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A 21-year-old female spotted seal (Phoca largha), with a swollen abdomen, had a five-month history of anorexia and vomiting. Ultrasonography revealed an extended mass with central necrotic foci in the right cranial abdomen. Computed tomography revealed an abdominal mass with a low-density central lumen and a pulmonary nodular lesion. Cytology of an abdominal specimen collected through fine-needle aspiration indicated a malignant tumor with round, atypical cells with large nuclei. Three days after diagnosis, necropsy revealed a 10-cm large, solid, whitish mass in the pancreatic parenchyma and multiple small nodules in the liver, spleen, mesentery, lungs, and mediastinal lymph nodes. Histopathological analysis showed prolific neoplastic cells with marked atypia and occasional keratinization. Immunohistochemistry revealed that the neoplastic cells were positive for cytokeratin AE1/AE3 antibody. Thus, the seal was diagnosed with squamous cell carcinoma, of presumed pancreatic origin, which had metastasized to multiple organs.

L-type amino acid transporter 1 (LAT1) is upregulated in various malignant tumors in humans. LAT1 expression correlates with the grade of cancer and prognosis. LAT1 is responsible for the supply of many essential amino acids to cancer cells. Inhibition of LAT1 reduces the amino acids that enter the cell and inhibits cancer cell growth. Therefore, novel anticancer drugs targeting LAT1 have attracted much attention in recent years. In this study, to explore the applicability of using LAT1 expression in intracranial tumors as a prognostic factor and therapeutic target, we investigated the expression of LAT1 in surgically resected primary and secondary intracranial tumor tissues from dogs and cats. Immunohistochemical analysis of LAT1 was performed on intracranial tumor tissue from 14 dogs and 3 cats. Primary intracranial tumors were seen in 10 dogs and included meningiomas, histiocytic sarcomas, pituitary tumors, and gliomas, and 9 out of 10 cases were positive for LAT1. Primary intracranial tumors were seen in 2 cats and included meningioma and lymphoma; both cases were positive for LAT1. Secondary intracranial tumors were positive for LAT1 in 3 out of 4 cases in dogs and 1 out of 1 in cats. Since the majority of intracranial tumors in dogs and cats were positive for LAT1, immunostaining for LAT1 is expected to be a prognostic indicator and therapeutic target in the future.

We report a case of systemic metastasis of malignant melanoma in a mini-Rex (Oryctolagus cuniculus). The animal presented with lameness of the right hind limb, swelling of popliteal lymph node, and a black mass on the first digit. Paralysis of hindlimbs and forelimbs, dysuria, and dysphagia progressed over time, and the rabbit died on day 35 from the first visit. At necropsy, many black lesions were observed in multiple organs including the marrow of most bones. Histopathologically, the tumor cells had highly atypical nuclei of various sizes and an abundant eosinophilic cytoplasm, and some cells contained melanin granules. These cells were positive for PNL2 and S-100, melanoma markers, by immunohistochemistry. This is the first report of malignant melanoma in a mini-Rex with severe malignancy and systemic metastasis including the bone marrow.

Background Feline leukemia virus (FeLV) is a retrovirus with global impact on the health of domestic cats and is usually examined by serology. In our daily clinical practice, we noticed that cats infected with FeLV often possess wavy whiskers (sinus hairs on the face). To investigate the relationship between wavy whiskers (WW) and FeLV infection, the association between the presence or absence of wavy changes in whiskers and serological FeLV infection was examined in a total of 358 cats including 56 cats possessing WW, using the chi-square test. The results of blood tests from 223 cases were subjected to multivariate analysis (logistic analysis). Isolated whiskers were observed under light microscopy, and upper lip tissues (proboscis) were subjected to histopathological and immunohistochemical analyses. Results The prevalence of WW was significantly correlated with FeLV antigen positivity in the blood. Of 56 cases with WW, 50 (89.3%) were serologically positive for FeLV. The significant association between WW and serological FeLV positivity was also confirmed by multivariate analysis. In WW, narrowing, degeneration, and tearing of the hair medulla were observed. Mild infiltration of mononuclear cells in the tissues, but no degeneration or necrosis, was found. By immunohistochemistry, FeLV antigens (p27, gp70 and p15E) were observed in various epithelial cells including the sinus hair follicular epithelium of the whisker. Conclusions The data suggest that the wavy changes in whiskers, a unique and distinctive external sign on a cat’s face, were associated with FeLV infection.

The aim of this study was to evaluate the impacts of ophthalmic findings obtained from both macroscopic examination and ocular ultrasonography when diagnosing bovine endophthalmitis. A newborn crossbreed (Japanese black and Holstein breeds) calf was suspected of visual impairment and central nervous system (CNS) symptoms, such as decreased activity and weak drinking performance. This calf was found to display macroscopic signs, such as clouded lens, convergent strabismus, and horizontal nystagmus, in both eyes. On ocular ultrasonography of both eyes, a V-shaped, thickened, hyperechoic structure was present in the anechoic vitreous humors, indicating retinal detachment. The animal died 4 days after the examination. Sepsis was evident in this case, as Escherichia coli was isolated from multiple organs. The autopsy and histological examination revealed meningitis, encephalitis, and secondary hydrocephalus in the CNS, and endophthalmitis and retinal detachment in both eyes. In this case, the ophthalmic findings did not provide definitive evidence for a diagnosis of endophthalmitis. However, this study indicated that retinal detachment might be an ultrasonographic finding that is suggestive of bovine endophthalmitis.

Background In cattle, the lingual diseases are primarily diagnosed postmortem by histopathological examination of the affected tongues obtained after the death or during necropsy. In humans, ultrasonography has been used to provide differential diagnoses, and for preoperative or intraoperative planning of glossectomy in various lingual diseases. This is a bovine clinical case report, in which ultrasonography for sudden swelling of the tongue, which was possibly caused by snake bite, was utilized as a preoperative indication to perform a glossectomy. Case presentation An eight-month-old female Japanese black calf presented with sudden swelling of the tongue with well-defined discoloration in the cranial region. A 10-MHz linear probe on a portable-type ultrasound machine (MyLabOne VET, Esaote Co., Genova, Italy) was applied to the ventral surface of the tongues in the affected case, and also in five healthy calves under sedation to observe normal tongues. Ultrasonography of the swollen tongue in this case revealed that the ventral lingual muscular layers were severely thickened compared with those of normal tongues. However, the muscle layers were regularly aligned with the echogenic muscular fibers. This resembled the lingual muscular architectures of normal tongues. Color-flow Doppler ultrasonography revealed that blood flow was weakened in the small peripheral vessels in the spaces between the lingual muscular structures, and was lacking in the deep lingual artery between the apex and base of the tongue. This finding was very different than that of normal tongues, which exhibited weakened or rich blood flows. Based on ultrasonographic findings, this case was treated with glossectomy. After recovery, the calf grew up normally with a normal appetite and rumination, and did not exhibit mouth pain behavior. Histopathologically, hemorrhagic necrotic changes, together with focal formation of fibrin thrombus in the lingual blood vessels in the affected tongue, were noted. Conclusions To the best of our knowledge, the present report is the first description of lingual ultrasonography performed in cattle. In this case, ultrasonography enabled visualization of decreased vascularity, which might be associated with hemorrhage or formation of fibrin thrombus in the suddenly swollen tongue presented.

Background Inflammatory pseudotumours (IPTs) are distinctive lesions consisting of myofibroblastic spindle cells and a variety of inflammatory cells. The aetiology of IPTs is unknown. Reports of IPTs in veterinary medicine have been scarse. Moreover, only one case of intradural extramedullary IPT has been previously reported. In this report, we introduce the first known case of canine IPT, which occurred in the parenchyma of the spinal cord. Case presentation A 10-year-old female Miniature Dachshund presented with a 2-month-long history of progressively worsening ataxia and tetraparesis. Neurological examination was consistent with a lesion involving the cervical spinal cord. Magnetic resonance imaging revealed an intradural space-occupying lesion in the region of the fourth cervical vertebra. Dorsal laminectomy and resection of the mass were performed. Histopathological examination revealed the proliferation of immature spindle cells (fibroblasts/myofibroblasts and glial cells) and a highly cellular mixture of neutrophils, macrophages and lymphocytic cells. The mass was located in the parenchyma of the spinal cord and was diagnosed as an IPT occurring in the parenchyma of the spinal cord. No causative pathogen was detected. The dog’s symptoms improved, during the first month after surgery. However, neurological symptoms, such as laboured breathing and dysuria, subsequently worsened and the dog died 42 days after surgery. Conclusions The present study describes a canine case of IPT occurring in the parenchyma of the spinal cord. The diagnosis and determination of the site of the mass was difficult solely based on preoperative imaging in the present case. The outcome of this case was poorer than that observed in cases of canine extramedullary IPT and human intramedullary IPT, in which the patients exhibited recovery. The prognosis after surgical resection cannot be decided from the present case alone. However, patients should be monitored for potential serious complications and recurrence.

Background Nasal abnormalities are rare in bovines. In humans, nasal deformities are mainly classified as proboscis lateralis or supernumerary nostrils. This report discusses the etiology of triple nostrils in a calf, based on computed tomography, magnetic resonance imaging, and endoscopy. Case presentation A female Holstein calf presented with triple nostrils. The following abnormalities were observed: (1) formation of a small and flat blind-ended middle nostril between the right and left nostrils; (2) presence of a hair-bearing surface on the muzzle; (3) abnormal curvature of the nasal septum, resulting in a narrower right nasal cavity due to transformation of the nasal bones; and (4) formation of a bone-like structure within the nasal septum. These findings were similar to those of supernumerary nostrils in humans. Conclusions To the best of our knowledge, this represents the first description of a calf with triple nostrils. The use of imaging modalities is necessary for investigating the etiology of triple nostrils.

A captured Japanese wild boar (Sus scrofa leucomystax) fetus was dicephalic. The fetus had two heads, but one body from the cranial neck region. Computed tomography imaging revealed that the two crania merged at the occipital bone, and the vertebral bodies between the atlas and the seventh thoracic vertebra were deformed. The fetus was found to have two tongues and laryngopharynges, but its esophagus and trachea were not duplicated. Each head contained a cerebrum and cerebellum, but the brains merged at the obex of the medulla oblongata, and the cervical spinal cord had duplicated ventral clefts. The heart was composed of three atria and four ventricles. This is the first report of a dicephalus with cardiac malformation in a wild boar.

A 13-year-old spayed female dog had a mass in the left auricle. Grossly, connection between the mass and original auricular cartilage was not recognized. The mass was unencapsulated and contained multiple islands of mature hyaline cartilage and neoplastic adipocytes. The neoplastic cells comprised predominant mature adipocytes, scattered lipoblasts and irregular round to spindle cells with moderate atypia. The atypical cells occasionally had lipid droplets. A diagnosis of well-differentiated liposarcoma (WDL) with chondroid metaplasia was made. This is the first report for liposarcoma with chondroid metaplasia in the auricle of domestic animals.

A 2-month-old female Holstein calf and a 5-month-old female Japanese black calf presented with gingival vascular hamartoma located in the interdental space between the second and third mandibular incisors in the right and left mandibles, respectively. On radiographic or computed tomographic images, osteolytic changes appeared within the mandibular bones adjacent to the masses. The masses were removed along with affected mandibular bone by using unilateral rostral mandibulectomy. After surgery, both cases exhibited a normal appetite and grew normally, with no cosmetic changes or recurrences. Unilateral rostral mandibulectomy can be applied for invasive gingival vascular hamartomas associated with osteolytic changes.

Patients with hypoglycemic coma show abnormal signals in the white matter on magnetic resonance imaging. However, the precise pathological changes in the white matter caused by hypoglycemic coma remain unclear in humans and experimental animals. This study aimed to reveal the distribution and time course of histopathological and immunohistochemical changes occurring in the white matter during the early stages of hypoglycemic coma in rats. Insulin-induced hypoglycemic coma of 15-30-min duration was induced in rats, followed by recovery using a glucose solution. Rat brains were collected after 6 and 24 hr and after 3, 5, 7, and 14 days. The brains were submitted for histological and immunohistochemical analysis for neurofilament 200 kDa (NF), myelin basic protein, olig-2, Iba-1, and glial fibrillary acidic protein (GFAP). Vacuolation was observed in the fiber bundles of the globus pallidus on days 1-14. Most of the vacuoles were located in GFAP-positive astrocytic processes or the extracellular space and appeared to be edematous. Additionally, myelin pallor and a decrease in NF-positive signals were observed on day 14. Microgliosis and astrogliosis were also detected. Observations similar to the globus pallidus, except for edema, were noted in the internal capsule. In the corpus callosum, a mild decrease in NF-positive signals, microgliosis, and astrogliosis were observed. These results suggest that after transient hypoglycemic coma, edema and/or degeneration occurred in the white matter, especially in the globus pallidus, internal capsule, and corpus callosum in the early stages.

Background Peritoneal mesothelioma is a rare abdominal disease; that occasionally occurs congenitally in younger calves. Cytologic examination of peritoneal effusion (PE) was utilized to diagnose this disease, and was not diagnostic. Diagnostic accuracy has been elevated by recent use of ultrasonography (US), despite most diagnoses have been obtained post-mortem in slaughter houses or during clinical necropsy. In humans, ante-mortem diagnosis is highly associated with clinical use of computed tomography (CT) and laparoscopy together with imaging-assisted biopsy. The present report evaluates the diagnostic applicability of CT and laparoscopy as well as US via the practical application of these imaging modalities in an affected calf, and compares the cytologic and histologic findings among in PE, and specimens obtained from fine-needle aspiration and core-needle biopsy. In addition, the present results were reviewed in comparison with those of previous bovine and human reports. Case presentation A 58-day-old male Japanese black calf presented first with scrotal swelling, followed by progressive abdominal distention. Abnormalities of the case included: 1) accumulation of anechoic PE inside the swollen scrotum and abdomen; 2) formation of multiple echogenic nodules within the peritoneal membrane based on US images; 3) presence of hyper-dense spots (suspected calcification) along the margins of the nodules; 4) anatomic connections between intra-abdominal nodular lesions and the swollen tunica vaginalis via the inguinal region based on CT images; 5) serosanguineous-colored and less-turbid characteristics of PE; and 6) formation of multiple nodules over all of the serosa of the rumen as well as the peritoneal wall based on laparoscopic views. Fine-needle aspiration and core-needle biopsy were successfully performed under US and laparoscopic observations, respectively. Histology findings of the core-needle biopsy specimen appeared more indicative (characterization of tubular structures comprised of cubical or columnar abnormal mesothelial cell linings) diagnostically of peritoneal mesothelioma than did findings of the fine-needle aspiration specimen. Conclusions To the best of our knowledge, this report is the first description of clinical applications of CT and laparoscopy to diagnose peritoneal mesothelioma in a calf. Laparoscopy enhanced the diagnostic accuracy due to clear gross visualization of the intra-abdominal abnormalities and applicability to imaging-guided core-needle biopsy.

In this study, we investigated the pathogenesis of Newcastle disease virus (NDV) in the chicken kidney. Twenty-six 32-day-old specific pathogen-free chickens were intranasally inoculated with the 9a5b NDV mutant isolate. Kidney tissue samples, collected at 6 and 12 hours postinoculation (hpi) and 1, 2, 3, 5, and 10 days postinoculation (dpi), were analyzed by histopathology, immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), and virus titration. Histopathologically, tubulointerstitial nephritis was detected in the renal cortex and predominantly in the medulla. Nephrotropism of 9a5b NDV was confirmed by IHC, RT-PCR, and virus isolation. Massive degenerative changes and infiltration of CD3-immunopositive cells accompanied replication of the 9a5b NDV isolate in chicken kidneys. In conclusion, pathological changes that were caused by NDV in chicken kidneys were similar to those caused by avian influenza virus, infectious bronchitis virus, and avian nephritis virus, and this highlights the importance of including NDV in the differential diagnosis of kidney disease in chickens.

Inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal signs in dogs. In humans, T helper cells have important roles in the pathogenesis of IBD. In contrast, no specific involvement of a distinct T cell subset has been described in canine IBD. The present study evaluated the gene and protein expression of cytokines of T cell subsets in duodenal mucosa from dogs with IBD. Relative quantification of interleukin (IL)-17A, interferon (IFN)-γ, IL-4 and IL-10 mRNA transcription was performed using duodenal mucosa from 27 IBD dogs and 8 controls. Duodenal mucosal IL-17A, IFN-γ and IL-10 protein levels were determined by ELISA in 15 IBD dogs and 8 controls. There was no significant difference in each cytokines mRNA transcription level between groups. There was no significant difference in IL-17A, IFN-γ and IL-10 protein expression levels between groups. Thus, there is no clear evidence for the involvement of distinct Th cytokine in the pathogenesis of canine IBD.

A 2-month-old male Japanese Black calf was presented with a 30-day history of progressive ataxia. Antemortem examination using computed tomography (CT) revealed narrowing of the disc spaces due to destruction of intervertebral structures between the first and second thoracic vertebrae and between the second and third thoracic vertebrae. Osteolysis was evident as irregular hypoattenuating lesions within the opposing end plates of the first, second and third thoracic vertebrae. Pseudomonas aeruginosa was detected as the causative bacteria, and discospondylitis was diagnosed. To the best of our knowledge, this is the first bovine case report describing the application of CT for the diagnosis of discospondylitis.

An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3(+)CD4(+) cells and LAG-3(+)CD8(+) cells were conserved whilst the number of MHC class II(+) cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4(+) and CD8(+) T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II(+) cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-γ and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection.

A 9-year-old male mixed breed cat showed chronic progressive neurological symptoms, which are represented by ataxia and seizures. At necropsy, spinal roots and spinal ganglions at the level of sixth cervical nerve to second thoracic nerve were bilaterally swollen and replaced by white mass lesions. Right brachial plexus and cranial nerves (III, V and VII) were also swollen. A mass lesion was found in the right frontal lobe of the cerebrum. Histologically, neoplastic lymphocytes extensively involved the peripheral nerves, and they infiltrated into the cerebral and spinal parenchyma according to the peripheral nerve tract. Immunohistochemically, most neoplastic lymphocytes were positive for CD20. The clinical and histological features in this case resemble those of neurolymphomatosis in humans.

A previous study showed that increases in chemokine expression and recruitment of antibody secreting cells (ASCs) in the CNS after intracerebral immunization contributed to the suppression of a neurotropic virus. In this study, intracerebral chemokine injection was used to investigate the usefulness of chemokines for controlling neurotropic viruses. Both CXCL12 and a cocktail chemokine (a mixture of CXCL9, 10, 12 and 13) attracted antigen-specific ASCs more strongly than CXCL9, 10 and 13 in an in vitro chemotaxis assay and in vivo intracerebral chemokine injection experiments. Mice pre-treated intracerebrally with CXCL12 and the cocktail chemokine showed an increased survival rate after intracerebral infection with rabies virus. These results suggest that intracerebral CXCL12 injection induces the migration of ASCs and suppresses the neuropathogenicity of rabies virus in the CNS.

Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4 + and CD8 + cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4 + T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8 + T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases.

Cardiomyopathies have been rarely described in rabbits. Here we report myocardial necrosis of the ventricular wall in rabbits with experimentally induced rabies. Myocardial lesions were found only in rabbits with brain lesions, and the severity of the cardiac lesions was proportional to that of the brain lesions. Neither the frequency nor the cumulative dose of anesthesia was related to the incidence or the severity of the myocardial lesions. The myocardial lesions were characterized by degeneration and/or necrosis of myocardial cells and were accompanied by contraction band necrosis, interstitial fibrosis, and infiltration of inflammatory cells. The brain lesions due to rabies virus infection were most prominent in the cerebral cortex, thalamus, hypothalamus, brainstem, and medulla. Rabies virus antigen was not found in the hearts of any rabbits. Based on these findings, the myocardial lesions were classified as neurogenic cardiomyopathy.

An adult swan goose (Anser cygnoides) kept in a zoological garden had gross hepatic enlargement with extensive ill-defined white foci. Microscopically, the hepatic lesions were composed of a mixture of adipocytes and myeloid cells. The goose was also affected with systemic amyloiclosis and there were areas of osseous metaplasia associated with deposition of amyloid within the liver.

Swine hemagglutinating encephalomyelitis virus (HEV) causes encephalomyelitis or vomiting and wasting disease in suckling piglets. Neurotoropism of the virus has been demonstrated in previous in vivo studies. In the present study, we investigated the infectivity and propagation of HEV in comparison with those of pseudorabies virus (PRV), another neurotropic virus, using dorsal root ganglia cells of newborn mice containing nerve cells and non-neuronal cells. HEV infected nerve cells but did not infect non-neuronal cells, whereas PRV infected both cell types. By using cytoskeletal inhibitors, it was suggested that propagation of HEV and PRV within and among nerve cells depended on microtubules and intermediate filaments of nerve cells, indicating that the viruses may be transported between the cell body and axonal terminals of neurons by fast axonal flow.

Canine mammary gland tumors (CMGTs), which are the most common neoplasms in sexually intact female dogs, have been suggested as a model for studying human breast cancer because of several similarities, including relative age of onset, risk factors, incidence, histological and molecular features, biological behavior, metastatic pattern, and responses to therapy. In the present study, we established a new cell line, the SNP cell line, from a CMGT. A tumor formed in each NOD.CB17-Prkdc(scid)/J mouse at the site of subcutaneous SNP cell injection. SNP cells are characterized by proliferation in a tubulopapillary pattern and are vimentin positive. Moreover, we examined miRNA expression in the cultured cells and found that the expression values of miRNA-143 and miRNA-138a showed the greatest increase and decrease, respectively, of all miRNAs observed, indicating that these miRNAs might play a significant role in the malignancy of SNP cells. Overall, the results of this study indicate that SNP cells might serve as a model for future genetic analysis and clinical treatments of human breast tumors.

The aim of this study was to investigate the effect of Newcastle disease virus (NDV) on the chicken pancreas. A virulent 9a5b mutant NDV isolate was inoculated intranasally into 32-day-old specific pathogen-free white Leghorn chickens. The pancreas was examined grossly and fixed for histopathological, immunohistochemical and electron microscopical investigations. Inflammatory changes were observed in the peripancreatic tissue at the early stage of infection (12 h post infection) and became more prevalent towards the end of the experiment. Multifocal areas of necrotizing inflammation were detected in the exocrine portion of the pancreas by 5 days post infection (dpi) and became more severe at 10 dpi. The endocrine islets were generally preserved, but slight degenerative changes were observed at 10 dpi. Immunohistochemically, NDV-nucleoprotein (NDV-NP) signals were detected in the peripancreatic tissues (associated with macrophages and other lymphoid cells) by 1 dpi. In the exocrine portion of the pancreas, NDV-NP signals were detected at 5 dpi and increased in intensity and distribution by 10 dpi. NDV particles were confirmed in the cytoplasm of exocrine acinar cells by transmission electron microscopy. CD3-positive cells were observed in the peripancreatic tissues earlier than in the pancreatic tissue. Moreover, in comparison with control chickens, insulin immunoexpression was unchanged, except on the last day of the experiment, when it was slightly reduced. The 9a5b NDV infection induced an inflammatory reaction and viral replication in the peripancreatic tissues earlier than in the pancreatic tissue. Furthermore, necrosis affected mainly the exocrine portion of the pancreas, while the endocrine portion was generally unaffected.

Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem. 26 , 34 Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. To evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b) and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. An asymmetric distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side 2 days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The results of this study, thus, demonstrate that neurotropic influenza virus travels to the CNS mainly via the vagus nerve.

One-hundred thirty-seven BALB/c mice were intranasally inoculated with neurotropic avian influenza A virus (H5N3). Thirty-nine of these mice died within 16 days post-inoculation (PID) and 98 of the mice recovered from the infection. To investigate whether viral antigens and genomes persist in the central nervous system (CNS) of recovered mice, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) methods were performed. Histopathologically, mild interstitial pneumonia and non-suppurative encephalomyelitis restricted to the basal part of the frontal lobe of the cerebrum, brain stem and thoracic spinal cord were observed in BALB/c mice until 40 PID. Small amounts of viral antigens were detected in the brain and spinal cord and some viral RNA segments (NA, NP, M, PA, HA, NS, PB1) were intermittently detected in the CNS until 48 PID. Immunosuppression of these mice by dexamethazone (DEX) treatment did not increase the frequency of detection of the lesions, viral antigens or genomes. These findings suggest that viral genomes of neurovirulent influenza virus persist with restricted transcriptive activity in the CNS of the mice even after clinical recovery from the infection.

Objective—To determine the expression of tight junction and adherens junction proteins in duodenal mucosa samples of dogs with inflammatory bowel disease (IBD). Animals—12 dogs with IBD and 6 healthy control Beagles. Procedures—Duodenal mucosa biopsy samples were endoscopically obtained from dogs with IBD and healthy control Beagles. The expression of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and β-catenin in the duodenal mucosa samples was determined by means of immunoblotting. The subcellular localization of E-cadherin in the duodenal mucosa samples was determined with immunofluorescence microscopy. Results—The expression of each claudin and β-catenin was not significantly different between control dogs and dogs with IBD. However, expression of E-cadherin was significantly lower in duodenal mucosa samples of dogs with IBD than it was in samples obtained from healthy control dogs. Results of immunofluorescence microscopy indicated decreased intensity of E-cadherin labeling in the tips of villi in duodenal mucosa samples obtained from 6 dogs with IBD, compared with staining intensity for other dogs. Conclusions and Clinical Relevance—Results of this study indicated expression of claudin-1, -2, -3, -4, -5, -7, and -8 and β-catenin was not significantly different between duodenal mucosa samples obtained from control dogs and those obtained from dogs with IBD. However, E-cadherin expression was significantly lower in the villus epithelium in duodenal mucosa samples obtained from dogs with IBD versus samples obtained from control dogs, which suggested that decreased expression of that protein has a role in the pathogenesis of IBD in dogs.

A 5-year-old male cross-breed dog with chronic rhinosinusitis was presented. Computed tomography (CT) revealed opacities consistent with soft tissue or fluid in the left nasal cavity and frontal sinus. A diagnosis of chronic rhinosinusitis with myxomatous polyps was based on the histology of biopsy samples, so prednisolone treatment was administered for 1 month. Although the clinical signs slightly improved, no changes were observed on the CT images. At 8 months after the initial presentation, the dog was treated with antivascular photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A and, 11 days later, CT revealed remarkably decreased soft tissue opacity; the facial swelling and sneezing also resolved. Three additional sessions of PDT were performed at 114, 210, and 303 days after the first PDT because of recurrence of clinical signs. The disease was well controlled for approximately 10 months by antivascular PDT.

The inhibitory receptor programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV) infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection.

Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.

To investigate the effect of antipyretics on the murine and poultry models of influenzal encephalitis, we injected large quantities of antipyretics, acetylsalicylic acid (aspirin) and dicrofenac sodium (voltaren). The effect of antipyretic treatment on the murine encephalitis model was unremarkable histologically and immunohistochemically. Whereas in chicks, CNS lesions consisting of perivascular cuffing and gliosis appeared only in those animals treated with the antipyretics and viral antigen was detected mainly in the nuclei of glial cells or vascular endothelia of voltaren-treated animals. We here demonstrate that antipyretic treatment aggravated the hematogenous spread of the influenza virus to the CNS in chicks, but did not affect the transneural infection in mice.

The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.