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.

A pathological study of sepsis associated with sarcoptic mange in raccoon dogs (Nyctereutes procyonoides) in Japan.

The pathological findings in Japanese raccoon dogs with sarcoptic mange infection associated with death from sepsis are described. Microscopical lesions of the skin were consistent with those described previously in wildlife populations with Sarcoptes infection, but secondary lesions were also present in the lungs, heart, kidneys, liver, spleen and brain of these animals. This infection was therefore very similar to "crusted scabies" or "Norwegian scabies" in man and was characterized by severe pathology and high mortality, with deaths frequently occurring due to sepsis.

Immunohistochemical demonstration of angiogenesis-associated homeobox proteins in canine vascular tumours.

Angiogenic homeobox genes regulate the behaviour of endothelial cells (ECs) during angiogenesis, so the aim of this study was to determine whether expression of these genes may be a determinant of malignancy in canine haemangiosarcoma (HSA). Homeobox proteins were evaluated immunohistochemically in tissue samples from canine HSAs (n=78), haemangiomas (HAs; n=30) and samples of granulation tissue (n=8). Active ECs in granulation tissue were positively labelled by antisera specific for HoxA9, HoxB3, HoxD3, HoxB7, Pbx1 and Meis1. Quiescent ECs in granulation tissue did not express HoxD3 and Pbx1. There were significantly more neoplastic cells positively labelled for HoxA9, HoxB3, HoxD3 and Pbx1 in HSA compared with HA. Almost all tumours were positive for HoxB7 and Meis1. HoxB3, HoxD3, Pbx1 and Meis1 proteins were detected in 80-90% of the HSAs, but in <20% of the HAs. Overall, homeobox protein expression in HSA appears to have a phenotype similar to that of active ECs in angiogenesis. The expression of homeobox genes associated with angiogenesis might be associated with the malignant growth of HSA.

Activation of matrix metalloproteinase (MMP)-2 by membrane type 1-MMP and abnormal immunolocalization of the basement membrane components laminin and type IV collagen in canine spontaneous hemangiosarcomas.

We performed immunohistochemical investigation of the basement membrane (BM) components, namely, type IV collagen and laminin, in 83 canine hemangiosarcomas (HSAs), 22 hemangiomas, and some granulation tissues (GTs). Additionally, we analyzed the expression and activities of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP (MT1-MMP) using the same samples by immunohistochemistry and gelatin zymography to investigate whether MMPs were associated with the BM degradation. In immunohistochemistry for the BM components, many HSAs showed discontinuous linear/negative immunoreactivity in the BM (type IV collagen: 49.4%/14.5%, laminin: 60.3%/10.8%, respectively). In contrast, almost all hemangiomas showed continuous staining in the BM (type IV collagen: 90.9%, laminin: 95.5%, respectively). Interestingly, positive cytoplasmic immunoreactivity for type IV collagen and laminin was observed in 97.6% and 91.6% HSA, respectively. Although MMP-9 immunoreactivity wasn't detected in neoplastic and active angiogenic endothelial cells (ECs), MMP-2 was detected in all ECs of GTs and in neoplastic cells of both vascular tumors. A strong immunoreactivity for MT1-MMP was observed in active angiogenic ECs in GTs and in neoplastic ECs in HSAs. However, almost all hemangiomas showed weak/negative immunoreactivity. In gelatin zymography, significantly strong activity of active MMP-2 was observed in HSAs, similar to that in active angiogenesis in GTs; however, weak/no activity of active MMP-2 was detected in hemangiomas. In canine HSA, neoplastic cells had active MMP-2, possibly activated by MT1-MMP, and discontinuous status of BM might be associated with activity of active MMP-2.

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.

Acute neuropathogenicity with experimental infection of equine herpesvirus 9 in common marmosets (Callithrix jacchus).

BACKGROUND: Equine herpesvirus 9 (EHV-9) is a new neurotropic equine herpesvirus which induced encephalitis in a variety of animals. However, there was no information on the susceptibility of EHV-9 in primates. METHODS: To assess the infectivity of EHV-9, four common marmosets (Callithrix jacchus) were inoculated by the nasal route with 10(6) plaque-forming units of EHV-9. RESULTS AND CONCLUSIONS: All of the inoculated animals exhibited various neurological signs progressing to collapse. Histologically, the affected animals had severe encephalitis characterized by neuronal degeneration and necrosis with intranuclear inclusion bodies, which extended from the olfactory bulb to the rhinencephalon and piriform lobe. Immunohistochemistry revealed EHV-9 antigens in degenerating neuronal cells. The nasal cavity had severe necrotizing rhinitis with prominent intra-nuclear inclusion bodies in the olfactory mucosa. These findings indicate that the marmosets are susceptible to EHV-9.

Teratoma of the umbilical cord in a giraffe (Giraffa camelopardalis reticulata).

A 12-year-old pregnant female giraffe (Giraffa camelopardalis reticulata) died approximately 2 months prior to her anticipated parturition date. At necropsy, a mass measuring approximately 20 x 36 x 20 cm was observed, attached to the umbilical cord, the latter being otherwise normal in appearance. Histologically, the mass contained 3 germinal tissue components with areas of squamous epithelium, respiratory epithelium, primitive neural tissues, glial tissue, peripheral nerve, adipose tissue, cartilage, and smooth muscle. Based on these findings, the tumor was diagnosed as a teratoma originating from the umbilical cord. This is possibly the second reported case of umbilical cord teratoma in animals.

Expression of vascular endothelial growth factor, basic fibroblast growth factor, and their receptors (flt-1, flk-1, and flg-1) in canine vascular tumors.

Expression of vascular endothelial growth factor (VEGF), its receptors (flt-1 and flk-1), and basic fibroblast growth factor (bFGF) in canine hemangiosarcoma (HSA) and hemangiomas was investigated by immunohistochemical analysis. In addition, expression of the mRNAs of VEGF, flt-1, flk-1, and flg-1 (a receptor for bFGF), was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) with cRNA probes. VEGF, bFGF, flt-1, and flk-1 were immunohistochemically detected in the neoplastic cells in HSAs; the staining intensity was stronger in HSAs than in hemangiomas. On the other hand, the neoplastic cells in hemangiomas exhibited very weak or no expression of VEGF, although they showed moderate expression of flt-1 and flk-1. The mRNAs of VEGF, flt-1, flk-1, and flg-1 were detected in the neoplastic cells in HSAs by ISH and RT-PCR. However, VEGF mRNA was not detectable in the neoplastic cells in hemangiomas by ISH, although it was detected in the inflammatory cells in the tumors by RT-PCR. Moreover, the HSAs that showed intense staining for flk-1 had a high proliferative activity, which was reflected as a high Ki-67 positive index. These results suggest that the expression of the growth factors and their receptors, especially flk-1, might be associated with the malignant proliferation of HSAs.

Morphological and immunohistochemical features of Cryptosporidium andersoni in cattle.

Light and electron microscopic features and immunohistochemical features of Cryptosporidium andersoni (C. andersoni) and host reaction in the mucosa were studied. Although the affected cattle demonstrated no apparent clinical signs, a severe infection of C. andersoni was observed in the abomasum. C. andersoni were round in shape, measured 6-8 microm in size and were mainly observed to be freely located in the gastric pits, being attached in occasional cases to the surface of the abomasum epithelium. Frequent inflammatory cells had infiltrated the lamina propria of the affected mucosa, and frequent mitotic figures were observed in epithelial cells at the dilated isthmus. To access the cell kinetics, the number of epithelial cells infected with C. andersoni were counted and compared with noninfected cattle. The number of gastric pit cells in infected cattle was significantly higher than that in the controls. The number of proliferative cells determined by the Ki-67 antigen in C. andersoni infected cattle was also significantly higher than that in the controls. Transmission electron microscopy and scanning electron microscopy revealed that the morphology of the C. andersoni organism was common to those of other Cryptosporidium spp. Immunohistochemically, several commercial antibodies against Cryptosporidium spp. showed positive reactions at the wall of these oocysts or parasitophorous vacuoles. This report is possibly the first to discuss the prominent hyperplasia of the abomasum mucosa, as well as morphologic features of C. andersoni in cattle.

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.

Expression of myogenic regulating factors, Myogenin and MyoD, in two canine botryoid rhabdomyosarcomas.

Myogenin and MyoD regulate the development of skeletal muscle, and their expressions are specific to the stages of myogenesis. Therefore, these myogenic regulatory proteins could be considered as sensitive and specific markers for rhabdomyosarcoma. In this report we investigated the immunohistochemical reactivities of myogenin and MyoD in two canine bladder botryoid rhabdomyosarcomas that were different in the degree of differentiation. MyoD was stained in the Ki-67 antigen-positive undifferentiated mesenchymal cells, which had proliferative activity similar to myoblasts differentiated from mesoblasts. In contrast, multinucleated neoplastic cells were positive for myogenin and alpha-sarcomeric actin but not for Ki-67 antigen, similar to the myotubes differentiated from myoblastic cells. The expressions of myogenin and MyoD were closely correlated to the histologic features of myogenic neoplastic cells.

Experimental infection of equine herpesvirus 9 in dogs.

Equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus, was inoculated intranasally at 107 plaque-forming units in five dogs to assess its pathogenicity. Dogs showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brains. Histologically, dogs had a fulminant nonsuppurative encephalitis characterized by severe neuronal degeneration and loss, with intranuclear inclusions, slight glial reactions, perivascular cuffing, and multifocal hemorrhage. The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed reactivity for EHV-9 antigen in neurons. All dogs had mild bronchopneumonia and various degrees of lymphoid necrosis. These findings indicate that dogs are fully susceptible to EHV-9 and that EHV-9 can cause fulminant encephalitis with high mortality in dogs, as in gazelles and goats.

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.

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.

Pathological changes of renal tubular dysplasia in Japanese black cattle.

Pathological studies were conducted on 91 Japanese Black cattle with a hereditary disease which induced growth retardation, long hooves and renal failure. In calves one to two months old, no gross abnormalities were observed in the kidneys, but microscopical examinations revealed immature epithelia which were arranged irregularly and not attached to the basement membranes in some proximal tubules. In animals three to 36 months old, the kidneys had shrunk perceptibly and had grey-white radial streaks; microscopically they showed severe interstitial fibrosis with round-cell infiltration in the outer zone of the medulla and cortex, and reductions in the numbers of glomeruli and tubules. In the fibrotic areas there were immature epithelia with an irregular arrangement, and the basement membrane of the tubules was thickened. It was concluded that renal tubular dysplasia was the primary lesion of the disease, and that interstitial fibrosis and reductions in the numbers of nephrons were secondary lesions.

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.

Differential effects of partial hepatectomy and carbon tetrachloride administration on induction of liver cell foci in a model for detection of initiation activity.

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CCl(4)) administration on induction of glutathione S-transferase placental form (GST-P)-positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative-quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and CYP 2E1 apoprotein amount by immunoblotting (experiment I) after PH or CCl(4) administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CCl(4) administration and induction of liver cell foci, the non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) was administered to 7-week-old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CCl(4) administration were drastically decreased at the 12-h time point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CCl(4) group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST-P-positive foci was related to cell kinetics in the PH group, with about a 6-h time lag between time for carcinogen administration giving greatest induction of GST-P-positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CCl(4) administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.

Avian-to-mammal transmission of an avian rotavirus: analysis of its pathogenicity in a heterologous mouse model.

It has been suggested that group A avian rotaviruses can be transmitted to mammals, but there is no direct evidence that such viruses induce disease in mammals. Suckling mice were orally inoculated with two avian rotaviruses. A pigeon rotavirus, PO-13, was found to induce diarrhea, but a turkey rotavirus, Ty-3, did not. The diarrhea induced by PO-13 was dependent on the age of the mouse. In histopathological examinations, antigens of PO-13 were sporadically detected in absorptive cells in the ileum, and lesions were observed as ballooning degenerations of absorptive cells in a region from the duodenum to the ileum. However, the rotavirus antigen was not detected in the majority of these degenerative cells. These results indicated that PO-13 could infect and induce diarrhea in suckling mice. This is the first evidence of an avian rotavirus being experimentally transmissible to a mammal.