Recognition of the Nor98 variant of scrapie in the Swedish sheep population.

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.     

Endoscopic sex determination and gonadal manipulation in Gulf of Mexico sturgeon (Acipenser oxyrinchus desotoi).

Seventeen Gulf of Mexico sturgeons (Acipenser oxyrinchus desotoi) underwent endoscopic sex determination, gonadal biopsy, and various reproductive surgeries as part of a conservation development plan. The fish were anesthetized with tricaine methanesulfonate (MS-222) buffered with sodium bicarbonate and maintained on a recirculating water anesthesia circuit. A 6-mm Ternamian EndoTip Cannula, placed through the ventral midline, midway between pectoral and pelvic fins, permitted the introduction of a 5-mm telescope. Swim bladder aspiration and CO2 insufflation of the coelomic cavity provided excellent observation. Second and third cannulae were placed under direct visual control, lateral and cranial or caudal to the telescope cannula. Sex determination was successfully performed in all fish; however, five of 17 sturgeons (29%) required endoscopic gonadal biopsy to confirm sex. Bilateral ovariectomy or orchidectomy was successfully performed in three males and four females. Unilateral ovariectomy and bilateral ligation of the müllerian ducts using an extracorporeal suturing technique was accomplished in an additional three females. No apparent morbidity was associated with the anesthesia or endoscopic surgery in any fish. The ability to safely perform minimally invasive reproductive surgery in fish may have important management and conservation benefits.     

Genetic polymorphism at CSN1S2 locus in two endangered sicilian goat breeds

In this study we surveyed two endangered Sicilian goat breeds (Girgentana and Argentata dell'Etna) for genetic polymorphism at the CSN1S2 locus. In a total of 537 goats, we detected CSN1S2^A, CSN1S2^B, CSN1S2^C° (CSN1S2^C + CSN1S2^E), CSN1S2^D, CSN1S2^F and CSN1S2^O alleles by means of various polymerase chain reaction (PCR), allele specific‐PCR (AS–PCR) and PCR‐restriction fragment length polymorphism (RFLP) reactions with the aim of improving the knowledge of the genetic resource of these two breeds. Three and five alleles, with six and twelve genotypes, were identified at CSN1S2 locus, in Girgentana and Argentata, respectively. Argentata dell'Etna showed a higher degree of genetic variation. The allelic and genotypic distribution seems to be significantly different (p < 0.001) in the two breeds. In Argentata the rare null allele (CSN1S2^O) was found at low frequency (0.033); this genetic peculiarity makes its preservation worthwhile.     

Genetic diversity analysis of two buffalo populations of northern India using microsatellite markers

The genetic diversity in two buffalo populations of northern India, the Bhadawari and the Tarai was assessed using a set of 22 heterologous (bovine) microsatellite markers. The average number of alleles across all loci in both populations was found to be 4.7, indicating that this set of 22 bovine microsatellite markers could be used to study genetic variation in buffalo species also. The overall polymorphic information content (PIC) value for these markers was 0.54. The average observed and expected heterozygosities for both populations were 0.59 and 0.64, respectively. Common alleles with varying allele frequencies in both populations also represented the genetic variability existing between Bhadawari and Tarai buffaloes. However the θ estimates for population differentiation indicated low levels of differentiation between the two populations. This was further supported by the low genetic distance (0.155) between Bhadawari and Tarai, which was calculated using Nei's standard genetic distance method. The present study on Bhadawari and Tarai populations represents a much‐needed preliminary effort that could be extended to other local buffalo populations of India as well.     

FC-17 Cutaneous papillomatosis and carcinomatosis in the highly endangered Western Barred Bandicoot

Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently being clinically tested in anticancer therapy. In order to analyse the immunostimulatory effects of NDV on bovine papillomatosis, we inoculated 14 cows subcutaneously with an attenuated vaccine containing the LaSota strain of NDV (LS-NDV). Four cows with papillomatosis served as controls. Serum samples were collected from each animal 1 h preinoculation and 7 and 21 days postinoculation. In inoculated cows on days 7 and 21, the mean antibody titres were log₂ 2.43 ± 0.92 and 5.57 ± 0.72, respectively, by haemagglutination inhibition, and the mean levels of TNFα were 5.80 ± 4.19 and 5.39 ± 2.66 ng/mL, respectively, by WEHI-164 cytotoxicity assay. Significant differences between inoculated and control animals were evident for antibody titres on day 21 and clinical scores on day 60. A correlation was evident between the TNFα activities and clinical scores on day 21. The clinical observations at day 60 showed that the papillomas in five cows had completely resolved (36%), one animal had no alterations on clinical appearance of the tumour (7%), and papillomas in eight cows had regressed (57%). In conclusion, these results demonstrated that inoculation of LS-NDV vaccine stimulates an antibody response and a limited increase in TNFα activity and may enhance clinical recovery in bovine papillomatosis.
Funding: Scientific Research Council of Akdeniz University.     

Pulmonary metastatectomy in the management of four dogs with hypertrophic osteopathy

The efficacy and outcome of pulmonary metastatectomy in the management of hypertrophic osteopathy (HO) secondary to metastatic osteosarcoma was retrospectively evaluated in four dogs. Metastatectomy was performed by subpleural enucleation, partial lung lobectomy or complete lung lobectomy through either a median sternotomy or thoracoscopically. Perioperative morbidity was minimal. Clinical signs associated with HO resolved within 24 h of pulmonary metastatectomy in all dogs. Durable remission of symptomatic HO was achieved in all dogs (range, 50–294 days), although recurrence of HO was noted in one dog, 246 days postmetastatectomy due to metastasis to the lungs and chest wall. Pulmonary metastatectomy resulted in a rapid and prolonged resolution of HO, and the clinical benefits of metastatectomy potentially exceed the morbidity associated with the surgical procedure.     

Monitoring response to aminobisphosphonate therapy in canine osteosarcoma using dual energy x‐ray absorptiometry (dexa)

Introduction: Palliative therapy is essential to improve the quality of life of dogs with osteosarcoma (OSA), when definitive therapy is not considered a valid option. Bisphosphonates, a novel class of antiosteoclastic drugs, are widely used in humans for several painful osteolytic conditions. Dual energy x‐ray absorptiometry (DEXA) is recognized as a reliable tool to measure bone mineral density (BMD), and to monitor treatment response to bisphosphonates in humans. A prospective evaluation of pamidronate, an injectable aminobisphosphonate, is ongoing in dogs with appendicular OSA. The potential value of DEXA for objective evaluation of BMD variations with palliative therapies is concurrently being assessed. Materials and Methods: Dogs with naturally occurring appendicular OSA treated with pamidronate constitute the patient population. A DEXA scan (QDR‐4500 W, Hologic, Bedford, MA) is performed on day 0 (baseline) and on every treatment day with pamidronate thereafter (every 28 days). For each dog, a whole body scan is performed, followed by a scan of the tumor, and contralateral normal bone. Three regions of interest are subsequently analyzed for BMD changes in tumor and normal bone. Statistical analysis was performed using Student t‐test and paired t‐tests, with significance being set at p < 0.05. Results: Nineteen dogs have been enrolled to date. Seven responders and 6 non‐responders have suitable data for analysis. A significant difference is observed (p = 0.04) between tumor BMD variations of responders and non‐responders at day 28 (mean variations +18.0% and ?4.6% respectively). The changes at day 28 are significant only in the responders (p = 0.038 vs p = 0.05 in non‐responders). When BMD of tumor and normal bone at day 84 is compared to day 0 in six responders, only tumor had a significant increase (p = 0.017 vs p = 0.279, respectively). Conclusions: Objective measurements of response to therapy are essential in pain palliation studies. Increased tumor bone BMD, as obtained by DEXA analysis, may correlate with subjective clinical improvement in pamidronate‐treated dogs with appendicular OSA.     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Cardiac troponin I in canine patients with lymphoma and osteosarcoma receiving doxorubicin: comparison with clinical heart disease in a retrospective analysis

The cumulative cardiotoxicity that occurs as a result of doxorubicin chemotherapy is irreversible and can affect both quality and quantity of life for the cancer patient. Cardiac troponin I (cTnI) is a sensitive and specific marker of cardiomyocyte death. The purpose of this retrospective study was to evaluate serum concentrations of cTnI in dogs with lymphoma or osteosarcoma given doxorubicin chemotherapy, and with known cardiac outcome, based on a minimum assessment by physical examination and thoracic radiography. Serum samples were also available for cTnI measurement from seven healthy dogs given intracoronary doxorubicin. Serial serum samples obtained before, during and after doxorubicin chemotherapy showed increased cTnI concentrations in some clinical patients following chemotherapy (P = 0.0083 compared to baseline), but this did not correlate with clinical signs of cardiomyopathy. In dogs that subsequently developed cardiomyopathy however, serum cTnI concentrations were elevated before clinical signs became evident (confirmed with echocardiography).