Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies.

During meat inspections in pigs, dystrophinopathies are among the muscle lesions targeted for disposal. In this study, the authors examined the lesions and the distribution of dystrophin expression in 25 pigs with dystrophinopathy. In addition, complementary deoxyribonucleic acid (cDNA) sequencing and western blotting were performed in 6 of the 25 cases, all of which were characterized by degeneration, necrosis, and fat replacement of muscle fibers. Comparing the results of immunohistochemistry with anti-dystrophin antibodies that recognized at different sites in the protein, the authors noted that the loss of dystrophin expression was most pronounced in the C-terminus-recognizing antibody (19/25 cases). The authors detected 5 missense mutations and 3 types of shortened transcripts generated by the skipping of exons in the cDNA, which were associated with the pathogenesis. One missense mutation had been reported previously, whereas the remaining mutations identified had not been previously documented in pigs. In the cases with shortened transcripts, normal-sized transcripts were detected together with the defective transcripts, suggesting that these mutations were caused by splicing abnormalities. In addition, they were in-frame mutations, all of which have similar pathogeneses of Becker muscular dystrophy in humans. These cases were 6 months of age and exhibited macroscopic discoloration, fatty replacement, and muscle degeneration, suggesting that the effect of these mutations on skeletal muscle was significant.

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.

Characteristic computed tomographic myelography findings in 23 Thoroughbred horses.

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

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals.

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with "exceptional" gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these "exceptional" lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.

A clinical case of single left ventricle in a Holstein calf.

A 15-day-old Holstein calf with lethargy and tachypnea presented to the Veterinary Teaching Hospital at Obihiro University of Agriculture and Veterinary Medicine for evaluation of suspected congenital heart defect. A Levine grade 6 systolic murmur was noted at right apical site auscultation and phonocardiogram also recorded systolic a murmur. Electrocardiography findings include increased R and S waves, R wave split, and negative T waves without arrhythmia. Echocardiography revealed a single ventricle with a trace of the right ventricular wall, atrioventricular valve regurgitation, and turbulent in a single ventricle. Arterial blood analysis showed a marked decrease in oxygen saturation of 78% and oxygen partial pressure of 44 mmHg. Post-mortem examination confirmed the diagnosis of a single left ventricle.

Severe calcification of systemic blood vessel walls caused by continuous hypercalcemia in a cat with congenital hypothyroidism.

A 97-day-old male Japanese domestic cat was diagnosed as congenital hypothyroidism. During the treatment, continuous hypercalcemia was detected. Although fluid therapy was performed, the cat died at the age of 1785 days. At autopsy, both parathyroid glands were enlarged, and elastic arterial walls were increased in thickness and hardness. Histopathological examination revealed hyperplasia of both parathyroid glands and interstitial fibrosis of bilateral kidneys. Severe calcification of the tunica media and tunica externa in systemic elastic and muscular arteries were also observed. These calcifications were considered to be due to renal secondary hyperparathyroidism. In the present case, hypothyroidism might have caused hyperparathyroidism through renal failure. In veterinary medicine, this is the first reported case of hypothyroidism accompanied with hyperparathyroidism.

Immunohistochemical phenotyping of macrophages and T lymphocytes infiltrating in peripheral nerve lesions of dourine-affected horses.

Dourine is a deadly protozoan disease in equids caused by infection with Trypanosoma equiperdum. Neurological signs in the later stage of infection may be caused by peripheral polyneuritis and related axonal degeneration. This neuritis involves T lymphocytes, B lymphocytes, and macrophages, and is observed in cases without obvious neurological signs. However, the pathogenesis of neuritis remains unclear. We identified M2 macrophages and CD8 T cells as the predominant phenotypes in neuritis of dourine-affected horses with or without neurological signs. In contrast, the populations of M1 macrophages and CD4 T cells were small. This result indicates that inflammation was chronic and suggests that dourine-associated neuritis occurs at the early stage of infection.

A clinical case of presumed cerebellar medulloblastoma in a Japanese Black calf with increased neuron-specific enolase in cerebrospinal fluid.

A 2-day-old Japanese Black male calf that presented with opisthotonos with spastic extension of all four limbs and nystagmus was presented. Evaluation of cranial neurology revealed a horizontal slow nystagmus and absence of menace response in the left eye. Necropsy revealed a mass located between the posterior margin of the cerebrum and anterior margin of the cerebellum, and continuously with the cerebellar lesion. The brainstem was severely compressed by those lesions. Original structures of the cerebellum were mostly replaced by grayish-white and brownish tissues. Those lesions were diagnosed as presumed cerebellar medulloblastoma by histopathological and immunohistochemical examination. As neuron-specific enolase in the cerebrospinal fluid which is a biomarker for neuronal damage was increased compared with healthy calves.

Intramedullary spinal nephroblastoma in a mixed breed dog.

A 1-year-old male mixed breed dog presented for the evaluation of progressive hindlimb paresis. Neurological examination indicated a spinal cord lesion between the 3rd thoracic and 3rd lumbar vertebrae. Magnetic resonance imaging (MRI) revealed an intramedullary spinal cord lesion located at the level of the 1st and 2nd lumbar vertebrae. Following cytoreductive surgery of the mass, palliative radiation therapy was administered. A diagnosis of nephroblastoma was made based on histological examination. After radiation therapy, the disappearance of the spinal lesion was confirmed by MRI. The dog was improved from gait abnormality and alive at 16 months postoperatively, with slight signs of neurological dysfunction.

Acute monoblastic leukemia in a feline leukemia virus-negative cat.

A 12-year-old female domestic short-haired cat was presented due to weight loss, anorexia, and tachypnea. Complete blood count revealed severe anemia, leukocytosis with massive undifferentiated blast cells, and thrombocytopenia. Bone marrow aspiration showed acute myeloid leukemia, subclassified as monoblastic leukemia (M5a) based on the outcomes of the cytochemistry examinations. The SNAP feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) test using whole blood was negative. In addition, FeLV/FIV proviral polymerase chain reaction test using bone marrow aspirate was also negative. Although the cat was treated with doxorubicin, cytosine arabinoside, and prednisolone, anemia did not improve without blood transfusion. The owner declined further treatment after 2 months, and the cat died a few days later.

Molecular diagnosis of bovine B-cell lymphoma using polymerase chain reaction for immunoglobulin heavy chain gene.

We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility as a method to diagnose bovine B-cell lymphoma. PCR for IgH gene rearrangement indicated monoclonal proliferation of B-cells in 24 of 35 cattle with B-cell lymphoma. In contrast, PCR for IgH gene rearrangement in lymph nodes and tumor tissues from 65 cattle diagnosed with tumors other than B-cell lymphoma and non-tumors revealed polyclonal population of B-cells. Sensitivity, specificity, positive predictive value, and negative predictive value for PCR for IgH gene rearrangement for bovine B-cell lymphoma were 68.6%, 100%, 100%, and 85.5%, respectively. Clonality analysis using PCR for IgH gene rearrangement may be useful for adjunctive diagnosis of bovine B-cell lymphoma.

Disseminated thymic B-cell lymphoma in a Holstein heifer.

A 19-mo-old Holstein heifer was inactive and dyspneic. Physical examination revealed wheezing, exophthalmos, a cervical mass, and lymphadenopathy. Cytology of the cervical mass and lymph nodes showed predominantly large atypical lymphocytes. Lactate dehydrogenase and thymidine kinase activities were elevated. Although nested PCR for bovine leukemia virus (BLV) using blood was positive, quantitative PCR showed a low number of provirus copies. Autopsy revealed enlargement of most lymph nodes examined, as well as white masses of various sizes in muscles of the left hindlimb and thoracic and abdominal organs. Histopathology revealed severe infiltration with neoplastic lymphocytes in these organs. The cervical mass was immune-positive for B-cell markers. The final diagnosis was thymic B-cell lymphoma with BLV infection.

Incidental induction of secondary bowel disorders in guinea pigs during a batch safety test of veterinary vaccines.

Veterinary vaccines are subjected to a safety testing using laboratory animals via intraperitoneal injection per batch. From April 2010 to March 2011, 7 guinea pigs in 4 batch tests exhibited unrecoverable weight loss and/or were found dead. Six guinea pigs had developed intussusception, whereas another one had developed an intestinal obstruction consequent to adhesion. A histopathology revealed that these lesions were associated with inflammatory foci. Other animals than the 7 guinea pig also developed similar inflammatory foci but did not develop bowel disorders. In the retesting of these batches, animals did not exhibited clinical signs, though inflammatory foci were detected. The clinical signs, detected in the primary test, might be due to bowel disorders secondary to an inflammatory response, rather than toxicity.

Polyradiculoneuropathy in dourine-affected horses.

Dourine is an equine protozoan disease caused by Trypanosoma equiperdum. Dourine-afflicted animals die after developing neurological clinical signs, such as unilateral paresis. The disease has been a problem for many years; however, the pathogenesis regarding the neurological clinical signs of dourine has been unclear. In the present study, we conducted a histopathological examination in order to investigate the mechanisms by which dourine-afflicted horses develop the accompanying neurological clinical signs. Four dourine-afflicted horses in Mongolia were evaluated. An apparently healthy horse exhibited multifocal neuritis without axonal or myelin degeneration. The other horses, which had obvious neurological clinical signs, also exhibited multifocal neuritis. In particular, the nerves that innervated areas associated with neurological clinical signs exhibited neuritis with demyelination in the latter horses. Inflamed, non-demyelinating nerves were infiltrated with B lymphocytes and T lymphocytes; while inflamed, demyelinating nerves were infiltrated with mononuclear phagocytes. Our observations revealed lesion progression in the nerves, such that polyradiculoneuropathy could explain the accompanying neurological clinical signs of dourine. To our knowledge, this is the first report to describe a pathogenic mechanism for the development of the neurological clinical signs found in dourine-afflicted horses.

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.

CD3 and CD20 co-expression in a case of canine peripheral T-cell lymphoma with prominent cardiac and peripheral nerve involvement.

An 8-y 9-mo-old male Pug dog was presented because of anorexia, hindlimb ataxia, vomiting, and progressive weight loss. Clinical examinations revealed atrophic hindlimb muscles with decreased postural reaction, enlargement of the cardiac silhouette and megaesophagus on radiograph, and reduced cardiac contractility on ultrasonography. The dog died 10 d after the initial examination, and an autopsy was performed. Grossly, the heart was enlarged, with multifocal-to-coalescing extensive plaque-like areas of discoloration on the epicardial surface. On cross-section, the ventricles were moderately dilated, and discoloration extended into the myocardium. Peripheral lymph nodes were of normal size. Histologically, atypical lymphoid cells replaced the myocardium and also extensively infiltrated peripheral nerve bundles in various organs. With immunohistochemistry, the neoplastic cells exhibited strong immunoreactivity for CD3 and CD20, and were negative for CD8, granzyme B, CD79α, and Pax5. Double-label immunofluorescence confirmed co-expression of CD3 and CD20 by the neoplastic cells. Molecular clonality analysis presented a clonal T-cell receptor gamma gene rearrangement. The case was diagnosed as a CD3+/CD20+ peripheral T-cell lymphoma with prominent cardiac and peripheral nerve involvement, indicating neurolymphomatosis.

Molecular and histopathological characterization of Cryptosporidium and Eimeria species in bats in Japan.

Bats are potential reservoirs of Cryptosporidium and Eimeria. The genus Cryptosporidium infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Many epidemiological studies in wild animals have been performed; however, most of them relied on only PCR-based detection because of the difficulty of performing pathological analyses. Accordingly, the natural host and pathogenicity of Cryptosporidium bat genotypes remain unclear. In this study, we captured Eptesicus nilssonii (Northern bats) in Hokkaido, Japan. Of the three intestinal samples obtained, two were positive for Cryptosporidium spp. and one was positive for Eimeria spp. The corresponding microorganisms were also confirmed histopathologically. We detected the novel Cryptosporidium bat genotype XII and Eimeria rioarribaensis in bat intestine.

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.

Histopathological study of encephalomalacia in neonatal calves and application of neuronal and axonal degeneration marker.

Five calves that had shown neurological symptoms within 9 days after birth were histopathologically diagnosed as encephalomalacia. Two calves showed bilateral laminar cerebrocortical necrosis and neuronal necrosis in the corpus striatum and hippocampus. Since the distributional pattern of the lesions was consistent with that of global ischemia in other species, the lesions were probably hypoxic/ischemic encephalopathy consistent with the history of dystocia and perinatal asphyxia. One calf also showed bilateral laminar cerebrocortical necrosis. However, the lesions were chronic ones, because the calf had survived for long time and necropsied at postnatal day 118. Additionally, the lesions did not involve the corpus striatum and hippocampus. The other two calves showed multifocal necrosis with vascular lesions characterized by fibrin thrombi, perivascular edema and perivascular hyaline droplets in the cerebral cortex, corpus striatum, thalamus, brain stem and cerebellum. Considering the age of onsets and histopathological appearance, it was possible that latter three calves were also hypoxic/ischemic encephalopathy, however, exact cause of them was not revealed. In all calves, degenerated/necrotic neurons showed positive reactions for Fluoro-Jade C and degenerated axons showed immunoreactivity for Alzheimer precursor protein A4. Therefore, these markers were applicable to examination of brain injury in neonatal calves.

The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis.

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10 days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.