Physico-chemical properties of calf-diarrhea coronavirus

Replication of calf diarrhea coronavirus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. Sensitivity to ether and chloroform indicated that the virus is enveloped, and this was confirmed by electron microscopic observation of the virion. The virus was readily inactivated by trypsin and sodium deoxycholate. The virus was labile at 50°C in diluted medium, but readily stabilized in the presence of MgCl₂. It was stable at pH 5 and 7, while a slight loss of infectivity was observed at pH 3. The virus was readily filtered through membrane filters of 200 and 100-nm pore sizes, but not through 50-nm filters. The buoyant density of the virion in CsCl was estimated to be 1.25 g/ml.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Localization of endothelin receptor subtype-A in the testis of rats, dogs, and monkeys

In order to evaluate the physiological roles of the testicular endothelin (Edn) signaling via Edn receptor subtype-A (Ednra) in mammals, the localization of Ednra was investigated by in situ hybridization and immunohistochemistry in the testis of rats, dogs, and monkeys. For in situ hybridization, a rat Ednra RNA probe which is highly homologous to the subcloned canine and monkey Ednra (88.7% and 87.9% identical, respectively) was used. Both Ednra mRNA and protein were detected in interstitial cells and cells in the basal compartment of the seminiferous tubules, mainly Sertoli cells, as well as spermatogonia and some early spermatocytes, but not spermatids. The localization pattern of Ednra was exhibited in a same manner among species, indicating that the physiological role of Edn signaling throughout Ednra was maintained in the mammalian testis.     

Magnetic resonance imaging of alveolar echinococcosis experimentally induced in the rat lung

Pulmonary alveolar echinococcosis (AE) caused by the metacestode of Echinococcus multilocularis is a lethal zoonosis and is a lesion secondarily induced by hematogenous dissemination from hepatic AE lesions. In the present study, a hematogenous pulmonary AE model was experimentally induced in rats by the injection of echinococcal larval tissue homogenate to the tail vein, and then the pathological and diagnostic aspects of pulmonary AE were examined by magnetic resonance imaging (MRI). Histological primary, mature and degenerated AE lesions were observed 5, 18 and 50 weeks after injection, respectively. These lesions were discriminated as signal-void, hypointense and hyperintense regions in T1-weighted MRI (T1WI), respectively. The change in signal intensity in T1WI might reflect the content of proteinaceous fluid as a result of AE cyst degeneration. Western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis provided evidence for AE infection in the early stage. T1WI in combination with Western blot analysis could possibility become definitive and early signs of hematogenous pulmonary AE infection.     

Occurrence of swine salmonellosis in postweaning multisystemic wasting syndrome (PMWS) affected pigs concurrently infected with porcine reproduction and respiratory syndrome virus (PRRSV)

Fourteen diseased pigs from four farms in which there had been an outbreak of salmonellosis were investigated. Granulomatous inflammation with depletion of lymphocytes was observed in the swollen lymph nodes in these pigs. Antigens to porcine circovirus type 2 (PCV2) were immunolabeled in the lesions along with detection of viral DNA as PCV2 by polymerase chain reaction (PCR). In addition, antigens to porcine reproductive respiratory syndrome virus (PRRSV) were immunodetected in the lungs and Salmonella Choleraesuis was isolated from the affected pigs. The nine salmonellosis affected pigs, five (55.6%) with salmonellosis and PMWS concurrently infected with PRRSV were much higher than those infected with salmonellosis and PMWS (22.2%) or with salmonellosis and PPPRV (22.2%).     

The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat

Increases in intracellular Ca2+ are required for oocyte activation and subsequent development. Calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in oocyte activation. However, how CaMKII is regulated during this process is not well characterized. We show here for the first time in rat oocytes that CaMKII is phosphorylated during oocyte activation. CaMKII phosphorylation was suppressed by KN93, a CaMKII inhibitor, but not KN92, which is the inactive analogue of KN93. Electrical stimulation of rat oocytes resulted in degradation of both cyclin B and Mos, presumably due a rise in Ca2+ induced by the electrical pulse. KN93 blocked the degradation of both proteins induced by the electrical pulse. Addition of a protein phosphatase inhibitor, okadaic acid (OA), further increased the amount of CaMKII and also increased the amount of phosphorylated enzyme. Importantly, in oocytes undergoing spontaneous activation, accumulation and phosphorylation of CaMKII also occurs in a time-dependent manner. Consistent with this, addition of KN93 inhibited spontaneous activation. Collectively, our results show that CaMKII is phosphorylated during oocyte activation and that this phosphorylation is involved in inactivation of p34cdc2 kinase and somewhat involved in degradation of Mos. Furthermore, CaMKII phosphorylation is negatively regulated by a protein phosphatase.     

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Comparison of cell populations derived from canine olfactory bulb and olfactory mucosal cultures

OBJECTIVE: To evaluate the numbers and proportions of olfactory ensheathing cells (OECs) in cell cultures derived from the olfactory bulb (OB) and olfactory mucosa of dogs. ANIMALS: 7 dogs. PROCEDURES: OB tissue and olfactory mucosa from the nasal cavity and frontal sinus were obtained from euthanatized dogs and prepared for cell culture. At 7, 14, and 21 days of culture in vitro, numbers and proportions of OECs, astrocytes, and fibroblasts were determined via immunocytochemistry. Antibody against the low-affinity nerve growth factor receptor p75 was used to identify OECs, antibody against glial fibrillary acidic protein was used to identify astrocytes, and antibody against fibronectin was used to identify fibroblasts. RESULTS: Cultured OECs derived from the olfactory mucosa of the nasal cavity and frontal sinus had similar characteristics. However, whereas OECs in the OB cell cultures constituted approximately 50% of the cells at 7 days and approximately 75% at 21 days the proportion of OECs in cultures derived from both mucosal types was much lower, with approximately 40% OECs at 7 days and approximately 25% at 21 days. Analysis of OEC numbers revealed that these changes were accompanied by corresponding decreases and increases in the population of cells with fibronectin receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although olfactory mucosal cell cultures yielded a sufficient number of OECs for spinal cord transplantation procedures in dogs, modification of culture conditions would be required to ensure that the derived cell population contained a sufficient proportion of OECs.