An Outbreak of Fatal Bordetella bronchiseptica Bronchopneumonia in Puppies.

Twenty-two newborn puppies that did not receive colostrum exhibited acute respiratory signs and died at a breeding facility. Pathological examinations were performed on four of the puppies. At necropsy examination, the lungs were firm and mottled dark red, consistent with acute bronchopneumonia. Histopathologically, there was marked infiltration of neutrophils and macrophages into the bronchi and alveoli, and gram-negative coccobacilli were attached diffusely to the cilia of bronchial mucosa. Immunohistochemistry for Bordetella bronchiseptica antigen revealed positive labelling of the bacterial agents. On electron microscopy, a large number of coccobacilli were observed attaching to the cilia of bronchial epithelial cells. Real-time polymerase chain reaction amplified a B. bronchiseptica gene from the affected lung tissue. Based on these findings, the four puppies were diagnosed with fatal B. bronchiseptica bronchopneumonia.

Proliferative Enteropathy caused by Lawsonia intracellularis in Chickens.

Proliferative enteropathy (PE) is an infectious disease caused by Lawsonia intracellularis (Li), an obligate intracellular bacterium. PE is endemic in swine herds and has been reported in a variety of mammals including horses, hamsters, rabbits, rats, guinea pigs, ferrets, foxes, dogs, sheep, deer and non-human primates. Avian cases are reported only in ratite birds, including emus and ostriches. Some studies show an absence of Lawsonia spp. infection in chickens. In this study, we performed morphological and bacteriological examinations on the intestines of two broiler chickens that had been condemned at a poultry slaughter plant in Japan due to intestinal haemorrhage, which was a result of focal coccidial enteritis. Histopathology revealed proliferation of the villous epithelium in the small and/or large intestines, especially the caeca, regardless of coccidial lesions. Warthin-Starry silver staining and immunohistochemistry using anti-Li monoclonal antibody revealed numerous bacteria and/or antigens in the villous epithelium. Scanning electron microscopy and transmission electron microscopy revealed the presence of curved rods, morphologically compatible with Li, in the apical cytoplasm of the epithelium. Polymerase chain reaction products specific for Li were amplified from DNA samples extracted from formalin-fixed and paraffin wax-embedded tissue. These results suggest that Li can cause PE, characterized by proliferation of the villous epithelium, in chickens.

Neuropathological findings in cats with clinically suspect but histologically unconfirmed feline spongiform encephalopathy.

Central nervous system (CNS) tissues from 192 cats with neurological signs were examined histologically, and tissues from 173 of them were later examined immunohistochemically as part of a survey to determine the prevalence of feline spongiform encephalopathy (FSE). One of the cats was from Norway and the others were from Great Britain. The most commonly recorded clinical signs were ataxia, behavioural changes and epilepsy, but none of the cats had histopathological evidence of FSE. The most common organic CNS lesions were non-suppurative encephalomyelitis in 28 per cent, neoplasia in 15 per cent and a heterogeneous group of degenerative encephalopathies in 9 per cent of the cats. A range of minor histological lesions of uncertain significance was also observed. No histological lesions were observed in the tissues of 63 (33 per cent) of the cats. Disease-specific prion protein (PrP(Sc)) was observed in only one of the 173 cats examined by immunohistochemistry.

Effects of antigen-retrieval pretreatments for immunohistochemical detection of Akabane viral antigen.

Effects of various antigen-retrieval pretreatment techniques for immunohistochemical detection of Akabane virus antigen were examined with formalin-fixed, paraffin-embedded tissues taken from mice intracerebrally inoculated with the virus. The results indicated that no pretreatment is necessary for the detection of the antigen, and excessive enzymatic pretreatment of sections may lead to false-negative results.

Natural infection with attaching and effacing Escherichia coli (O 103:H-) in chicks.

The existence of natural infection with attaching and effacing Escherichia coli (AEEC) in chicks was reported. Numerous Gram-negative bacilli were attached to the enterocyte in association with lesions characterized on a wavy appearance of the mucosal surface in the intestines of six chicks. Immunohistochemically, these bacteria reacted positively with antiserum to Escherichia coli (O 103). By electron microscopy, numerous colibacilli were seen to be closely attached to the surface membranes of the enterocytes. In regions of bacterial attachment, almost all cell microvilli were effaced, and some of the remaining ones were elongated and/or disoriented. Part of the cell membrane formed a cup invagination and pedestal-like protrusion associated with the attached bacteria. A concentration of electron-dense material was seen beneath the adherent organisms. Bacteriologically, numerous E. coli (O 103:H-) were isolated from the jejunal contents of two chicks and the E. coli did not produce verotoxin or enterotoxin. The characteristic lesions could also be induced in the cecal mucosa of young chicks experimentally inoculated with the isolated E. coli. This is the first report of natural infection in chicks with AEEC.

A study on the origin of adenocarcinoma in fowls using immunohistological technique.

When tumor tissues collected from 12 hens suffering from so-called adenocarcinoma were stained with antisera by the indirect immunofluorescent method, all of the tissues appeared to contain ovalbumin. We suggest that most adenocarcinomas in the thoracoabdomen of mature hens are oviductal in origin.

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens.

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.

Comparative pathology of HPCD pigs infected with wild-type and ara-T-resistant strains of Aujeszky's disease virus.

Fourteen 1- to 4-week-old hysterectomy-produced and colostrum-deprived (HPCD) pigs were inoculated intranasally with wild-type and ara-T-resistant strains of Aujeszky's disease virus (ADV), and the pathological lesion induced by the two strains was compared. The wild-type strain (YS-81) led to high mortality, and the pigs developed multifocal necrosis throughout the body and encephalitis. In comparison, the ara-T-resistant strain (YS-81TR) of the virus killed only 1-week-old HPCD pigs inoculated with 10(6.0) PFU per ml of the virus and did not kill pigs more than 2-weeks of age. The latter revealed consistently severe pneumonitis on post-inoculation day (PID) 14. Results of the present study indicated that the ara-T-resistant strain of ADV was less virulent for HPCD pigs than the parental wild-type strain of ADV and that it was able to grow better in the lung than in any other tissue.

Immunohistological demonstration of spread of Aujeszky's disease virus via the olfactory pathway in HPCD pigs.

The spread of Aujeszky's disease virus (ADV) from nasal mucosa via the olfactory pathway was studied in HPCD pigs. ADV antigen was detected in the epithelial cells, nasal gland cells, olfactory nerve cells and peripheral nerve fibres in the nasal cavity and in neuroglial cells in the olfactory bulb. Results indicate that the olfactory pathway is one of the most important neuronal pathways of ADV infection in pigs.

Demonstration of viral antigen and immunoglobulin (IgG and IgM) in brain tissue of pigs experimentally infected with haemagglutinating encephalomyelitis virus.

Haemagglutinating encephalomyelitis virus (HEV) was inoculated either orally or intranasally into ten 3-day-old gnotobiotic piglets. All infected pigs showed inappetence and listlessness, but there were no clinical signs of nervous disorder. Severe encephalomyelitis, characterized by neuronophagia, focal gliosis and perivascular cuffing, was observed in the brain stem and cerebral cortex. Nasally infected pigs, in particular, developed lesions in the area of the stria olfactoria and tractus olfactorius. Coincident with the encephalitic changes, HEV antigen was observed first in the trigeminal ganglion cells and then in degenerating neurones. Immunoglobulin (IgG and IgM)-containing cells were also found in perivascular cuffs and glial foci. They appeared at first on PID 7 and after that increased in number. These findings suggest that these encephalitic lesions are a specific immune response to HEV following its multiplication in the central nervous system.

Immunopathology in Aujeszky's disease virus-infected pigs exposed to fluctuating temperatures.

Pigs exposed to fluctuating temperatures (high, 30 +/- 2 degrees C; low, 4 +/- 1 degrees C) were intranasally inoculated with Aujeszky's disease virus (ADV). ADV-infected pigs, exposed to the fluctuating temperatures, showed severe clinical signs and ADV in the nasal secretions persisted longer than in the ADV-infected control pigs kept at the normal temperature (20 +/- 2 degrees C). High concentrations of ADV were isolated from nasal secretions on the 1st day after inoculation of the virus. Pathologically, all ADV-infected pigs had non-suppurative encephalitis and trigeminal ganglionitis. The lesions were more widely distributed in pigs exposed to fluctuating temperatures than in infected control pigs. Two infected pigs given the stress had severe malacic foci in the frontal lobe and four of them had prominent interstitial pneumonia. In the pigs exposed to fluctuating temperatures, a significant number of immunoglobulin-containing cells, especially IgM-containing cells, did not respond to ADV infection. A significant (P < 0.01) difference in the number of IgG- and IgM-containing cells was observed between the ADV-infected pigs exposed to the fluctuating temperature and ADV-infected control pigs, respectively. These results demonstrated that the stress of fluctuating temperatures enhanced the susceptibility to ADV infection.

Immunohistological study of encephalomalacia in pigs infected with Aujeszky's disease virus.

Seven hysterectomy-derived colostrum-deprived pigs aged 4 weeks were inoculated intranasally with 10(3) plaque-forming units (1 ml) of the Yamagata YS-81 strain of Aujeszky's disease virus. One pig died and five developed encephalomyelitis and trigeminal ganglionitis. Three pigs killed on days 12-16 showed prominent malacic degeneration. Associated with the malacic foci were many lysosome-positive cells. IgG- and IgM-containing cells in the perivascular cuffs and glial nodules were first detected on day 7, after which they increased in number. They were thought to be closely associated with the presence of neutralizing antibody. These findings suggest that inflammatory cells in the brain are of haematogenous origin.

Immunopathological and ultrastructural studies on the tonsil of gnotobiotic pigs infected with strain 67N of haemagglutinating encephalomyelitis virus.

The 67N strain of haemagglutinating encephalomyelitis virus (HEV) was inoculated orally or nasally into ten 3-day-old gnotobiotic piglets. Tonsillar changes characterized by epithelial degeneration and lymphatic cell infiltration occurred in the crypt from post-inoculation days (PID) 5 to 7, and HEV was isolated from the tonsil on PID 3 and 5. Ultrastructurally, many HEV particles were found in the cisternae, cytoplasmic vesicules and extracellular spaces of crypt epithelium. Immunohistologically, many HEV particles were observed in the crypt epithelial cells in the early stage of infection. Thereafter, a large number of immunoglobulin (IgG, IgM and IgA)-containing cells were observed in the tonsil. The appearance of IgM-containing cells was coincident with the detection of neutralizing antibody in the serum. These findings suggest that HEV replication in the crypt epithelium stimulates the proliferative response of immunoglobulin (IgG, IgM and IgA)-producing cells.

Pathological changes in HPCD pigs with prednisolone induced recrudescence of pseudorabies virus.

In HPCD pigs inoculated with PRV, latent PRV could be reactivated in-vivo by the administration of large doses of prednisolone 3 months after the primary infection. In two pigs, virus shedding was without clinical signs of disease, whereas depression of circulating lymphocytes was prominent. Reactivation of PRV was also demonstrated by cultivation of the brain cortex on the 7th day and the mandibular lymph node on the 9th day after the prednisolone began treatment. Coincident with the virus isolation, characteristic lesions were observed in 2 pigs in the central nervous tissues and mandibular lymph nodes and these were composed of cell necrosis and eosinophilic intranuclear inclusion bodies. Cells containing the intranuclear inclusion bodies had immature and mature PRV particles. Results of the present study with HPCD pigs indicated that the lesions in the brain and lymph node accompanied by eosinophilic intranuclear inclusion bodies were pathogonomonic lesions induced by reactivation of PRV.

Detection of reactivating pseudorabies virus in tissue by immunoperoxidase technique.

Reactivation of pseudorabies virus (PRV) was induced in pigs by prednisolone treatment. The virus was re-isolated from nasal secretions of four, brain cortex of two and mandibular lymph node of three out of 12 pigs, respectively. The characteristic lesion of recurrently infected pigs was focal necrosis in the brain cortex and mandibular lymph node, accompanied by reactivating virus particles in the degenerating cells. Coincident with the pathological lesions, PRV antigen was detected in two of the brain cortex and six of the mandibular lymph node specimens by immunoperoxidase staining. These results suggest that the immunoperoxidase technique was more sensitive than virus isolation for demonstrating the reactivity of PRV in recurrently infected pigs.

Neosporosis in the aborted fetus and newborn calf.

Protozoal encephalitic lesions were found in four aborted fetuses and one dead newborn calf. The organism was identified as Neospora caninum by immunoperoxidase. The brain lesions were of two forms. One was observed in three fetuses of 5 months gestation and was characterized by multifocal necrosis. The other was found in a 7-month fetus and in a newborn calf, and showed severe infiltration with macrophages and plasma cells containing IgG. This association, between the age of fetus and inflammation, may reflect development of the immune system in bovine fetuses.

Congenital spongiform change in the brain stem nuclei of a domestic kitten.

This report describes a novel spongiform change in the brain stem nuclei of a 9-month-old mixed breed kitten with neurological signs. Histologically, vacuoles were found in perineuronal spaces and neuropil, with mild to moderate astrocytosis in the brain stem nuclei. Vacuoles were not observed in the cytoplasm of neurons and no evidence of neuronal loss was found. Ultrastructurally, there were intramyelinic vacuoles with separation of lamellae at intraperiod lines and larger spaces formed by coalescence of ruptured vacuoles. Immunohistochemically, abnormal accumulation of prion protein (PrP) was not detected in the brain stem lesions. This is, to our knowledge, the first report of a feline spongiform change localized in the brain stem nuclei.

Effects of dexamethasone on the pathogenesis of porcine circovirus type 2 infection in piglets.

To investigate the effect of immunosuppression on porcine circovirus type 2 (PCV2) infection, hysterectomy-produced, colostrum-deprived piglets were inoculated with the virus by the intranasal or intraperitoneal route, with or without dexamethasone (DEX) treatment. Eleven piglets aged 8 days were divided into four groups, namely group A (four animals given PCV2), B (three given PCV2 with DEX), C (two given sterile medium with DEX) and D (two given sterile medium). No significant clinical signs were observed. Histopathological and immunohistochemical examination revealed granulomatous inflammation and PCV2 antigen in the lymphoid tissues of group B piglets, but not in the other three groups. Flow cytometric analysis of peripheral blood lymphocytes showed a reduced number of CD4+ T cells in DEX-treated piglets (groups A and C). No differences between groups were observed in respect of the number of B cells, serum IgG concentration, or PCV2 antibody titre. These results indicate that DEX influenced the pathogenic effects of PCV2 infection in lymphoid organs, and that suppression of cell-mediated immunity may play a role in the aetiology of postweaning multisystemic wasting syndrome.

In vitro isolation and characterisation of a bovine Neospora species in Japan.

Eleven aborted bovine fetuses and five calves suspected as having neosporosis were necropsied and tissues from these animals were inoculated into bovine cardiopulmonary aortic endothelial cells and monkey kidney cells and maintained at 37 degrees C with 5 per cent CO2. Neospora tachyzoites were observed in one cell 49 days after inoculation. The isolated parasite (JPA1) was morphologically identical to the previously reported bovine Neospora species (BPA1) and confirmed by its strong antigenic reactivity with bovine control antisera to Neospora species and its lack of reactivity with Toxoplasma gondii and Sarcocystis cruzi antisera. This is the first bovine Neospora species isolate in Asia and further studies with this isolate are now expected.

Preparation and applicability of Sarcocystis cruzi antigens and their anti-S. cruzi rabbit sera for serodiagnosis of bovine sarcocystosis.

Sarcocystis cruzi antigens and their antisera were prepared for the seroimmunological diagnosis of bovine sarcocystosis. Fresh S. cruzi cysts directly removed from cardial muscle of slaughtered cattle were digested with trypsin to release bradyzoites. By twelve cycles of freezing at-22 degrees C and thawing at 37 degrees C, bradyzoites were let to leach out soluble material. The soluble antigens were inoculated four times to rabbits at a dose of 343 micrograms protein and anti-S. cruzi sera were prepared with blood of the rabbits. Gel immunodiffusion test showed no cross-reaction between the present antigens and any of anti-Toxoplasma gondii, -Hammondia hammondi and -Besnoitia wallacei rabbit sera. Avidin-biotin complex immunoperoxidase (ABC) technique with the present anti-S. curzi rabbit sera showed clear positive reaction against S. cruzi cysts in the muscular sections of infected cattle.