重组高繁殖力疫苗株H5N2(H5/PR8)病毒的制备和鉴定

作为禽流感疫苗株,不但要求其具有良好的免疫原性,而且要求在生产中具有良好的生长特性,现用的A/Turkey/England/N28/73(H5N2)疫苗株,血凝价28,生长滴度较差.本研究利用自然重组法,预制备一株高繁殖力的预备疫苗株,使它的表面基因血凝素(HA)和神经氨酸酶(NA)来源于A/Turkey/England/N28/73(H5N2)毒株,并使其保持原有的良好的免疫原性,内部基因来源于流感病毒PR8,A/Puerto Rico/8/34(H1N1)毒株,并使其重组病毒赋有了流感病毒PR8高繁殖力的特性.实验结果表明,已获得一株高繁殖力的重组流感病毒H5/PR8,血凝价达到211,生长繁殖特性明显增强.抗原性分析显示,H5/PR8重组病毒与亲本毒株H5N2抗原性无明显差异;致病性分析发现,H5/PR8重组病毒的致病能力较亲本毒株H5N2有明显下降.这为疫苗株的改良奠定了坚实的基础,对禽流感灭活疫苗的生产具有重大意义.     

Canine T-cell lymphomas: a morphological,immunological, and clinical study of 46 new cases

The aim of this study is to report 46 new cases of canine T-cell lymphomas among a series of 140 lymphomas studied by immunophenotyping (incidence 32.8%). According to the updated Kiel classification adapted to the canine species, 13 were classified as low-grade and 33 as high-grade lymphomas. Among the low-grade lymphomas, five were small clear-cell lymphomas, three were pleomorphic small-cell lymphomas, and five mycosis fungoides. Among the high-grade cases, there were 11 pleomorphic mixed-, small-, and large-cell lymphomas, 6 pleomorphic large-cell lymphomas, 11 lymphoblastic lymphomas, and 5 unclassifiable high-grade plasmacytoid lymphomas. The cytohistologic features were highly suggestive of a T-cell phenotype on the basis of cell morphology (irregular nuclei and clear cytoplasms) (30/46 cases), a T-cell zone pattern, and the presence of hyperplastic postcapillary venules (22/46 cases). All 46 cases were CD3+ CD79a-, and among 34 cases investigated for CD4 and CD8 expression, 13 were CD4+CD8-, 13 were CD8+CD4-, and 8 were CD4CD8 double positive or double negative. The pleomorphic mixed lymphomas were mainly CD4+CD8- (6/7) and the lymphoblastic lymphomas were double positive or double negative (6/8). The main clinical, hematologic, and biochemical features were generalized (28/46) or regional lymphadenopathy (16/46), hepatosplenomegaly (15/46), extranodal involvement (11/46), mediastinal mass (9/46), and leukemia (8/46), which were mainly present in cases of lymphoblastic lymphomas and hypercalcemia (16/46).     

Hepatoblastoma in a cat

Hepatoblastomas are neoplasms that originate from putative pluripotential stem cells of the liver. A hepatic mass from an 8-year-old Abyssinian cat was composed of cords and sheets of neoplastic cells, with scattered rosettes and small ductal structures. Most neoplastic cells had a pale eosinophilic cytoplasm and a round to ovoid nucleus. The tumor also had short spindle cells with an oval nucleus. Immunohistochemically, neoplastic cells were weakly positive for embryonic hepatocellular markers, such as alpha-fetoprotein and cytokeratin (CK) 8/18, but negative for the hepatocellular marker Hepatocyte Paraffin 1. The cells were also positive for CD56/neural cell adhesion molecule and for the biliary epithelial markers CK 7, CK 8/18, CK CAM5.2, and vimentin, but negative for CK 20. Some neoplastic cells expressed neuroectodermal or neuroendocrine markers, such as protein gene product 9.5 and synaptophysin, but were negative for chromogranin A and not argyrophilic by the Grimelius technique. The cat died soon after the biopsy without clinical improvement.     

Steroid—receptor interaction in a canine anal adenoma

An anal adenoma from a dog has been examined for cytosol binding proteins for androgens and oestrogens. Sucrose-density gradient analysis revealed low capacity binding moieties for testosterone with sedimentation coefficients of 2S and 8S, for 5α-dihydrotestosterone (8S) and for oestradiol-17β (4S and 8S). In each case it was shown that the steroids could be translocated to the nuclei and were found there to be protein-bound. The dissociation constants for 5α-dihydrotestosterone and oestradiol-17β were 5×1 × 10^-10M and 2×6 × 10^-10M respectively and the corresponding receptor site concentrations were 9 and 38 fmol/mg cytosol protein.
The possible significance of these findings with regard to the induction and maintenance of such tumours is discussed.     

Evaluation of a percutaneously controlled hydraulic occluder in a rat model of gradual venous occlusion

OBJECTIVE: To evaluate the efficacy of a percutaneously controlled hydraulic occluder (HO) in a rat model of gradual venous occlusion. STUDY DESIGN: Experimental study. ANIMALS: Ten male Sprague-Dawley rats. METHODS: HOs and perivascular transit time ultrasound flow probes were applied to the caudal vena cava (CVC). Occluders in group I rats (n=6) were gradually attenuated over 8 weeks, whereas occluders in group II rats (n=4) were not attenuated. Vena caval blood flow measurements were performed weekly in all rats for 8 weeks. After euthanasia, the CVC was evaluated grossly and by histopathology. RESULTS: Premature occlusion of the vena cava occurred during the 1st week in 3 rats (1 in group I, 2 in group II) because of kinking of the vena cava between the HO and the flow probe. For the remaining rats, mean blood flow in group I decreased significantly from 40.71 mL/min/kg at baseline to 4.68 mL/min/kg at 8 weeks (P=.0094, power=0.91). Group II rats maintained vena caval blood flow at all times during the study. Complete occlusion of the vena cava was confirmed at necropsy in all group I rats. CONCLUSIONS: Gradual occlusion of the CVC of rats was achieved with HOs over 8 weeks. This model is limited by the size of the experimental animals and comparatively large implants. CLINICAL RELEVANCE: HOs may provide a method for gradual occlusion of congenital portosystemic shunts.     

Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.     

Skin pustules and nodules caused by Actinomyces viscosus in a horse

A 12-year-old Arabian stallion was evaluated for a refractory skin problem of 2 years' duration. Skin nodules and pustules, which would rupture, had developed over the right lumbar muscles. Anaerobic culturing from the pustules yielded Actinomyces viscosus, and histologic examination of biopsy specimens revealed globular eosinophilic structures. Concomitant treatment with isoniazid (8 mg/kg of body weight, q 24 h for 8 weeks), trimethoprim-sulfadiazine (30 mg/kg, q 24 h for 8 weeks), and sodium iodide solution (66 mg/kg, every 1, 2, or 4 weeks, for 32 weeks) resolved the condition.     

Malignant fibrous histiocytoma (MFH) cells and macrophages/histiocytes have a common antigen recognized by a monoclonal antibody risen against a rat MFH-derived cloned cell line

A monoclonal antibody (B9) was generated by using a rat malignant fibrous histiocytoma (MFH)-derived cloned cell line (MT-8) as the immunogen. Immunohistochemically, B9 reacted specifically with a cytoplasmic antigen of MT-8 cells. Furthermore, B9 immunolabeled another MFH-derived cloned cells (MT-9) and histiocytic sarcoma cells, as well as macrophages/histiocytes in normal and diseased tissues of rats. These findings suggest the presence of a common antigen recognized by B9 between MFH cells and macrophages/histiocytes. This suggests that MFH cells may express histiocytic nature.     

Characterization of a novel activation antigen on porcine lymphocytes recognized by monoclonal antibody 5A6/8

In this report, we describe the characterization of a novel activation antigen on porcine lymphocytes recognized by mAb 5A6/8. This antigen was detected on B and T cells 24 h after treatment with various stimuli. It was also found on alveolar macrophages, and at low levels on untreated monocytes. MAb 5A6/8 precipitated two bands of 45 and 50 kDa under non-reducing conditions, and of 22 and 28 kDa under reducing conditions. The cellular distribution, expression kinetics and/or molecular size of the 5A6/8 antigen differ from those of other known lymphocyte activation antigens. MAb 5A6/8 was able to inhibit lymphocyte proliferative responses driven by different stimuli, suggesting a role for this molecule in the events that lead to lymphocyte activation.     

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 10⁹ and 0.86 × 10⁹, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 10⁴ IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.     

Evaluation of Propofol as a General Anesthetic for Horses

This study provides baseline information on the potential use of propofol as a general anesthetic for horses. Using a Latin square design, propofol (2, 4, and 8 mg/kg) was administered intravenously on three separate occasions to six mature horses. Information about anesthetic induction, duration, and recovery was recorded along with results of rectal temperature, heart rate, respiratory rate, pHa, Paco₂ and Pao₂. Statistical analysis included a mixed model analysis of variance, a general linear model analysis and least square means test for post hoc comparisons. A P <.05 was considered significant. The quality of induction of anesthesia varied from poor to good. Two horses were not recumbent following the lowest dose of propofol. Brief paddling limb movements occurred occasionally and unpredictably after recumbency induced by all three doses. During recovery, horses were uniformly calm and coordinated in their moves to stand. Duration of recumbency (minutes) was dose related; 15.05 ± 1.58 (±±SD) following 2 mg/kg, 31.06 ± 5.56 following 4 mg/kg, and 47.85 ± 13.63 following 8 mg/kg. During recumbency at all doses, heart rate significantly increased from a predrug value of 40 ± 6 beats per minute. Substantial respiratory depression, characterized by a significant decrease in respiratory rate (from 11.7 ± 2.9 to 3.7 ± 1.6 breaths per minute) and increased Paco₂ (from 44.5 ± 2.5 to 52.7 ± 8.0 mm Hg) was seen only after 8 mg/kg. A significant decrease in Pao₂ was observed throughout the recumbency induced by 8 mg/kg, and also at 3 and 5 minutes following induction of anesthesia with 4 mg/ kg propofol. At 5 minutes after injection, Pao₂ was 87.4 ± 13.8 and 58.1 ± 17.0 mm Hg after 4 and 8 mg/kg, respectively. The results of this study do not favor the routine use of propofol as a sole anesthetic in otherwise unmedicated horses.     

Pharmacokinetics of mirtazapine and its main metabolites in Beagle dogs: a pilot study

Mirtazapine (MRT) is a human antidepressant drug mainly metabolised by the cytochrome P450 enzyme system to 8-OH mirtazapine (8-OH) and dimetilmirtazapine (DMR). The drug is usually administered to dogs with anorexia according to doses extrapolated from humans, although it could also have applications as an antidepressant and analgesic in this species. The aim of this study was to assess the pharmacokinetics of MRT and its metabolites, DMT and 8-OH. Six healthy male Beagle dogs were administered MRT orally (20 mg/dog) and plasma MRT and metabolite concentrations were evaluated by high performance liquid chromatography with fluorescence detection. The pharmacokinetic profiles of MRT and DMR were similar (detected from 0.25 up to 10 h), while 8-OH (detected from 0.50 up to 10 h) attained the highest concentrations. The mean half-life of MRT was 6.17 h with a clearance of 1193 mL/h/kg. The study showed that MRT has a different pharmacokinetic profile in the dog compared to other species.     

Spinal meningiomas in dogs: Description of 8 cases including a novel radiological and histopathological presentation

Clinical, imaging, and histological features of 8 canine spinal meningiomas, including a cervical cystic meningioma with imaging and intraoperative features of an arachnoid cyst, are described. All meningiomas were histologically classified and graded following the international World Health Organization human classification for tumors. Six meningiomas were located in the cervical spinal cord. Myelography showed intradural/ extramedullary lesions in 3/4 cases. Magnetic resonance imaging revealed hyperintense intradural/extramedullary masses on pre-contrast T1-weighted and T2-weighted images with homogeneous contrast enhancement in 7/8 cases. One dog had a cerebrospinal fluid-filled subarachnoid cavity dorsal to the cervical spinal cord. A spinal arachnoid cyst was diagnosed on imaging, but the histopathological study of the resected tissue revealed a grade I meningothelial cystic meningioma. There were no differences in outcome associated with tumor grade and surgical treatment (6/8). Cystic meningioma should be considered in the differential diagnosis of intraspinal cystic lesions, and biopsy is necessary for definitive diagnosis.     

Combined pharmacokinetics of gentamicin in pony mares after a single intravenous and intramuscular administration

Healthy mature pony mares (n = 6) were given a single dose of gentamicin (5 mg/kg of body weight) IV or IM 8 days apart. Venous blood samples were collected at 0, 5, 10, 20, 30, and 45 minutes and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, 30, 36, 40, and 48 hours after IV injection of gentamicin, and at 10, 20, 30, and 45 minutes and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, and 30 hours after IM injection of gentamicin. Gentamicin serum concentration was determined by a liquid-phase radioimmunoassay. The combined data of IV and IM treatments were analyzed by a nonlinear least-square regression analysis program. The kinetic data were best fitted by a 2-compartment open model, as indicated by residual trends and improvements in the correlation of determination. The distribution phase half-life was 0.12 +/- 0.02 hour and postdistribution phase half-life was 1.82 +/- 0.22 hour. The volume of the central compartment was 115.8 +/- 6.0 ml/kg, volume of distribution at steady state was 188 +/- 9.9 ml/kg, and the total body clearance was 1.27 +/- 0.18 ml/min/kg. Intramuscular absorption was rapid with a half-life for absorption of 0.64 +/- 0.14 hour. The extent of absorption was 0.87 +/- 0.14. Kinetic calculations predicted that IM injections of 5 mg of gentamicin/kg every 8 hours would provide average steady-state serum concentrations of 7.0 micrograms/ml, with maximum and minimum steady-state concentrations of 16.8 and 1.1 micrograms/ml, respectively.     

Impact of Giardia vaccination on asymptomatic Giardia infections in dogs at a research facility

Feces were collected from 107 asymptomatic dogs at a research facility in Guelph, Ontario. The prevalence of Giardia infection was 11% (12/107). To assess the effectiveness of Giardia vaccination for treatment of Giardia carriage, 9 additional asymptomatic Giardia antigen-positive dogs were brought into the facility. The Giardia antigen-positive dogs were then randomly allocated to receive either vaccine (n = 10) or a saline placebo (n = 10). Feces were then monitored monthly for 6 mo for Giardia antigen and Giardia cysts. At weeks 4, 8, 12, and 16 following vaccination, there were more Giardia-positive dogs in the vaccinated group (10/10, 9/10, 9/10, 8/10, respectively) compared with the controls (7/10, 7/10, 8/10, 4/10, respectively). At week 20, an equal number of dogs (5/10) were Giardia positive, and at week 24, fewer dogs were positive in the vaccinated group than in the control group (2/10 versus 5/10, respectively). However, there was no significant difference between the 2 groups. Vaccination was, therefore, not an effective treatment for asymptomatic canine Giardia infections in this setting.     

Characterization of a feline T-cell-specific monoclonal antibody reactive with a CD5-like molecule.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.     

The pharmacokinetics of maropitant, a novel neurokinin type-1 receptor antagonist, in dogs

Maropitant is the first NK1 receptor antagonist developed to treat and prevent emesis in dogs; it is administered by subcutaneous (s.c.) injection at 1 mg/kg, or orally (p.o.), in tablet form, at either 2 or 8 mg/kg depending on indication. The absolute bioavailability of maropitant was markedly higher (90.7%) following s.c. injection than after oral administration (23.7% at the 2 mg/kg dose and 37.0% at the 8 mg/kg dose). First-pass metabolism contributes to the low bioavailability of maropitant following oral administration. The difference in bioavailability between the two oral doses reflects the nonlinear kinetics characterizing the disposition of maropitant within the 2-8 mg/kg dose range. Systemic clearance of maropitant following intravenous (i.v.) administration was 970, 995 and 533 mL/h.kg at doses of 1, 2 and 8 mg/kg, respectively. Nonproportional kinetics were observed for p.o. administered maropitant at doses ranging from 2 to 16 mg/kg but dose proportionality was demonstrated at higher doses (20-50 mg/kg). Linearity was also demonstrated following s.c. administration at 0.5, 1 and 2 mg/kg. Maximum plasma drug concentration (Cmax) occurred 0.75 h (tmax) after s.c. administration at 1 mg/kg, and at 1.7 and 1.9 h after oral administration of 8 and 2 mg/kg doses, respectively. The apparent terminal half-life of maropitant was 7.75, 4.03 and 5.46 h after dosing at 1 mg/kg (s.c.), 2 mg/kg (p.o.) and 8 mg/kg (p.o.), respectively. Feeding status had no effect on oral bioavailability. Limited accumulation occurred following once-daily administration of maropitant for five consecutive days at 1 mg/kg (s.c.) or 2 mg/kg (p.o.). At the dose of 8 mg/kg (p.o.) once daily for two consecutive days, the mean AUC(0-24h) (second dose) was 218% that of the first dose value. Urinary recovery of maropitant and its main metabolite was minimal (<1%), thus supporting the evidence that maropitant clearance is primarily hepatic.     

1株跨种裂解李氏杆菌噬菌体的分离鉴定

本研究旨在获得天然李氏杆菌噬菌体,提供防治李氏杆菌病新型生物制剂,减少抗微生物药物的使用,抑制病原菌耐药性的产生。利用产单核细胞李氏杆菌作为宿主菌对屠宰场污水进行双层琼脂平板法筛选,获得噬菌体,并对其进行透射电镜观察、生长特性检测(温度、pH、一步生长曲线、最佳感染复数、有机溶剂影响)、基因组酶切鉴定和全基因组测序分析。分离出的产单核细胞李氏杆菌噬菌体中,选取1株裂解性强,遗传稳定的噬菌体进行后续试验,并命名为LP8;经电镜观察为肌尾科噬菌体,可以跨种裂解18株产单核细胞李氏杆菌和5株威尔斯李氏杆菌,确定LP8的最佳感染复数为1、最适生长温度为45 ℃、最适pH为7;在6种有机溶剂中,仅异戊醇可导致LP8活性丧失;对提取的基因组进行酶切鉴定,确定为双链DNA;LP8全基因组测序结果表明,基因组大小为87 038 bp、含有120个编码基因、编码基因的累计长度为76 326 bp、编码基因的平均长度为636 bp、编码区域长度占基因组的比例为87.69%。本试验分离出产单核细胞李氏杆菌噬菌体,并对其噬菌能力和应用价值进行鉴定。分离出的LP8噬菌体相较于李氏杆菌的噬菌体,细菌裂解能力更强,适应环境范围更广。本研究为实验室后续建立产单核细胞李氏杆菌噬菌体库和产单核细胞李氏杆菌噬菌体的其他应用提供了良好的基础。     

Clinical, immunological and histopathological findings in a subpopulation of dogs with pododermatitis

Abstract   Clinical, immunological and histopathological findings in 20 adult dogs of varying breeds with chronic (≥ 6 months) inflammation confined to the pedal skin were compared over a 2-year period with those of a group of age-matched controls ( n  = 20). All affected dogs were pruritic but systemically well. Lesions were present on all four feet in 18/20 cases. Affected feet were characteristically erythematous, swollen, painful and alopecic. Sinus tracts were evident in 4/20 dogs. Despite a methodical series of diagnostic tests, no underlying cause was identified. None of the dogs responded to antimicrobial therapy administered for 8 weeks, none had evidence of ectoparasitism and none satisfied the criteria for atopic dermatitis. There was no response to a dietary trial using a novel protein source. The condition was characterized histopathologically by epidermal hyperplasia, hyperkeratosis, spongiosis, dermal oedema and perivascular aggregates of lymphocytes and plasma cells. Clinical signs did not correlate with histopathological findings. Affected dogs had significantly elevated serum IgG and IgM concentrations. The results of lymphocyte proliferation assays and phenotypic studies to determine the relative percentage of CD3⁺, CD4⁺, CD8⁺ and CD21⁺ lymphocyte subsets, and the ratio of CD4⁺/CD8⁺ cells were not significantly different between groups. No age, sex or seasonal predilections were noted. All dogs subsequently responded to immunosuppressive doses of prednisolone or cyclosporin. The term immunomodulatory-responsive lymphocytic–plasmacytic pododermatitis is proposed to denote what may be a previously unrecognized condition in some dogs with pododermatitis of undetermined aetiology.     

Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight

In MRL/MpJ mice, there is a genetic mutation of exonuclease 1 (Exo1), in which the exon 9 is sometimes deleted. In the present study, to check the generation of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B, but was present little or very weakly in pCX/Ex/EIE/M. Next, the same spliced band was demonstrated in the pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the dell1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-1 and tr-2 Exo1).