Diagnosis and treatment of presumptive pyelonephritis in an Asian elephant (Elephas maximus).

A 37-yr-old female Asian elephant (Elephas maximus) presented with anorexia, restlessness, and dark-colored urine. Urinalyses showed hematuria, leukocyturia, isosthenuria, proteinuria, granular casts, and no calcium oxalate crystals. Bloodwork revealed azotemia. Urine culture revealed a pure growth of Streptococcus zooepidemicus resistant to sulfamethoxazole-trimethoprim but susceptible to cephalosporins. A presumptive diagnosis of pyelonephritis was made based on bloodwork, urinalysis, and urine culture. The animal was treated with intravenous ceftiofur, and intravenous and per rectum fluids were given for hydration. The elephant's attitude and appetite returned to normal, the abnormal blood parameters resolved, and urinary calcium oxalate crystals reappeared after treatment, supporting presumptive diagnosis. Follow-up ultrasonography revealed an abnormal outline of both kidneys with parenchymal hyperechogenicity and multiple uterine leiomyomas.     

Cloning and characterization of a bovine genomic fragment homologous to epidermal growth factor genes

Epidermal growth factor (EGF) is a potent mitogen for a variety of cell types. The 53-amino acid mature EGF protein is encoded by sequences in exons 20 and 21 of a gene spanning over 110 kb. In this study, we report the cloning and characterization of 7.5 kb of bovine genomic sequence homologous to exon 19 through 21 from EGF genes from other mammalian species. The cloned gene fragment had an unusual sequence composition in the form of an in-frame TGA codon in the coding sequence. The sequence was expressed at low levels in kidney tissue and the corresponding cDNA contained the TGA codon. The level of similarity between the bovine exonic sequence and the human, porcine, murine, feline, and canine corresponding sequences varied from 64% to 73%; however, when only sequences encoding the mature EGF protein were compared, the level of similarity between the bovine sequence and the sequence from these species was 59% to 66%. The sequence similarity of the deduced mature protein was lower (34% to 39%) than the sequence similarity of the deduced propeptide. Although the cloned sequences could originate from a bovine EGF pseudogene, the possibility exists that they originate from the functional EGF gene. An as yet unidentified mechanism to by-pass the stop codon would allow the synthesis of a functional EGF protein. Alternatively, the cloned sequence could originate from an EGF-like gene.     

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.     

Variance component estimation on the frequency of pathologic changes in the navicular bones of Hanoverian Warmblood horses

The results of a standardized radiological examination of 3748 young Hanoverian Warmblood horses selected for sale at auction as riding horses were used to quantify the influence of systematic effects on and to estimate genetic parameters for the prevalence of pathologic changes in the navicular bones. Radiographic findings in the navicular bones of the front limbs were analyzed as all‐or‐none traits. The pathologic changes were mostly classified as slight [PCN(I); 14.9%], less often as moderate [PCN(II); 5.3%] or severe [PCN(III); 1.8%]. Date and year of auction had a significant influence on the prevalence of documented radiographic findings. The prevalence of PCN(I) was further significantly dependent on the examiner, the type and the quality of auction. PCN(II) was significantly more prevalent in male than in female horses. The age, the anticipated suitability and the region of origin of the horses did not have any significant influence on the prevalence of pathologic changes in navicular bones. A higher percentage of genes of the Hanoverian and the Holstein Warmblood horse increased the probability of PCN(I) classification. A significant influence of the sire was found for PCN(I) and PCN(II), and of the male founder for PCN(II) and PCN(III). The female founder was significant only for PCN(II). In general, radiographic findings of any severity in front left and right navicular bones were significantly correlated with each other. Restricted maximum likelihood (REML) was used for the estimation of genetic parameters. The analyses were performed multivariately in linear animal and sire models including height at withers as a separate trait. Heritability estimates for the prevalence of PCN(I), PCN(II) and PCN(III) of horses of both sexes ranged between h² = 0.09 and 0.21. When distinguishing between findings in males and females, somewhat implausible estimates were obtained for PCN(II) in females, which might have been caused by their low prevalence. The additive genetic correlations between the investigated traits indicated that radiographic findings consistent with navicular syndrome have a uniform genetic pattern in males and in females, and irrespective of their severity. However, their genetic correlation to height at withers was found to be inconsistent and, therefore, not to be utilizable for selection.     

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.     

Noninvasive monitoring of doxorubicin cardiotoxicity: a pilot study of two dogs

The cardiotoxicity of anthracycline anticancer agents, most notably doxorubicin, limits their use in treating a wide variety of cancers. Currently available methods for assessment of anthracycline‐induced cardiotoxicity (AIC) often lack specificity and sensitivity. The purpose of this pilot investigation was to determine noninvasive diagnostic methods that are predictive for AIC. Two mongrel dogs were anesthetized for injection of doxorubicin (22.5 mg) directly into the left coronary artery. Doxorubicin injection was repeated 2 weeks later. Dogs were followed for 12 weeks or until heart failure was documented. Dogs were monitored before and at serial time points after doxorubicin using echocardiography and signal‐averaged electrocardiography as well as ambulatory ECG monitoring. Blood was also collected for measurement of cardiac troponin I and T concentrations. Doxorubicin was readministered at 6 and 12 weeks after the second injection, as interim data analysis did not reveal adequate evidence of cardiomyopathy. Dog‐1 appeared to be more sensitive than Dog‐2 to AIC based upon gradual changes in variables of cardiac function and earlier time to failure (113 days). In contrast, Dog‐2 had inconsistent changes in cardiac function and a longer time to failure (139 days). Dog‐1 also had greater peak plasma cardiac troponin I levels (17.96 ng/ml) as compared to peak levels in Dog‐2 (4.08 ng/ml). In addition, Dog‐1 had increased ventricular extrasystolic contractions (VE)(0/day at baseline, 443/day one week prior to death), whereas dog‐2 showed very few VE's throughout. Spectral analysis of ECG data also revealed a significant decrease over time in total spectral power in Dog‐1 from 37,148 at baseline to 603 msec²/Hz one week prior to death, indicating decreased parasympathetic tone. Endomyocardial biopsies revealed minor changes despite significant clinical abnormalities. These data indicate differences in susceptibility to AIC between Dog‐1 and Dog‐2, and suggest a role for these noninvasive measures in monitoring the heart during chemotherapy.