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.

Experimental infection of pregnant cows with noncytopathogenic bovine viral diarrhoea virus between days 26 and 50 postbreeding.

The effect of bovine viral diarrhoea virus (BVDV) infection on early pregnant cows between 10 and 24 days after virus inoculation at day 26 of pregnancy was determined. Four cows were inoculated intravenously with either BVDV (treated, n=3) or growth medium (control, n=1). The treated cows were euthanized on either day 10, 17 or 24 post-infection and the control cow was euthanized on day 24 post-infection. The level of serum 2-5A synthetase increased in all of the three treated cows. Progesterone levels decreased to below 1.0 ng/ml between 10 and 22 days after inoculation in two of the three treated cows and the embryos/foetuses of two cows died. Therefore, BVDV may be a cause of early embryonic or feotal loss in early pregnant cows and serum 2-5A synthetase may be useful as an indicator of viral infection in cows.

Experimental infection of early pregnant cows with bovine viral diarrhea virus: transmission of virus to the reproductive tract and conceptus.

The objective of this study was to chronologically investigate the effect of bovine viral diarrhea virus (BVDV) infection on early pregnant cows before placenta formation. Six cows were intravenously inoculated with either BVDV (treated, n=4) or growth medium (control, n=2) (day 0) at day 26 of pregnancy. Two treated cows and one control cow were euthanized on day 3 post-infection and the remaining animals were euthanized on day 6. BVDV was isolated from maternal tissues such as lymphoid or reproductive tissues of treated animals on days 3 and 6 post-infection. Additionally, one treated cow autopsied on day 6 post-infection had evidence of infectious BVDV in the allantoic membranes, allantoic fluid and embryos. In three treated cows, a significant decline in progesterone concentration was also observed post-infection while in control cows they remained constant. Therefore, BVDV can infect bovine embryos before placenta formation and may affect progesterone profiles in cows during early pregnancy.

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 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.

Abscess-forming inflammatory granulation tissue with Gram-positive cocci and prominent eosinophil infiltration in cats: possible infection of methicillin-resistant Staphylococcus.

We occasionally encounter feline cervical or mesenteric lesions diagnosed histopathologically as abscess or inflammatory granulation tissue with eosinophil infiltration. Gram-positive cocci accompany the lesions. In the present study, such lesions obtained from 27 cats were examined to evaluate the histopathologic features and the nature of the causative bacteria. The average age was 7.3 +/- 3.5 years. No sex predilection was observed. Most frequent locations of the lesions included the abdominal cavity with/without mesenteric lymph nodes (11/27, 41%) and subcutaneous tissue or lymph nodes of the neck (9/27, 33%). Common clinical presentation was a localized mass. Grossly, the lesions contained abscesses in the center and were surrounded by fibrous tissue. Microscopically, the necrotic zone contained bacterial colonies. Large numbers of eosinophils and macrophages infiltrated the area surrounding the necrotic tissue. The surrounding connective fiber-rich granulation tissue demarcated the eosinophilic abscess. The bacteria were Gram-positive cocci in 23 of the 27 cats and were positive for anti-staphylococcus antiserum in 19 of the 23 cats. In 15 out of 17 lesions, the colonies expressed immunoreactivity to penicillin-binding protein 2', which is a drug-resistance gene product of methicillin-resistant Staphylococcus (MRS) species. These findings suggest strongly that MRS causes this type of infectious lesion.

Encephalomyelitis associated with akabane virus infection in adult cows.

Between August and September 2000, five 2-7-year-old cows in Korea exhibited neurologic signs and were diagnosed as infected with Akabane virus based on the results of histopathology, immunohistochemistry, serology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss was noted in the brain and the ventral horn gray matter of the spinal cord. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigen was detected in the brain and spinal cord, mainly in degenerating neurons and glial cells. RT-PCR analysis revealed a target band of expected size in four cows. This is the first report on an outbreak of natural Akabane virus infection in adult cattle.

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.

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.

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.

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells.

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.

Supplementary report on experimental autumn crocus (Colchicum autumnale L.) poisoning in cattle: morphological evidence of apoptosis.

Previously we reported that tissue destruction characterized by the presence of karyopyknotic, karyorrhectic and mitotically arrested cells was seen in alimentary epithelial cells and lymphocytes in the lymphoid and hemopoietic systems of cattle experimentally administered with autumn crocus (Colchicum autumnale L.). This report deals with the mechanism of acute cellular injury following experimental autumn crocus poisoning in cattle as demonstrated by the in situ DNA strand break analysis and electron microscopy. The analyses revealed that cellular injury caused by autumn crocus was closely associated with apoptosis.

Characterization of antibodies raised against bovine-PrP-peptides.

To analyze the antigenicity of peptides derived from bovine prion protein (PrP) cDNA, we immunized rabbits with four synthetic peptides and compared the immunoreactivity of antibodies to PrPs from various species by immunoblotting and immunohistochemistry. Two of the antibodies reacted strongly with all PrPs. The other antibodies, raised against overlapping peptides close to two glycosylation sites, did not recognize PrPSc-mouse but did recognize PrPSc-sheep which contains two sugar residues and PrPCJD with or without a sugar residue. Our results suggest that these antibodies may have species-specificity for both glycosylation status and amino acid sequences of the protein. In conclusion, we identified two regions in bovine-PrP which appear suitable for raising antibodies that detect various kinds of PrPs, and one region (Ab103-121) which appears suitable for raising antibodies that detect several species of PrPs. These antibodies may be useful for diagnosing prion diseases and for researching their pathogenesis.

Pathological and immunological findings of athymic nude and congenic wild type BALB/c mice experimentally infected with Neospora caninum.

Neospora is a cyst-forming coccidian parasite that causes abortions and neuromuscular disorders in a wide variety of mammals. Japanese bovine isolate JPA1 was inoculated intraperitoneally into BALB/c nu/ nu (athymic nude) and BALB/c (congenic wild type) female mice to examine the distribution of parasites and resistance mechanisms to Neospora infection. All the athymic nude mice died within 28 days after intraperitoneal injection of 2 x 10(5) JPA1 tachyzoites, whereas all the congenic wild type mice survived without exhibiting any clinical signs. Tachyzoites were identified in the uterus and pancreas and later spread to many other organs. Most tachyzoites identified in the necrotic foci were localized in the epithelium of the venules and capillaries. Nude mice developed high level of serum interferon-gamma and interleukin-6 as infection proceeded. Inflammatory response to Neospora infection might be mediated by Th1-type dependent cellular immunity.