Non-suppurative and necrotizing testicular arteritis in the male reproductive organs of a heavy draft horse.

Equine testicular arteritis commonly occurs as a consequence of the migration of nematode larvae or equine arteritis virus (EAV) infection. However, testicular arteritis without evidence of these infections has been reported, and the underlying pathogenesis remains unclear. We encountered testicular arteritis without evidence of nematode or EAV infection in a 3-year-old male heavy draft horse with scrotal enlargement. Grossly, the volume of the pampiniform plexus was markedly increased due to edema. Histologically, non-suppurative and necrotizing testicular arteritis, characterized by lymphocyte infiltration and fibrinoid necrosis of the arterial walls, was diffusely observed in the spermatic cord, pampiniform plexus (most severe), testis, and epididymis. We were unable to identify the cause of arteritis, such as a viral infection or autoimmune abnormality.

Chronic otitis externa with heat shock protein 70-positive intranuclear inclusion bodies in the ceruminous gland epithelium of a Chihuahua dog.

A Chihuahua dog showed persistent itching in the right ear canal. Anti-inflammatory medicines and prednisolone were ineffective and total ear canal ablation was performed. Histological diagnosis was chronic otitis externa. Eosinophilic intranuclear inclusion bodies (Cowdry type A and full-type) were occasionally observed in the ceruminous gland epithelium. The inclusion bodies were negative for nucleic acid and ultrastructurally composed of fibrous structures (approximately 10 nm in width). Viral infection was initially suspected, but polymelase chain reaction tests did not detect the expected viral genes. Immunohistochemistry revealed that the inclusion bodies were positive for heat shock protein 70 (HSP70), suggesting that these bodies could be protein aggregates including HSP70. The etiology of this lesion has not been elucidated, but chronic inflammation may influence the cytoplasm-to-nuclear transportation of HSP70. To the best of our knowledge, this is the first report of canine chronic otitis externa with HSP70-positive intranuclear inclusion bodies.

Spontaneous pulmonary adenocarcinoma in a common marmoset (Callithrix jacchus).

A seven-year-old female common marmoset (Callithrix jacchus) presented with weight loss. Imaging revealed a left thoracic mass, confirmed at necropsy. Histology and immunohistochemistry suggested a well-differentiated pulmonary adenocarcinoma. No evidence of local lymphovascular invasion or distant metastasis was observed. This is the first report of pulmonary adenocarcinoma in marmosets.

Molecular characterization of Trichomonas gypaetinii isolated from the upper alimentary tract of Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, Japan.

Recent phylogenetic and morphologic studies of Trichomonas spp. suggests that there are more than 3 species that infect the upper alimentary tract of wild birds, which include T. gallinae, T. stableri, and T. gypaetinii. In this study, investigations were conducted on the prevalence of trichomonads in the upper alimentary tract of 12 Steller's sea eagles (Haliaeetus pelagicus) and 18 white-tailed sea eagles (H. albicilla). All birds were rescued from the wild and kept at a rehabilitation facility in Hokkaido, Japan, for variable durations and did not show any symptoms of trichomonosis. The ITS1-5.8SrRNA-ITS2 (ITS) genomic region of Trichomonas spp. was detected from 29 samples by PCR, and flagellates were confirmed from 4 samples by culture. Morphologic observations and measurement recordings were conducted under a light microscope on trophozoites obtained from the cultured isolates. Genomic sequences of the ITS, 18S ribosomal RNA (18S rRNA), Fe-hydrogenase, and RNA polymerase II largest subunit (Rpb1) regions were determined by direct sequencing, and phylogenetic analyses were conducted with previously published sequences of Trichomonas spp. All isolates were concluded as T. gypaetinii based on morphologic and molecular characterizations of the ITS and 18S rRNA genes. This is the first study to isolate T. gypaetinii from Haliaeetus eagles and further provide novel sequences of the Fe-hydrogenase and Rpb1 genes of T. gypaetinii. Both genomic regions also confirmed that T. gypaetinii belong to independent clusters from other Trichomonas spp.

Morphological analyses of nephrin expression in progressive glomerulonephropathy of common marmosets.

In this study, we focused on nephrin, one of the key molecules within the slit diaphragm of podocytes, as although there have been reports on its expression in humans and rats, their presence in common marmosets has not been reported. We investigated nephrin expression and changes in glomeruli, depending on the development of spontaneous progressive glomerulonephropathy in common marmosets. Nineteen common marmosets at two to ten years of age were evaluated. The kidney was examined by microscopy with hematoxylin and eosin and immunohistochemical staining for nephrin. The lesions were classified into three grades according to a renal lesion grading system reported previously. The nephrin-positive area was measured by morphometric analysis, and the nephrin-positive ratio was calculated. Nephrin expression was observed along the glomerular capillary loop in a continuous linear pattern in renal lesion grades 0 to 2 and either discontinuous linear or coarse granular pattern in grade 3. Nephrin expression tended to decrease significantly depending on the grade of renal lesions. Alteration in nephrin expression has been suggested to play an important role in the progression of renal lesions.

Case report: The case of a 17 kg ovarian granulosa cell tumor in a Breton draft mare.

Granulosa cell tumor (GCT) is a benign tumor which affects the mare's ovaries. In this report, a case of unilateral GCT in an ovary, which weighed 17.04 kg, of a 9-year-old Breton draft mare is described. A transrectal ultrasonography exam revealed a unilateral multi-cystic enlarged ovary. Laparoscopic ovariectomy was difficult due to enlargement of blood vessels in the ovarian broad ligament. The mare was necropsied, and the pathological changes in the GCT-affected ovary and unaffected ovary were evaluated. The ovarian mass in the GCT-affected ovary had a cribriform pattern and was positive for anti-Müllerian hormone (AMH) and its receptor (AMHR2). The contralateral ovary showed no follicular development and was negative for AMH. AMHR2 was positively expressed in stromal cells. The AMH concentration in plasma was 4,210 ng/ml. This is the first report showing the presence of AMH (2,210 ng/ml) in ascites fluid, and it also shows that laparoscopic ovariectomy might not be suitable for larger ovaries affected by a GCT. Ultrasonographic, endocrine, and histopathological analyses were helpful for making a definitive diagnosis of GCT in this mare.

Systemic Amyloid A Amyloidosis in Stejneger's Beaked Whales (Mesoplodon stejnegeri).

Stejneger's beaked whales (Mesoplodon stejnegeri) are one of the lesser known species of mammals, with little information available on their population status or incidence of diseases. Recent pathologic investigations on stranded and bycaught wild cetaceans around Hokkaido, Japan, revealed an unusually high incidence of systemic amyloidosis in this species, warranting further investigation. The objective of this study was to further characterize the systemic amyloidosis of Stejneger's beaked whales by retrospective histopathologic analyses of tissues from animals that stranded in Japan between 1994 and 2018. Various tissues from 35 individuals were examined histologically with hematoxylin and eosin, Congo red, and immunohistochemistry for amyloid A (AA), in which 12 (34%) were diagnosed with systemic amyloidosis. The organs with the highest severity of amyloid deposition were the stomach and intestine. The type of amyloid was confirmed as AA of approximately 9 kDa by 2-dimensional gel electrophoresis with extracted amyloid from the liver and subsequent Western blotting with an antiserum against AA peptide. There were no statistically significant associations between amyloidosis and sex, body condition of the whales, or the presence of chronic inflammation. The high prevalence of this disease might be of concern for overall population numbers, and continued pathologic monitoring of stranded animals is necessary throughout its distributional range.

Organ blood flow in response to infusion of arginine vasopressin in premature fetal sheep.

BACKGROUND: Arginine vasopressin (AVP) infusion has been shown to be a useful strategy for the management of systemic perfusion failure in premature infants. Our objective was to determine the characteristics of the blood flow redistribution induced by AVP infusion in premature fetal sheep. METHODS: Nine sheep fetuses at 99 to 113 days of gestation were continuously infused with AVP. Measurement of blood flow to individual fetal organs was performed using a colored microsphere technique, with measurements performed at 30 min before and 90 min after the initiation of AVP infusions. RESULTS: The AVP infusion significantly increased blood flow to the medulla oblongata (P < 0.05), and significantly decreased flow to the adrenal glands (from 492.0 ± 239.6 to 364.9 ± 143.3 mL/min/100 g, P < 0.05) and heart (from 592.6 ± 184.5 to 435.6 ± 137.4 mL/min/100 g, P < 0.05). The infusion significantly increased the vascular resistance in adrenal glands, kidneys, ileum, colon, heart, and cerebellum. In the brain, except for the cerebellum, no significant increase in resistance was identified. CONCLUSIONS: There was no significant response to AVP infusion in cerebral blood flow in mid-gestation fetal sheep. Our observations suggest that, under AVP stimulation, the blood flow to the adrenal glands and myocardium might be decreased due to an increase in vascular resistance.

Rhinocerebral Zygomycosis Due to a Lichtheimia ramosa Infection in a Calf: Neural Spread Through the Olfactory Nerves.

Here, we report a case of rhinocerebral zygomycosis due to a Lichtheimia ramosa infection in a calf. A histopathological examination revealed that a fungus had invaded the brain through the olfactory nerves. Lichtheimia ramosa was detected by polymerase chain reaction analysis of DNA extracted from formalin-fixed paraffin-embedded samples of the affected tissue. This is the first case of rhinocerebral zygomycosis to involve cattle. Also, this is the first such case to involve fungal invasion into the central nervous system through the cranial nerve itself, rather than through perineural tissue.

Prominent hepatic ductular reaction induced by Oschmarinella macrorchis in a Hubbs' beaked whale Mesoplodon carlhubbsi, with biological notes.

Beaked whales are among the least known group of cetaceans, and information regarding their pathology and parasitology is especially scarce. We describe a case of significant parasitism by a trematode found in the liver of an adult male Hubbs' beaked whale Mesoplodon carlhubbsi that stranded in Hokkaido, Japan. Post-mortem examinations revealed a localised area of discolouration restricted to the hilar region of the left hepatic lobe, where spindle-shaped trematodes occupied the dilated and hypertrophic bile ducts. Histologically, the intrahepatic bile ducts were characterised by adenomatous hyperplasia with goblet cell metaplasia of the biliary epithelium. Findings in the adjacent hepatic parenchyma included pseudocarcinomatous ductular reactions obliterating hepatocytes, a histomorphology not previously reported in marine mammals. Morphological identification of the trematode corresponded to Oschmarinella macrorchis, which has only been reported once in a Stejneger's beaked whale, M. stejnegeri. PCR amplification and sequencing analyses of the parasite's mtDNA ND3, 18S and 28S rRNA regions generated novel gene sequences. Environmental contaminant levels were measured to explore its potential relationship with the parasitism but there was no conclusive association. A high level of polychlorinated biphenyl (30000 ng g-1 lipid weight) was detected in the blubber of this individual, when compared to those of 3 other male Hubbs' beaked whales stranded in Japan. Stomach contents were also analysed, indicating the presence of various squid species and unidentified fish. Our results contribute to the knowledge of a little-known beaked whale and provide evidence for the first time of the pathobiological response caused by O. macrorchis.

Fatal Pulmonary and Cerebellar Zygomycosis due to Rhizomucor pusillus in a Ringed Seal (Pusa hispida).

A 4-year-old captive ringed seal (Pusa hispida) was treated with subcutaneous antibacterial injections for pus exuding wounds in the skin and associated blubber following a bite attack. Three months after the incident, the animal presented nystagmus and died the following day. At necropsy, there was a 25 × 18 × 25 mm well-delineated, opaque nodular mass in the lung, besides the skin ulcers and localized areas of discoloration in the blubber correlating with the bite wound and injection sites. Histopathology of the pulmonary mass demonstrated severe eosinophilic inflammatory infiltration among numerous intralesional fungal hyphae. The hyphae were irregularly branched, broad and aseptate, consistent of zygomycosis. Magnetic resonance imaging was conducted on the head, which was initially frozen intact, revealing diffuse areas of hyperintensity in the cerebellum. Restricted histopathologic examination of the cerebellum showed severe granulomatous inflammation well spread within the neuroparenchyma, associated with abundant intralesional fungal hyphae similar to those appreciated in the pulmonary mass. Molecular analyses of the fungi in the pulmonary and cerebellar tissue identified the etiologic agent in both sites as Rhizomucor pusillus. The likely route of infection is through inhalation of R. pusillus spores or fragmented hyphae from the environment that developed into an initial pulmonary infection, becoming the source of hematogenous dissemination to the cerebellum. The skin and blubber lesions likely contributed to immunosuppression. Zygomycosis is uncommon in pinnipeds, and the present report emphasizes the importance of considering zygomycete dissemination even when the primary focus is highly confined.

Histological Properties of Main and Accessory Olfactory Bulbs in the Common Hippopotamus.

The olfactory system of mammals comprises a main olfactory system that detects hundreds of odorants and a vomeronasal system that detects specific chemicals such as pheromones. The main (MOB) and accessory (AOB) olfactory bulbs are the respective primary centers of the main olfactory and vomeronasal systems. Most mammals including artiodactyls possess a large MOB and a comparatively small AOB, whereas most cetaceans lack olfactory bulbs. The common hippopotamus (Hippopotamus amphibius) is semiaquatic and belongs to the order Cetartiodactyla, family Hippopotamidae, which seems to be the closest extant family to cetaceans. The present study evaluates the significance of the olfactory system in the hippopotamus by histologically analyzing the MOB and AOB of a male common hippopotamus. The MOB comprised six layers (olfactory nerve, glomerular, external plexiform, mitral cell, internal plexiform, and granule cell), and the AOB comprised vomeronasal nerve, glomerular, plexiform, and granule cell layers. The MOB contained mitral cells and tufted cells, and the AOB possessed mitral/tufted cells. These histological features of the MOB and the AOB were similar to those in most artiodactyls. All glomeruli in the AOB were positive for anti-Gαi2, but weakly positive for anti-Gαo, suggesting that the hippopotamus vomeronasal system expresses vomeronasal type 1 receptors with a high affinity for volatile compounds. These findings suggest that the olfactory system of the hippopotamus is as well developed as that of other artiodactyl species and that the hippopotamus might depend on its olfactory system for terrestrial social communication.

A Parallelized Pumpless Artificial Placenta System Significantly Prolonged Survival Time in a Preterm Lamb Model.

An artificial placenta (AP) is an arterio-venous extracorporeal life support system that is connected to the fetal circulation via the umbilical vasculature. Previously, we published an article describing a pumpless AP system with a small priming volume. We subsequently developed a parallelized system, hypothesizing that the reduced circuit resistance conveyed by this modification would enable healthy fetal survival time to be prolonged. We conducted experiments using a premature lamb model to test this hypothesis. As a result, the fetal survival period was significantly prolonged (60.4 ± 3.8 vs. 18.2 ± 3.2 h, P < 0.01), and circuit resistance and minimal blood lactate levels were significantly lower in the parallel circuit group, compared with our previous single circuit group. Fetal physiological parameters remained stable until the conclusion of the experiments. In summary, parallelization of the AP system was associated with reduced circuit resistance and lactate levels and allowed preterm lamb fetuses to survive for a significantly longer period when compared with previous studies.

Autopsy imaging for cardiac tamponade in a Thoroughbred foal.

Autopsy imaging (Ai), postmortem imaging before necropsy, is used in human forensic medicine. Ai was performed using computed tomography (CT) for a 1-month-old Thoroughbred foal cadaver found in a pasture. CT revealed pericardial effusion, collapse of the aorta, bleeding in the lung lobe, gas in the ventricles and liver parenchyma, and distension of the digestive tract. Rupture in the left auricle was confirmed by necropsy; however, it was not depicted on CT. Therefore, Ai and conventional necropsy are considered to complement each other. The cause of death was determined to be traumatic cardiac tamponade. In conclusion, Ai is an additional option for determining cause of death.

Quantitative evaluation of cervical cord compression by computed tomographic myelography in Thoroughbred foals.

Five Thoroughbred foals (age, 8-33 weeks; median age, 31 weeks; weight, 122-270 kg; median weight, 249 kg) exhibiting ataxia with suspected cervical myelopathy (n=4) and limb malformation (n=1) were subjected to computed tomographic (CT) myelography. The areas of the subarachnoid space and cervical cord were measured on transverse CT images. The area of the cervical cord was divided by the area of subarachnoid space, and stenosis ratios were quantitatively evaluated and compared on the basis of histopathological examination. The sites with a ratio above 52.8% could have been primary lesion sites in the histopathological examination, although one site with a ratio of 54.1% was not a primary lesion site. Therefore, in this study, a ratio between 52.8-54.1% was suggested to be borderline for physical compression that damages the cervical cord. All the cervical vertebrae could not be scanned in three of the five cases. Therefore, CT myelography is not a suitable method for locating the site of compression, but it should be used for quantitative evaluation of cervical stenosis diagnosed by conventional myelography. In conclusion, the stenosis ratios determined using CT myelography could be applicable for detecting primary lesion sites in the cervical cord.

Morphometric analysis of progressive changes in hereditary cerebellar cortical degenerative disease (abiotrophy) in rabbits caused by abnormal synaptogenesis.

We previously investigated rabbit hereditary cerebellar cortical degenerative disease, called cerebellar cortical abiotrophy in the veterinary field, and determined that the pathogenesis of this disease is the result of failed synaptogenesis between parallel fibers and Purkinje cells. In this study, longitudinal changes in the development and atrophy of the cerebellum of rabbits with hereditary abiotrophy after birth were morphometrically examined (postnatal day [PD] 15 and 42) using image analysis. Although development of the cerebellum in rabbits with abiotrophy was observed from PD 15 to PD 42, the growth rate of the cerebellum was less than that in normal rabbits. In rabbits with abiotrophy, the number of granular cells undergoing apoptosis was significantly higher at PD 15 and dramatically decreased at PD 42. The number of granular cells did not increase from PD 15 to 42. The synaptogenesis peak at PD 15 occurred when the largest number of apoptotic granular cells in rabbits with abiotrophy was observed. Although 26% to 36% of parallel fiber terminals formed synaptic junctions with Purkinje cell spines, the remainder did not at PD 15 and 42. The rate of failure of synaptogenesis in the present study might be specific to this case of abiotrophy. Morphometric analysis revealed detailed changes in development and atrophy in animals with postnatal cerebellar disease occurring soon after birth.

Lambda-carrageenan treatment exacerbates the severity of cerebral malaria caused by Plasmodium berghei ANKA in BALB/c mice.

BACKGROUND: There is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood-brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of λ-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei. METHODS: Five-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with λ-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given λ-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood-brain barrier integrity. RESULTS: Plasmodium berghei ANKA-infected BALB/c mice treated with λ-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with λ-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the λ-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice. CONCLUSION: The results of this study indicate that the administration of λ-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.

Cerebral ischemia or intrauterine inflammation promotes differentiation of oligodendroglial precursors in preterm ovine fetuses: possible cellular basis for white matter injury.

White matter injury in premature infants is known to be major cause of long-term neurocognitive disability, but the pathogenic mechanism remains unclear, hampering our ability to develop preventions. Periventricular leukomalacia is a severe form of white matter injury. In the present study, we explored the effects of cerebral ischemia and/or intrauterine inflammation on the development of oligodendroglia in the cerebral white matter using chronically instrumented fetal sheep. Each fetus received one of three insults: hemorrhage, inflammation and their combination. In the hemorrhage group, 40% of the fetoplacental blood volume was acutely withdrawn, and 24 hours after removal, the blood was returned to the fetus. The inflammation group received intravenous granulocyte-colony stimulating factor and intra-amniotic endotoxin and thus suffered from necrotizing funisitis and chorioamnionitis. The inflammatory hemorrhage group underwent acute hemorrhage under the inflammatory state. The sham group received no insults. Importantly, periventricular leukomalacia was not detected in the sham and the inflammation groups. Differentiating oligodendroglia at various developmental stages were identified by immunohistochemical analysis with specific antibodies. No difference in the density of oligodendroglial progenitors was detected among the four groups, whereas oligodendroglial precursors were significantly reduced in the three insult groups, compared to sham control. Moreover, the density of immature oligodendroglia was higher in the inflammation group and the inflammatory hemorrhage group, while the density of mature oligodendroglia was highest in the hemorrhage group. We propose that cerebral ischemia or intrauterine inflammation induces the differentiation of oligodendroglial precursors in preterm fetuses, eventually resulting in their exhaustion.

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

Monoclonal proliferation of B-cells with two integration sites of bovine leukemia virus proviral DNA in cattle with enzootic bovine leukosis.

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.