viruses were widespread throughout the season, G2d viruses endemically circulated in Northeast Japan after January 2022. Deep sequence analysis showed that the four HPAIVs isolated at the beginning of winter had both N8 and N1 subtypes of neuraminidase genes. Environmental water-derived G2a HPAIV, A/water/Tottori/NK1201-2/2021 (H5N8), has unique polymerase basic protein 1 and nucleoprotein genes, similar to those of low pathogenicity avian influenza viruses (LPAIVs). These results indicate that multiple H5 HPAIVs and LPAIVs disseminated to Japan via transboundary winter migration of wild birds, and HPAIVs with novel gene constellations could emerge in these populations. Cross-neutralization test revealed that G2a H5N8 HPAIVs were antigenically distinct from a G2b H5N1 HPAIV, suggesting that antibody pressure in wild birds was involved in the transition of the HPAIV groups during the season.

To our knowledge, ours is the first case of applying a 3D-printed prosthetic beak to an Oriental stork (Ciconia boyciana). A stork in captivity underwent several surgeries for beak fractures, but the lower-mandible fractures failed to be repaired. Therefore, we applied a patient-specific beak prosthesis of titanium alloy and nylon. Because the prosthetic beak could not be maintained due to mandible and soft-tissue inflammation, the stork was euthanized. Still, we confirmed typical behavior and feeding for ~3 months after surgery. This report highlights some of the challenges we encountered and identifies process improvements required for a more successful surgery.

Cold agglutinin disease, one of the serological classifications of immune-mediated hemolytic anemia, is caused by the production of autoantibodies that react with erythrocytes at low temperatures. A captive bottlenose dolphin presented with regenerative and hemolytic anemia. Anticoagulated whole blood was agglutinated at room temperature (approximately 18°C), with reversal of agglutination on warming to 37°C, indicating the presence of cold agglutinin. Based on these findings, this animal was diagnosed with cold agglutinin disease. Clindamycin, doxycycline, and prednisolone were administered orally to treat the infection and immune-mediated hemolytic anemia. Anemia gradually improved after initiation of pharmacotherapy, and erythrogenesis slowed as erythroblasts disappeared and reticulocyte count decreased in peripheral blood. This represents the first report of cold agglutinin disease in a cetacean.

A 7-year-old female sugar glider showed a well-defined, homogeneous, hypoechoic mass in the caudal abdominal cavity on ultrasound. Exploratory laparotomy revealed both uteri were severely dilated by viscous, purulent exudate. The patient underwent ovariohysterectomy with removal of bilateral vaginal canals. Antibiotic therapy was initiated postoperatively. Histopathological findings were consistent with suppurative metritis, leading to a clinical diagnosis of pyometra. Despite abscess formation in the uterine stump or central vaginal canal and bilateral renal pelvic/ureter dilations postoperatively, the sugar glider survived more than 270 days. Unfortunately, surgical and medical treatments performed in this case did not prove curative. The outcome of this case highlights the importance of further accumulation of cases to define more appropriate therapies for pyometra in sugar gliders.