In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

An epidemiological analysis was conducted on production records in Hokkaido, Japan, to investigate the potential association between improved milk quality and longevity outcomes. The study found significant variations in herd somatic cell count levels and chronic subclinical mastitis morbidity based on geographical area and herd size. The analysis also revealed a positive correlation between herd somatic cell count and chronic subclinical mastitis morbidity. Although the hypothesis of a causal link between milk quality and longevity was examined, no such association was found. However, intensive assistance for identified high-risk areas and farms is expected to enhance overall milk quality.

Immune responses in bovine clinical mastitis (CM) probably differ depending on the causative pathogen and disease severity. The observational study aimed to investigate whether both factors are associated with the dynamics of immune indicators, including somatic cell score (SCS), white blood cell count (WBC), serum albumin/globulin (A/G) ratio, and differential somatic cell count (DSCC). We collected blood and milk samples 0, 3, 5, 7, 14, and 21 days after CM occurred in 38 cows, and grouped the cases (n=49) by disease severity and pathogen. We analyzed data using a linear mixed model considering the effects of pathogens and severity, calculated estimated-marginal means for indicators at each time point, and compared the means between groups. The dynamics of WBC varied depending on both pathogen and severity. WBC changed drastically in either severe or coliform-caused CM, slightly elevated in streptococcal mastitis, but unchanged in staphylococcal mastitis. This possibly relates to the deficiency in innate immune response toward staphylococci. The A/G ratio also changed depending on severity, as it dropped sharply only in severe CM. We observed a non-linear relationship between DSCC and SCS, possibly due to mammary epithelial cells shedding in milk when CM occurred. When cows recovering from Streptococcus dysgalatiae mastitis, DSCC decreased while SCS remained high, suggesting a healing process requiring more macrophages. Our results demonstrate that both the severity and pathogen are associated with immune responses in CM, providing insights into mastitis pathogenesis.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

The vertebral heart scale (VHS) is the most common method used for the objective evaluation of heart size, however, VHS cannot evaluate the entire heart area. This retrospective study aimed to evaluate the development and feasibility of vertebral heart area ratio (VHAR=heart area/the fourth thoracic vertebra [T4] body area) as a new cardiac measurement method. A total of 125 dogs considered without clinical heart abnormalities in the pre-anesthetic examination, measurements of the VHS and VHAR using thoracic radiography were compared with computed tomography measurements of the vertebral cardiac volume ratio (VCVR=cardiac volume/T4 body volume) and investigate whether the VHAR values differed between observers. The mean cardiac and T4 body volumes were 116.99 ± 108.07 cm³ and 0.92 ± 0.91 cm³, respectively. The mean values of observers 1 and 2 were 9.9 ± 0.7 v (VHS), 42.64 ± 27.94 cm² (heart area), and 1.37 ± 0.96 cm² (T4 body area). Intraclass coefficients were the highest for the heart area, followed by the T4 body area and VHS. The VHAR showed a moderate correlation with VHS in observers 1 (r=0.671) and 2 (r=0.633). The VCVR showed a more positive correlation with VHAR (r=0.573) than with VHS (r=0.426). These results indicated that VHAR could be used as a complement to VHS for heart size measurement, and the high degree of observer agreement for the measurements indicated the measurement reproducibility of VHAR.

Here, we present a case of severe glomerular fibrin thrombosis in a dog with lymphoma. A 3-year-old neutered male Chihuahua presented with acute kidney injury, hypoalbuminemia, and transudate ascites. The dog showed symmetric enlargement of the spleen, which was diagnosed as B-cell lymphoma based on cytology and polymerase chain reaction tests. The dog died after intensive care, and the kidneys were removed for histopathological examination. Light microscopy, immunofluorescence, and electron microscopy analyses were performed for renal pathology; however, the findings did not support the evidence of protein-losing nephropathy. Instead, the endocapillary accumulation of fibrin thrombi was prominent in most glomeruli. A diagnosis of severe glomerular fibrin thrombosis was established, and hypoalbuminemia was considered the underlying cause of kidney damage.

Suncus murinus is gaining prominence as a laboratory animal; however, there is no generally accepted method for microbiological monitoring. This study aimed to apply non-serological microbiological monitoring of laboratory mice for S. murinus and identify the subdominant species obtained by culture methods for microbial assessment. Culture and PCR were used to test S. murinus for the laboratory mice test panels including 10 bacterial species and orthohantaviruses, all of which were negative. The species that grew sub-dominantly in rectal feces were identified as Aeromonas hydrophila, which is pathogenic to mammals. These results indicate that microbiological monitoring should be used to detect pathogens directly from S. murinus, not from sentinel animals, due to the host-specific microbial environment.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Cats normally play a role of the definitive host in which the plerocercoid (sparganum), the second larval form of Spirometra spp., develops into an adult in the intestines. However, some cases of cats with visceral or subcutaneous sparganosis were sporadically reported worldwide. We herein documented the discovery of a sparganum in abdominal cavity of a domestic cat during a surgery of dystocia. The larva was molecularly identified as Spirometra mansoni, belonging to Type I, that was recently misidentified to be S. erinaceieuropaei in several Asian countries. This is the first report for sparganum of S. mansoni in the cat. The future study is necessary to provide further insights into the species of Spirometra causing sparganosis and spirometrosis in humans and other animals.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine tumor, and more than 90% of feline MCC cases test positive for Felis catus papillomavirus type 2 (FcaPV2). In the present study, basal cell markers p40, p63, and p73 and the stem cell marker SOX2 and cytokeratin 14 (CK14) were immunohistochemically examined in normal fetal, infant, and adult feline skin tissues. The expression of these proteins was examined in tumors positive for FcaPV2, including MCC, basal cell carcinoma (BCC), Bowenoid in situ carcinoma (BISC), and squamous cell carcinoma (SCC). Infant and adult feline skin tissues had mature Merkel cells, which were CK14-, CK18+, CK20+, SOX2+, synaptophysin+ and CD56+, while fetal skin tissue had no mature Merkel cells. MCC was immunopositive for p73, CK18, and SOX2 in 32/32 cases, and immunonegative for CK14 in 31/32 cases and for p40 and p63 in 32/32 cases. These results indicate that MCC exhibits different immunophenotypes from Merkel cells (p73-) and basal cells (p40+, p63+, and SOX2-). In contrast, all 3 BCCs, 1 BISC, and 2 SCCs were immunopositive for the basal cell markers p40, p63, and p73. The life cycle of papillomavirus is closely associated with the differentiation of infected basal cells, which requires the transcription factor p63. Changes in p63 expression in FcaPV2-positive MCC may be associated with unique cytokeratin expression patterns (CK14-, CK18+, and CK20+). Furthermore, SOX2 appears to be involved in Merkel cell differentiation in cats, similar to humans and mice.

Our previous study indicated that cytotoxicity of intraepithelial lymphocytes is a poor prognostic factor in feline intestinal T-cell lymphoma (FITL), but the effect of cytotoxic lymphocytes on mucosal epithelium is still unknown. Thus, we investigated the association between cytotoxic lymphocytes and mucosal epithelium in 71 cases of feline intestinal T-cell lymphoma (FITL): epithelial injury, basement membrane injury, cleaved-caspase-3 positivity of epithelial cells, and the number and Ki67 positivity of intraepithelial lymphocytes in granzyme B (GRB)+ and GRB- FITLs were evaluated. Epithelial injury score and the number of intraepithelial lymphocytes in granzyme B (GRB)+ FITL were significantly higher than those of GRB- FITL (P<0.05, P<0.05), but no significant differences were found in the basement membrane injury score, the percentage of cleaved-caspase-3+ epithelial cells, and the percentage of Ki67+ intraepithelial lymphocytes. There was a significant correlation between the epithelial injury score and the number of intraepithelial lymphocytes (P<0.05), but no significant correlation was observed between the epithelial injury score and Ki67+ percentage of intraepithelial lymphocytes. Because epithelial cell cleaved-caspase-3 positivity was observed in FITL, regardless of GRB expression in lymphocytes, GRB-mediated apoptosis may not contribute to epithelial injury in FITL. The association between increased number of intraepithelial lymphocytes and epithelial injury suggests that intraepithelial lymphocytes infiltration may contribute to epithelial injury in FITL.

After an Egyptian fruit bat (Rousettus aegyptiacus) in a zoo became emaciated and died, a necropsy revealed multiple nodules on the liver and lung surfaces. Microscopy revealed that the liver nodules consisted of neoplastic hepatocytes and showed metastasis in the lung lobes. Most of the neoplastic cells in the liver and lung showed positive labeling for HepPar-1, cytokeratin 19, glypican-3, and Ki-67. Hepatocellular degeneration and necrosis were diffuse in the liver parenchyma. Berlin blue staining revealed large amounts of iron in normal and neoplastic cells. Based on these pieces of evidence, this case was diagnosed as hepatocellular carcinoma with hemochromatosis. This is believed to be the first report of hepatocellular carcinoma in an Egyptian fruit bat that has been immunophenotypically examined in detail by pathological examination.

Rabbit Fibroma is a Leporipoxviral disease and is considered the third most common cutaneous neoplasm in pet rabbits. Two domestic rabbits (Oryctolagus cuniculus) were submitted to the veterinary clinic due to the presence of a nodule on the lip. Histologically, epithelial cells of the epidermis and hair follicles showed mild to moderate ballooning degeneration, spongiosis, and several eosinophilic intracytoplasmic inclusion bodies. The dermis was expanded by atypical spindle cells that also showed eosinophilic intracytoplasmic inclusion bodies. The tissues were evaluated by using transmission electron microscopy. In both cases, keratinocytes exhibit several electron dense and pleomorphic intracytoplasmic viral particles consistent with Poxviruses. To our knowledge, this is the first case report of Rabbit Fibroma Virus infection in Domestic Rabbits in Mexico.

Primary cardiac tumors in animals are very rare. The purpose of this report was to describe the first case of a cardiac tumor comprising a malignant peripheral nerve sheath tumor and spontaneous atrial osseous metaplasia in a Corriedale sheep. Histologically, the tumor in the bilateral atrial pericardium consisted of dense cellular components comprising tumor cells and a sparse cellular area, and non-neoplastic mature bone tissue. The tumor cells were spindle-shaped, round, or polygonal, and proliferating, with fascicular, storiform, palisading, and sheet patterns. Immunohistochemically, the tumor cells were positive for vimentin, S-100, occasionally positive for myeline basic protein, glial fibrillary acidic protein, neurofilament, neuron specific enolase, and neuron growth factor receptor suggesting that they originated from the nervous system. On the basis of these findings, the final diagnosis was a malignant peripheral nerve sheath tumor and spontaneous atrial osseous metaplasia.

The effects of an extended photoperiod (EP) on body composition of Thoroughbreds colts and fillies from December at one year old to April at two years old were investigated. Seventy-three Thoroughbreds reared and trained in Hidaka Training and Research Center, Japan Racing Association, Hokkaido were used. Forty-one horses were under the EP conditions from December 20 to April 15, and the 32 horses were under natural light alone as the control group. Body weight (BW), rump fat thickness (RFT), fat free mass (FFM) and percentage of fat (%F) were used as parameters of body composition. The present study revealed that BW and FFM increased with age in the EP group in colts. In fillies, BW increased with age in both the EP and the control group, however FFM increased with age only in the EP group. From December to April, only colts had a higher rate of increase in both BW and FFM in the EP group than in the control group. However, according to the mean rates of increase in FFM from January to March, the EP group was significantly higher than the control group in both sexes. Furthermore, monthly increase rate of FFM in March was significantly higher in the EP group than in the control group in both sexes. These results suggests that EP treatment to young Thoroughbreds in training at Hokkaido, which is shorter daylength in winter, accelerate the increase of FFM, representing muscle mass.

A retrospective study involving 14 pet rabbits histopathologically diagnosed with malignant peripheral nerve sheath tumors (MPNSTs) was conducted. The age at diagnosis was 4–12 years, with a median age of 8.6 years. All rabbits had solid subcutaneous tumor masses in varied locations. Surgical excision of the tumors was performed in all cases. Recurrence was observed in 10 cases (71%), and postoperative metastasis to the lung was suspected in 4 cases (29%). The postoperative mean and median survival times were 11 months and 9 months, respectively. Hence, MPNSTs should be considered in the differential diagnosis for subcutaneous masses in rabbits and it is essential to inform the owners of the potentially high recurrence and metastasis rates.

A 5-year-old neutered female mixed cat presented with reduced activity and ataxia of the hind limbs. Computed tomography and magnetic resonance imaging revealed an extradural mass compressing the spinal cord on the dorsal aspects from the 7th to 8th thoracic vertebra. Dorsal laminectomy was performed on the 7–8th thoracic vertebra and the cyst was totally removed, giving full resolution of the clinical signs. The cyst was diagnosed as a dermoid cyst. To our knowledge, this is the first report of feline dermoid cyst compressing the spinal cord that was diagnosed antemortem. The prognosis is favorable when the cyst is completely resected.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a mouse model with defined point mutation in primary receptor for alphaherpesviruses, nectin-1, by the CRISPR/Cas9 system. It has become clear that phenylalanine at position 129 of nectin-1 is important for binding to viral glycoprotein D (gD), and mutation of phenylalanine 129 to alanine (F129A) prevents nectin-1 binding to gD and virus entry in vitro. Here, to assess the antiviral potential of the single amino acid mutation of nectin-1, F129A, in vivo, we generated genome-edited mutant mouse lines; F129A and 135 knockout (KO). The latter, 135 KO used as a nectin-1 knockout line for comparison, expresses a carboxy-terminal deleted polypeptide consisting of 135 amino acids without phenylalanine 129. In the challenge with 10 LD₅₀ PRV via intranasal route, perfect protection of disease onset was induced by expression of the mutation of nectin-1, F129A (survival rate: 100% in F129A and 135 KO versus 0% in wild type mice). Neither viral DNA/antigens nor pathological changes were detected in F129A, suggesting that viral entry was prevented at the primary site in natural infection. In the challenge with 50 LD₅₀ PRV, lower but still strong protective effect against disease onset was observed (survival rate: 57% in F129A and 75% in 135 KO versus 0% in wild type mice). The present results indicate that single amino acid mutation of nectin-1 F129A provides significant resistance against lethal pseudorabies.

Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.

A 22-year and 9-month-old female Grevy’s zebra (Equus grevyi) showed signs of polyuria, polydipsia, glucosuria, and muscle atrophy. Blood tests revealed hyperglycemia, hypertriglyceridemia, electrolyte imbalance, high levels of adrenocorticotropic hormone (ACTH) and cortisol, and low levels of hormones secreted by the pituitary pars distalis. Pathological examinations revealed a pituitary gland tumor and bilateral adrenal cortical hyperplasia. Pituitary tumor cells showed immunoreactivity for α-melanocyte-stimulating hormone and ACTH. The deposition of amyloid β was observed in the parenchyma and vascular walls of the cerebrum. The zebra showed clinical signs of pituitary pars intermedia dysfunction and was histopathologically diagnosed with pituitary gland melanotroph adenoma. This case report provides insight into neoplastic and endocrine diseases associated with the aging of a zebra.