Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Nasal neuroendocrine carcinoma in a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus).

Neuroendocrine carcinoma was diagnosed in the left nasal cavity of a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus). Microscopically, the tumour consisted of sheets of anaplastic cells separated by narrow zones of fibrovascular stroma. The neoplastic cells had varying numbers of cytoplasmic granules stained by the Grimelius method. Immunohistochemically, the neoplastic cells were variably labelled for cytokeratin AE1/AE3, vimentin, chromogranin A and S-100. Ultrastructurally, some of the neoplastic cells had cytoplasmic membrane-bound dense-core granules of approximate diameter 140-240nm. The tumour had infiltrated the cerebrum and metastasized to the pituitary gland, mandibular and pulmonary lymph nodes, lungs, thyroid gland and adrenal glands.

Expression of the anti-apoptotic factors Bcl-2 and survivin in canine vascular tumours.

To investigate whether anti-apoptotic factors play a role in the malignant growth of canine haemangiosarcomas (HSAs), 83 HSAs and 22 haemangiomas were examined immunohistochemically for bcl-2 and survivin expression. Additionally, bcl-2 and survivin mRNA expression was quantified by semiquantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunolabelling for bcl-2 was observed in 50 of the 83 HSA samples (60.2%) but in none of the haemangiomas. The average survivin positive index was 24.7% in the HSAs and 0.6% in the haemangiomas. In contrast to the high average value for survivin mRNA expression, which was approximately six times that for the haemangiomas, no significant difference was observed between HSAs and haemangiomas for the average bcl-2 mRNA expression level. The discrepancy between bcl-2 mRNA and bcl-2 protein expression requires further investigation, but the results suggest that malignant proliferation in canine HSAs is associated with bcl-2 and survivin expression.

Summation of initiation activities of low doses of the non-hepatocarcinogen 1,2-dimethylhydrazine in the liver after carbon tetrachloride administration.

Summation of initiation by low doses of the indirect-acting non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) after proliferative stimulation with a necrogenic dose of carbon tetrachloride (CCl4) was investigated in terms of the induction of glutathione S-transferase placental form (GST-P) positive liver cell foci. Cell kinetics of liver after CCl4 i.g. treatment were examined with bromodeoxyuridine (BrdU) labeling (experiment I). To assess the correlation between cell proliferation and induction of liver cell foci, DMH (10 mg/kg i.g.) was administrated to 7-week-old male F344 rats at 12, 24, 36, 48, 60, 96 h after CCl4 i.g. and initiated populations expanded using the resistant hepatocyte model (experiment IIA). Subsequently, effects of repeated administration (10 mg/kg, four times, i.g.) of DMH were compared with the results of a single administration (40 mg/ kg, i.g.) with the same total dose (experiment IIB). In experiments I and IIA, the numbers and areas of GST-P-positive foci increased with the BrdU labeling index at the time of DMH treatment (maximum after 60 h). In experiment HB, repeated exposure of DMH at 10 mg/kg, four times resulted in significant (P<0.05) increase in number and area of GST-P-positive foci compared with the single administration (40 mg/kg). Thus, multiple low dose treatments during cell proliferation might be most effective for detection of weak initiation activity.

Proprioceptive innervation of the masticatory muscles in Pinché.

This study of the masticatory muscles of the primate showed that the temporal muscle contained 107 muscle spindles, 45 in the horizontal portion and 62 in the vertical portion; the masseter muscle contained 70, 58 in the profundus portion and 12 in the superficial portion; the medial pterygoid muscle contained 15; the lateral pterygoid muscle contained 6; and the zygomaticomandibular muscle contained 9. The muscle spindles were located around the coronoid process and mandibular ramus.

Muscle spindle supply to the human jaw muscle.

Histological study of the human jaw muscle revealed that the temporal muscle displayed 342 muscle spindles, 208 in the horizontal and 134 in the vertical portion; the masseter muscle contained 114 spindles, 91 in the superficial and 23 in the profound portion; the medial pterygoid muscle had 59; and the lateral pterygoid muscle contained 6, four in the upper head and two in the lower head. These data suggest that the extensive mobility of the temporomandibular joint, and the maintenance of mandibular posture during mastication and speech are strongly influenced by proprioceptive mechanisms.

In vitro attenuation of highly virulent infectious bursal disease virus: some characteristics of attenuated strains.

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

S-100 immunoreactive cells in the spleen and bursa of Fabricius of broiler chickens.

The distribution of S-100 protein in spleens and bursae of Fabricius of broiler chickens with various diseases was investigated by an immunohistochemical method with antiserum to bovine S-100 protein. S-100 protein-positive cells were found in six cases of 34 chickens. In spleens, S-100 protein was detected in reticular cells in the ellipsoids of the sheathed arteries and eosinophilic granulocytes. Follicular dendritic cells in germinal centres were also S-100 protein-positive. In the bursa, reticular cells of the follicles and eosinophilic granulocytes in the follicular cortex and surface epithelium were positive for S-100 protein. The evidence suggests that S-100 protein may be involved in diseases of chickens.

Expression of cyclin kinase inhibitor p27(Kip1) in skin tumours of dogs.

Skin tumours (n=148) of epidermal or hair follicle origin were examined immunohistochemically to determine the expression of p27(Kip1)(p27), a cyclin-dependent kinase inhibitor (CDKI), and of Ki-67. In normal skin, a large number of basal cells of the epidermis and hair follicles were positive for Ki-67 and many suprabasal epithelial cells were positive for p27. Most of the hair matrix cells were positive for Ki-67 but negative for p27. Hair papillae were strongly positive for p27. Squamous cell carcinomas had a p27 positive index (PI) significantly lower than that of trichoepitheliomas (P<0.005), basal cell tumours (P<0.05) and intracutaneous cornifying epitheliomas (P<0.001). In contrast, Ki-67 PIs of squamous cell carcinomas and pilomatrixomas were significantly higher than those of trichoepitheliomas, basal cell tumours and intracutaneous cornifying epitheliomas (P<0.01 to P<0.001). No significant difference was observed between the Ki-67 PI values of squamous cell carcinomas and pilomatrixomas. The results suggested that p27 is capable of suppressing cell proliferation in the differentiation of normal canine skin. In spite of being a benign neoplasm, pilomatrixomas had a low p27 expression; this may be a reflection of the proliferative potential of the hair matrix. The expression of p27 may be a useful marker for the analysis of cell kinetics.

Proliferative activity of canine mast cell tumours evaluated by bromodeoxyuridine incorporation and Ki-67 expression.

The proliferative activity of 91 canine mast cell tumours was assessed on the basis of the Ki-67 positive index (Ki-67 PI) and mitotic index (MI) and, in 15 cases, also by the labelling index of bromodeoxyuridine (BrdU; an analogue of tritiated thymidine) incorporated in vivo into S-phase cells. BrdU and Ki-67 were detected immunohistochemically. The tumours were graded histologically (I, II or III). The BrdU labelling index (BrdU LI) tended to increase as the grade became higher. In terms of the mean values of Ki-67 PI, significant differences were found between histological tumour grades I and II (P < 0.01) and between grades II and III (P < 0.01). In terms of mean MI, grades I and II were found to differ significantly (P < 0.05). With Spearman rank correlation coefficient and linear regression analysis, the BrdU LI and Ki-67 PI showed a highly significant correlation. This strong correlation indicated that Ki-67 was, like BrdU, a useful marker for proliferative potential in canine mast cell tumours; moreover, its use did not require the prior administration of any reagent to the live animal.

Subclinical renal oxalosis in wild-caught Japanese macaques (Macaca fuscata).

Various degrees of crystal deposition were found in the kidneys of 12 out of 59 Japanese macaques (Macaca fuscata) caught in the wild in Gifu, Japan. The needle- or rod-shaped crystals, which were radially arranged and occurred in the lumen and epithelium of the renal (mainly the proximal) tubules, were birefringent under polarized light. They stained with alizarin red S at a pH of 7.0 but not 4.2, and were identified as calcium oxalate. The morphological features of the renal lesions were similar to those previously reported in oxalate poisoning, and it was believed that the macaques ingested the oxalate in plants.

Spontaneous aortic arteriosclerosis in layer chickens.

Aortas of 21 grower chickens (both sexes) aged 30-130 days and of 51 layer hens aged 500 days were examined histopathologically. Aortic sclerotic lesions were classified histopathologically into six stages. As early as 30 days, the abdominal aortas of growers had cellular-fibrous plaques that became more severe with advancing age. The majority of the abdominal aortas of all the 500-day-old layers had a conspicuous increase of collagen fibres, with atheromatous lesions in 23 (45.1%). These sclerotic changes were most severe in the distal aortas.

Experimental infection with equine herpesvirus 9 (EHV-9) in cats.

The pathogenicity for cats of EHV-9, a new neurotropic equine herpesvirus, was assessed by intranasal inoculation with 10(6) plaque-forming units. Four cats killed 4, 5, 6 or 10 days after inoculation showed neurological signs consisting of hyper-excitability and aggressiveness, followed by tremors, occasional convulsions, and depression. Histologically, the cats showed severe encephalitis characterized by neuronal degeneration and loss, intranuclear inclusions, perivascular cuffing and gliosis in the cerebrum. A positive immunohistochemical reaction for EHV-9 antigen was seen in degenerating neuronal cells. The lesions extended from the olfactory bulb to the rhinencephalon and hippocampus. All cats had rhinitis, with or without intranuclear inclusion bodies in the nasal mucosa, and interstitial pneumonia. These findings indicate that the cat, like certain other species such as the goat, is susceptible to experimental infection with EHV-9, and may be at risk from natural infection.

Assessment of proliferative potentials of canine osteosarcomas and chondrosarcomas by MIB-1 immunohistochemistry and bromodeoxyuridine incorporation.

The proliferative potential of 17 canine osteosarcomas (OSs) (13 osteoblastic, two anaplastic, one fibroblastic and one chondroblastic), 18 chondrosarcomas (CSs) (13 mesenchymal and five ordinary), three osteomas, and one chondroma was evaluated immunohistochemically by labelling Ki-67 antigen with MIB-1 antibody, and incorporated bromodeoxyuridine (BrdU) with anti-BrdU antibody. The location of BrdU-positive cells in OSs and CSs was similar to that of MIB-1 positive cells, and the mean value of the BrdU labelling index (BrdU LI) and the MIB-1 positive index (MIB-1 PI) in each case were significantly correlated (rs = 0.942, P < 0.05 with Spearman rank correlation coefficient; r = 0.779 P < 0.05 with linear regression analysis). The mean MIB-1 PI of OSs was 29.5%, which was approximately 2.5 times that of CSs, and the highest MIB-1 PI was 34.8% +/- 1.8 S.E.M. in areas without osteoid. In CS cases, the survival rate after 24 months was significantly higher than in OS cases. The high MIB-1 PI therefore supports the view that OSs are clinically more aggressive than CSs in dogs. On the other hand, the highest MIB-1 PI values of mesenchymal CS components occurred in transitional areas, which were composed of poorly differentiated cells embedded in a myxomatous matrix between the chondroidal and mesenchymal regions. The MIB-1 PI was 21.3% +/- 3.0 S.E.M. P < 0.001 in transitional areas. Proliferative markers may be useful in diagnosis and prognosis.

Neuropathological study of gazelle herpesvirus 1 (equine herpesvirus 9) infection in Thomson's gazelles (Gazella thomsoni).

Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomson's gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.

Spontaneous vascular mineralization in the brains of cows.

The frequency of occurrence and the characteristics of vascular mineralization in the brains of cows were studied. Mineralized lesions were found in the pallidal arteries in eight of 13 cows. They appeared as basophilic deposits in the arterial walls, were sheath-like or tubular in shape, and were accompanied by intimal oedema or fibrous thickening. The deposits were positive for periodic acid-Schiff, von Kossa's, alizarin red and Berlin blue stains. Elemental analysis revealed the presence of large amounts of phosphorus, calcium and zinc, as well as small amounts of iron, copper and sodium.

Spontaneous globoid mineralization in the cerebellum of rats.

Cerebellar globoid mineralization in two rats was examined by light and electron microscopy, and by X-ray microanalysis. The mineralization was round to oval in shape; it varied in size and was positive for the periodic acid-Schiff and von Kossa reactions. Ultrastructurally, a concentric lamellar structure was prominent in moderately electron-dense depositions. Elemental analysis revealed the presence of large amounts of calcium and phosphorus, and small amounts of zinc, potassium and aluminum.

Eosinophilic neuronal inclusions in the thalamus of ageing B6C3F1 mice.

The age-related incidence of thalamic neuronal inclusions in the brains of aged B6C3F1 mice and their histopathological and ultrastructural features were studied. Round to oval or rod-shaped eosinophilic inclusions, which were frequently observed in the neurons of the thalamic area, were stained positively with phosphotungstic acid haematoxylin. The inclusions were detected first at 32 weeks of age and the incidence and severity were higher in older animals, all of the mice being affected after 58 weeks of age. Ultrastructurally, the inclusions appeared as sheaves of parallel osmiophilic filaments in the perikaryon of the neurons in the thalamus. Thus thalamic neuronal inclusions in mice seem to be age-related, but their significance remains unclear.

Immunohistochemical demonstration of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in cattle tissues.

Bromodeoxyuridine (BrdU), a non-radioactive thymidine analogue, was administered to 15 cattle at a dosage of 1-10 mg/kg intravenously or intraperitoneally to demonstrate S-phase cells in the tissues. The organs and tissues were fixed in 10% neutral buffered formalin or in 70% ethanol, sectioned, denatured with hydrochloric acid, and treated with monoclonal antibody against BrdU. Immunohistochemical methods were used to "visualize" BrdU-labelled nuclei. BrdU-positive cells were satisfactorily demonstrated in both formalin- and ethanol-fixed tissues of animals given doses of 2 mg/kg or over, by either route of administration. Large numbers of BrdU-positive cells indicative of active cell production were found in the basal region of the stratified squamous epithelium, the neck between gastric pits and gastric glands in the abomasum, and the crypts of Lieberkühn of the small and large intestines. Moderate numbers of positive cells were observed amongst inflammatory cells in cases of nephritis and in granulation tissue. Numerous positive cells were detected in leukaemia cells. The study showed that BrdU can be used to measure proliferative S-phase cells in cattle, as in human beings, mice and rats.

Effects of atropine sulfate on rat harderian glands: correlation between morphological changes and porphyrin levels.

We examined the correlation between histopathological changes and porphyrin levels in the Harderian glands of male rats after treatment with atropine sulfate. After a single administration of atropine sulfate (250 mg/kg/day), the porphyrin levels in the Harderian glands gradually increased, beginning from 2 hr after administration, and at 36 hr reached a maximum level, which was about 7 times higher than that of the control animals. Histopathologically, the Harderian glands showed marked luminal dilatation and a brownish pigment accumulation in the lumina 6 hr after a single dose. Under daily repeated administrations of atropine sulfate (250 mg/kg/day), the highest porphyrin levels in the Harderian glands were observed 24 hr after the third dose, and were about 9 times higher than those of the control animals. However, beginning from one week after the initial dose, much lower peak porphyrin levels were observed 6 hr after each dose. The maximum porphyrin levels were only twice as high as those of the control animals, and they returned to the control levels 24 hr after each atropine dose. Histological examinations of the Harderian glands revealed that repeated administrations of atropine sulfate induced the same histopathological changes observed after a single atropine administration, and that no aggravated dilation of the lumina or pigmentation in the lumina appeared after such repeated administrations. The degree and incidence of the histopathological changes observed correlated well with the porphyrin levels. Some animals showed a degeneration of the glandular epithelium after 4 weeks of treatment, and the frequency increased slightly after 13 weeks of treatment. The present study suggests that atropine suppresses the expulsion of secretory materials, including porphyrin, from the glandular lumen of the Harderian glands, and thereafter an excessive accumulation of porphyrin induces luminal dilatation. These changes were gradually reduced by repeated administrations. The degeneration of the glandular epithelium after repeated administration might be a consequence of retention of an excessive accumulation of porphyrin.