Centronuclear Myopathy with Abundant Nemaline Rods in a Japanese Black and Hereford Crossbred Calf.

Histopathological examination was performed on skeletal and diaphragmatic muscles from an 8-month-old male crossbred calf showing abnormal gait and tremor of the hindlimbs. There were numerous round fibres with centrally placed nuclei forming nuclear chains in longitudinal sections, associated with interstitial fibrosis or adipose tissue infiltration. On nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining, some muscle fibres in severe lesions showed a spoke-like appearance due to a radial arrangement of sarcoplasmic strands. Additionally, increased NADH-TR activity in the subsarcolemmal structures, appearingas ring-like or necklace-like forms, were observed. Transmission electron microscopy revealed dilated sarcoplasmic reticulum and variably shaped electron-dense inclusions consisting of myofibrillar streams. Another prominent feature was the existence of numerous nemaline rods within muscle fibres; these were stained red by Gomori's trichrome stain. Immunohistochemistry revealed that the nemaline rods showed strong immunoreactivity with α-actinin and desmin antibodies. Electron microscopically, these structures were composed of dense-homogeneous material and continuous with the Z disk. The case was diagnosed as centronuclear myopathy with increased nemaline rods.

Clinical experience of using multidetector-row CT for the diagnosis of disorders in cattle.

To determine the clinical usefulness of multidetector-row CT for the diagnosis of disorders in cattle, images were obtained from 27 cattle, which were then subjected to postmortem and histopathological examinations. The cattle were divided into three categories of disorder: neurological (18 cases), skeletal (four cases) and other (five cases). In five cattle, which were suspected to have brain diseases, no abnormalities were identified by either CT or histopathological examination. Eight types of lesions were detected by CT in the cattle with neurological and vestibular disorders. The diseases diagnosed included hydrocephalus (three cases), intracranial arachnoid cysts (three cases), otitis media (five cases), cerebral abscess (one case), meningoencephalocele (one case), porencephaly (one case), bicephalus (one case) and rupture of the spinal cord (one case). Lesions were identified in all the cattle with skeletal disorders, including luxation (two cases), fracture (two cases), spondylosis (one case) and congenital disorders of the skeletal system (one case). Morphological disorders in the eyes (one case), nasal cavity (two cases), frontal sinuses (one case), thyroid glands (two cases), lung fields (two cases) and abdominal organs (two cases) were diagnosed by CT.

Infection of different strains of mice with Lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy.

This report describes intestinal lesions in five strains of mice infected orally with Lawsonia intracellularis-infected tissue homogenates from rabbits or pigs (RLI and PLI). BALB/cA, C3H/HeJ, C57BL/6J and ICR mice were susceptible to infection with RLI, whereas only C3H/HeJ, C57BL/6J and ICR strains were susceptible to PLI. In susceptible mice, crypt epithelial hyperplasia occurred in association with an inflammatory reaction, as in proliferative enteropathy (PE) in other species. The intestinal changes in the infected mice varied from mild to severe. Unlike rabbit or porcine PE, in which the changes are confined to the ileum, the lesions in mice were located in the caecum. Immunolabelling of L. intracellularis antigen was abundant in early infection when the epithelial hyperplasia was mild or absent. When the hyperplasia had become severe, however, immunolabelling was weak. For this reason, it is suggested that transitory infection of the epithelium induces epithelial hyperplasia. Genetic differences between mouse strains appeared to play an important role in the response to L. intracellularis infection. Moreover, the susceptibility of BALB/cA mice to RLI but not to PLI suggests that there are significant biological differences between L. intracellularis isolates from rabbit PE and porcine PE.

Species-specificity of a panel of prion protein antibodies for the immunohistochemical study of animal and human prion diseases.

Monoclonal antibodies to the prion protein (PrP) have been of critical importance in the neuropathological characterization of PrP-related disease in men and animals. To determine the influence of species-specific amino-acid substitutions recognized by monoclonal antibodies, and to investigate the immunohistochemical reactivity of the latter, analyses were carried out on brain sections of cattle with bovine spongiform encephalopathy, sheep with scrapie, mice infected with scrapie, and human beings with Creutzfeldt-Jakob disease (CJD) or Gerstmann-Sträussler-Sheinker disease (GSS). Immunoreactivity varied between the antibodies, probably as the result of differences in the amino-acid sequence of the prion protein in the various species. Some monoclonal antibodies against mouse recombinant PrP gave strong signals with bovine, ovine and human PrP(Sc), in addition to murine PrP(Sc), even though the amino-acid sequences determined by the antibody epitope are not fully identical with the amino-acid sequences proper to the species. On the other hand, in certain regions of the PrP sequence, when the species-specificity of the antibodies is defined by one amino-acid substitution, the antibodies revealed no reactivity with other animal species. In the region corresponding to positions 134-159 of murine PrP, immunohistochemical reactivity or species-specificity recognized by the antibodies may be determined by one amino acid corresponding to position 144 of murine PrP. Not all epitopes recognized by a monoclonal antibody play an important role in antigen-antibody reactions in immunohistochemistry. The presence of the core epitope is therefore vital in understanding antibody binding ability.

p53 mutation as a potential cellular factor for tumor development in enzootic bovine leukosis.

Mutations of p53 in the lymphocytes from peripheral blood and from tumoral lymph nodes in six naturally occurring bovine leukemia virus (BLV)-infected cows were examined. A point mutation of the p53 gene was found in three of six (50%) BLV-infected cows. These p53 gene mutations resulted in amino acid substitutions of codons 144, 167 and 241. The BLV-infected cow in the tumor stage had abnormally proliferating monoclonal B-lymphocytes having the p53 mutation. However, the mutation was not found in somatic cells, except for tumor cells. These results show that p53 mutation plays an important role in the pathogenesis of BLV-induced neoplasms, and that the B-lymphocyte bearing p53 mutations may be a target cell for tumor formation of enzootic bovine leukosis.

Relationship between liver disorders and protection against Eimeria stiedai infection in rabbits immunized with soluble antigens from the bile of infected rabbits.

Soluble antigens exist in the bile of rabbits infected with Eimeria stiedai (E. stiedai) in the acute phase, and rabbits immunized with the antigens show resistance against the infection. In this study, the liver function of rabbits immunized either with the soluble antigens or PBS were examined following the parasite challenge. Rabbits immunized with PBS shed a number of oocysts and showed an increase in r-glutamyltransferase (GGT) activity and a decrease in blood Indocyanine green (ICG) clearance. However, rabbits immunized with the soluble antigens shed a lower number of oocysts and showed a transient increase of alanine-aminotransferase (ALT) activity on Day 8 post-challenge (p.c.). The blood Indocyanine green clearance of the rabbits showed no change throughout the experiment. By histopathological observation of the liver, a number of merozoites were found in the biliary ducts on Day 8 post-challenge in the non-immunized rabbits. In contrast, a number of lymphocytes and neutrophilic leukocytes assembled around the biliary ducts of the immunized rabbits, but few parasites were found there on Day 8 post-challenge. These results suggest that the soluble antigens stimulate local immune reactions, for example around the biliary ducts, resulting in elimination of the parasite's development.

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection.

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.

Specific IgA antibody response to coproantigens of Cryptosporidium parvum in serum and saliva of calves after experimental infection.

The antibody response to coproantigens of Cryptosporidium parvum was examined in saliva and sera of calves experimentally infected with C. parvum. Coproantigens of C. parvum with approximate molecular masses of 17, 15 and less than 14kDa were found in the feces of infected calves on day 3 or later, and 60 and 23kDa coproantigens observed between days 4 and 9 post-infection, respectively. The antibody reactivity to the coproantigens was mainly attributable to IgA class antibodies in saliva and was detectable during the convalescent phase of infection. A 15kDa protein isolated from the feces of infected calves by immunoaffinity adsorption using a monoclonal anti C. parvum antibody was recognized by IgA antibodies present in the saliva during the convalescent phase of infection. These results suggest that this coproantigen may be released from C. parvum sporozoites and may induce IgA antibody production in the mucosal immune system of infected calves.

Increase of Th1 type cytokine mRNA expression in peripheral blood lymphocytes of calves experimentally infected with Cryptosporidium parvum.

The expression of the messenger RNA of interleukin-12 (IL-12), interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 was examined by reverse-transcriptase polymerase chain reaction in peripheral blood lymphocytes of calves that were orally inoculated with Cryptosporidium parvum oocysts. In all of the calves, gene expression of interleukin-12, interleukin-6, and interferon-gamma was observed at delivery and this expression was repressed within the next 24h. In calves inoculated with C. parvum, mRNA expression of interleukin-12 and interferon-gamma was noticed on day 3 post-inoculation (p.i.) and increased in the convalescent phase of the infection, whereas in non-inoculated calves no mRNA expression was detectable up to the end of the experiment. No mRNA expression of interleukin-4 or 6 was detected during the experiment. Our observations suggest that systemic Th1 type immune responses are induced in calves infected with C. parvum and may be available for evaluation of the control of the infection.

Diffuse retinochoroiditis due to Baylisascaris procyonis in Monglian gerbils.

Baylisascaris procyonis, raccoon roundworm, causes a severe retinal lesion in humans. The lesion is termed as diffuse unilateral subacute neuroretinitis (DUSN). To understand the pathogenesis of B. procyonis in gerbils, we inoculated 17 embryonated eggs/g body weight of B. procyonis into 15 male Mongolian gerbils, Merionis ungiculatus, and monitored their fundi with an ophthalmoscope. Six of 15 gerbils (40%) showed severe retinitis with a sinuous track due to larval movement. The lesions extended across nearly half of the affected fundi. Histopathological examination revealed perivasculitis in the optic disk region, inflammatory proliferation of the pigment cells, and vitreitis in most cases. These findings were similar to those in human cases of DUSN, suggesting that gerbils might be a useful model for understanding the pathogenesis of B. procyonis infection in humans.

Naturally-occurring Neospora caninum infection in an adult sheep and her twin fetuses.

Neospora caninum tissue cysts were found in the brains of surgically delivered twin fetuses at 119 days of gestation. In the brains of both fetuses, there was an inflammatory reaction involving perivascular cuffings of mononuclear cells, glial nodules. The dam of these fetuses died because of metritis. Histopathological examination of the ewe revealed N. caninum tissue cysts and focal gliosis with mononuclear cell cuffings. A N. caninum-specific DNA fragment was detected in a brain homogenate of the ewe by the polymerase chain reaction method. This is the first report of N. caninum infection in twin ovine fetuses and in an adult sheep.

Eimeria stiedai merozoite 49-kDa soluble antigen induces protection against infection.

A soluble antigen isolated from Eimeria stiedai merozoites with a molecular mass of 49 kDa was detected in the bile of infected rabbits. Rabbits immunized with the antigen shed a lower number of oocysts than did nonimmunized rabbits postchallenge (p.c.). The immunized rabbits showed a marked and transient increase of alanine-aminotransferase (ALT) activity on day 8 p.c. The blood indocyanine green (ICG) clearance and r-glutamyltransferase (GGT) activity showed no change throughout the experiment However, nonimmunized rabbits showed a gradual increase of ALT and GGT in the plasma and a delay of ICG p.c. Many merozoites were observed in the biliary ducts of the nonimmunized rabbits on day 8 p.c. using standard histology. In contrast, in the immunized rabbits, many inflammatory cells were observed around the biliary ducts, but there were few parasites in the tissue. These results suggest that the 49-kDa soluble protein antigen detected in the bile of the infected rabbits was a merozoite-specific antigen, and the immune reaction to the antigen may induce protective effects against the infection.

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.

Peripheral neuropathy in German shepherd dogs.

Three aged (10-year-old) German Shepherd Dog litter mates, separately reared, were affected with familial and adult onset peripheral neuropathy. They developed clinical signs, unsteady gait of their hind legs with progressive muscular weakness at almost the same time. The main lesions were systemic neurogenic muscular atrophy, segmental demyelination and Wallerian degeneration of the peripheral nerve fibres. Histochemically, collateral ramification and multiple terminal arborization were observed in terminal axons of motor neurones in the muscles. These changes were attributed to a dying-back process.

Calcifying epithelial odontogenic tumours in small domesticated carnivores: histological, immunohistochemical and electron microscopical studies.

Histological, immunohistochemical and electron microscopical studies revealed one feline and four canine calcifying epithelial odontogenic tumours in 115 oral tumours over a 10-year period. The tumours consisted of islands and sheets of odontogenic epithelium of varying size within a stroma of fibrous connective tissues. The tumour cells were pleomorphic with variable amounts of eosinophilic cytoplasm and large hyperchromatic, polymorphic nuclei with prominent nucleoli. Clusters of keratinized tumour cells ("shadow cells") were frequently seen within the islands and sheets. The multiple spherules of homogeneous eosinophilic material stained positively with Congo red and Dylon stains and produced an apple green birefringence under polarization microscopy, indicative of amyloid. Mineralized foci were scattered throughout the tumour masses and in the homogeneous spherules. Immunohistochemically, the tumour cells reacted with anti-human keratin antibody, but not with anti-human vimentin or anti-chicken desmin antibodies. The homogeneous spherules did not react with anti-human keratin, anti-human vimentin, anti-chicken desmin, anti-amyloid A, anti-laminin or anti-human collagen (type I, III, IV) antibodies. Ultrastructurally, the cytoplasm of tumour cells was abundant and contained a large number of electron-dense bundles of tonofilaments. The homogeneous spherules consisted of fine filaments measuring about 10-12 nm in diameter.

Immunohistochemical analysis of equine pulmonary granular cell tumours.

Histopathological and immunohistochemical examinations were made on four female horses aged 9-12 years with pulmonary granular cell tumours (GCTs). The tumours, which were multiple, of varying size, firm and off-white in colour, surrounded the bronchi and bronchioles. Metastatic lesions were not detected. The tumour cells had abundant eosinophilic cytoplasm filled with prominent coarse eosinophilic granules. Immunohistochemically, these tumour cells reacted uniformly with vimentin and S100 antibodies. Most were immunolabelled by antibodies against glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and protein gene product 9.5 (PGP9.5), and a few cells were positive with Leu7 antibody. However, the tumour cells did not react with antibodies against neurofilament protein (NF), cytokeratin (CK), chromogranin, alpha1 antichymotrypsin (AACT), myoglobin, desmin, alpha-actin or alpha-smooth muscle actin (alpha-SMA). These immunohistochemical properties of tumour cells support the hypothesis that equine pulmonary GCTs are derived from Schwann cells of the peripheral nervous system in peribronchial and peribronchiolar tissues. GFAP, MBP, Leu7 and PGP9.5 antibodies should help to distinguish equine granular cell tumours from other tumours.

Malignant retroperitoneal paraganglial tumour in a cow.

A large primary retroperitoneal sublumbar neoplasm was identified in an 11-year-old Holstein cow, with metastases to the lungs, kidneys and lymph nodes. The tumour cells proliferated in a characteristic endocrine pattern, were argyrophilic and positive for neuron-specific enolase, and had membrane-bounded intracytoplasmic granules. In addition, the cells were occasionally positive for cytokeratin and had desmosome-like intercellular junctions. The primary tumour mass was diagnosed as a malignant paraganglial tumour of the aortico-sympathetic ganglion (organ of Zuckerkandl), and was considered to contain primitive cells with epithelial differentiation.

Immunohistochemical demonstration of bovine viral diarrhoea virus antigen in the pancreatic islet cells of cattle with insulin-dependent diabetes mellitus.

The pancreatic islets were studied in seven cattle with insulin-dependent diabetes mellitus (IDDM) associated with persistent bovine viral diarrhoea virus (BVDV) infection. BVDV antigen was detected immunohistochemically in the pancreatic islet cells. There was a decrease in the size and number of islets, vacuolar degeneration of residual islet cells, and lymphocytic insulitis. The atrophied islets were composed of small uniform cells with limited amounts of cytoplasm, containing a small number of insulin- and chromogranin-positive granules. Enlarged islets consisting of islet cells with vacuolated cytoplasm were also frequently observed. Many of the vacuolated islet cells differed from the cells of normal islets in containing fewer cytoplasmic insulin- and chromogranin-positive granules. Mild lymphocytic insulitis was observed frequently in enlarged islets but rarely in atrophied islets. Immunoreactivity with BVDV antibody was found in the acinar cells of the pars exocrina in all seven cattle and in the residual cells of the islets of Langerhans of four cattle. BVDV antigen-positive cells were seen more frequently in the enlarged islets than in the atrophied islets. Some islets with lymphocytic infiltrates showed a small number of antigen-positive cells. These findings suggest that autoimmune IDDM was induced by persistent BVDV infection, resulting in gradual destruction of the islet beta cells.

Histopathological and immunohistochemical studies of intracranial nervous-system tumours in four cattle.

Intracranial nervous-system tumours were diagnosed in three of 1092 bovine necropsy specimens submitted to the Department of Veterinary Pathology, Obihiro University between April 1983 and March 1996. A fourth case was a referral from the Department of Veterinary Pathology, Rakuno Gakuen University. Histopathological examination revealed four types of tumour: intracranial malignant peripheral nerve sheath tumour (MPNST), choroid plexus papilloma, differentiated fibrillary astrocytoma and anaplastic (malignant) astrocytoma. Immunohistochemically, the intracranial MPNST was strongly positive for S-100 protein and vimentin, and in places weakly positive for glial fibrillary acid protein (GFAP). The choroid plexus papilloma was strongly positive for epithelial membrane antigen (EMA), keratin, S-100 protein and vimentin, and positive for GFAP in places. The cytoplasm and fibrous component in the differentiated fibrillary astrocytoma were strongly positive for S-100 protein and GFAP. The anaplastic (malignant) astrocytoma was strongly positive for vimentin, S-100 protein and keratin in the cytoplasm and fibrous processes, and weakly positive for GFAP and EMA in places. Myelin basic protein (MBP) and synaptophysin showed a weak positive reaction in the marginal areas of the tumour.

Hereditary dilated cardiomyopathy in Holstein-Friesian cattle in Japan: association with hereditary myopathy of the diaphragmatic muscles.

This report deals with the pathology and genetic basis of dilated cardiomyopathy in 10 Holstein-Friesian cows aged 3-6 years, a disease similar to that reported in Simmental-Red Holstein and Holstein-Friesian cattle in several other countries. The main clinical signs were associated with systemic circulatory failure, and at necropsy the animals showed cardiomegaly, severe congestion and fibrosis of the liver, and systemic cardiac oedema. Histologically, hypertrophy and vacuolation of the cardiac muscle fibres and severe fibrosis were noted. Electron microscopically, the sarcoplasm of the hypertrophic fibres was seen to be filled with fine structures of low electron-density, together with thin filamentous material, suggesting myofibrillar lysis. The mitochondria showed increased size, an abnormal cristae pattern and vacuolation due to partial loss of cristae. Pedigree analysis of the affected cattle indicated an autosomal recessive mode of inheritance. The family line of this cardiomyopathy overlapped with that of hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle, the pathological aspects and inheritance mode of which were reported previously. The available evidence suggested a genetic association between these two pathologically distinct diseases.