Improvement of anemia associated with chronic renal failure by recombinant human erythropoietin treatment in ICR-derived glomerulonephritis (ICGN) mice

The ICR-derived glomerulonephritis (ICGN) mouse, a novel inbred mouse strain with a hereditary nephrotic syndrome, develops severe anemia associated with chronic renal failure. To reveal the pathogenic mechanism of anemia in ICGN mice, we subcutaneously administered recombinant human erythropoietin (rhEPO; 5 IU/mouse/day) or saline for 5 days to ICGN mice. In terminal-stage ICGN mice with severe anemia, rhEPO significantly increased hematocrit (Ht), red blood cells (RBC) and hemoglobin levels. Endogenous EPO levels in peripheral blood were reduced by rhEPO injection. No histopathological changes in bone marrow and kidneys were induced by rhEPO injection. Insufficiency of EPO may cause anemia in ICGN mice.     

Use of computer tomography for imaging of Crassicauda grampicola in a Risso's dolphin (Grampus griseus).

A mature male Risso's dolphin (Grampus griseus) stranded along the coasts of Friuli Venezia Giulia, northeast Italy, in May 2001. Parasitic infection with Crassicauda grampicola is often found in the tympanic bullae and pterygoid sinuses in many of the Risso's dolphins examined from the same area. For this reason, it was decided to perform computed tomography of the head to assess this imaging technique for the diagnosis of crassicaudosis in dolphins. A full postmortem examination confirmed the pathologic findings of the computed tomography scan. This technique can be considered a useful adjunct in the diagnosis of cranial crassicaudosis in live dolphins.     

Platelet vacuoles in a dog with severe nonregenerative anemia: evidence of platelet autophagy

A 13‐year‐old neutered male English Springer Spaniel was presented to The Ohio State University Veterinary Medical Center for evaluation of severe anemia. Upon blood smear review, approximately 50% of the platelets contained single to multiple variably sized clear vacuoles. Transmission electron microscopy of the platelets revealed hallmark features of autophagy, including membrane‐lined vesicles and vacuoles containing membrane whorls and degrading organelles. While autophagy has been demonstrated in a wide range of eukaryotic cells for decades, reports of platelet autophagy are lacking. This case report illustrates atypical platelet vacuolation with electron microscopic features characteristic of autophagy.     

Echocardiographic evaluation of the bottlenose dolphin (Tursiops truncatus).

Safe and effective echocardiography would represent a valuable tool for marine mammal veterinarians and physiologists evaluating the dolphin heart. Unfortunately, conventional ultrasound technology (transthoracic echocardiography) has been limited by logistic, anatomic, and behavioral challenges. Five mature male Atlantic bottlenose dolphins (Tursiops truncatus) were trained for echocardiographic imaging (four for both transthoracic and transesophageal imaging, and one for only transthoracic imaging). It was noted that transesophageal image quality transiently improved when the dolphins spontaneously exhaled. Subsequently, dolphins were conditioned to hold their breath following forced exhalation, and imaging proceeded during such behavioral breath holds. Over 25 transthoracic and 100 transesophageal echocardiographic studies were performed, including both two-dimensional imaging and color flow mapping. Transthoracic imaging yielded poor-quality images of only small portions of the heart. In contrast, transesophageal imaging, which improved dramatically with behavioral breath holding following exhalation, yielded consistently high-quality images of the entire heart (mitral, tricuspid, aortic, and pulmonary valves, atrial and ventricular septa, left and right atria, left and right ventricles, and ascending aorta and main pulmonary artery). Color flow mapping demonstrated mild tricuspid regurgitation in all dolphins, and mild aortic regurgitation in one dolphin found to have a pedunculated mass arising from the sinutubular junction just above the aortic valve. There were no complications in nonsedated dolphins. The heart of the bottlenose dolphin can be safely, effectively, and reproducibly evaluated with the use of transesophageal echocardiography in conjunction with behavioral breath holding following forced exhalation. This approach, and the normative echocardiographic data generated from this work, lays the foundation for future echocardiographic studies of cetaceans.     

Clinical efficacy and safety of recombinant canine erythropoietin in dogs with anemia of chronic renal failure and dogs with recombinant human erythropoietin-induced red cell aplasia

The efficacy and safety of recombinant canine erythropoietin (rcEPO) therapy was evaluated in 19 dogs with anemia of chronic renal failure (group 1) and 6 dogs with chronic renal failure and recombinant human erythropoietin (rhEPO)-induced red cell aplasia (group 2). Hematocrit (Hct) and absolute reticulocyte count (ARC) were monitored weekly for the first 8 weeks, CBC (including ARC) and serum iron profiles were evaluated monthly, and serum biochemical analyses were performed every 2 months for 6 (group 2) to 12 (group 1) months. For group 1 dogs, median Hct and ARC increased significantly during the 1st week of rcEPO treatment, and median Hct was sustained at >35% after week 5. In contrast, median Hct and ARC for group 2 did not change significantly with rcEPO treatment, even with doses greater than those used in group 1. Nevertheless, 2 (33%) of the 6 dogs in group 2 developed erythroid hyperplasia, reticulocytosis, and increases in Hct with rcEPO treatment. Although median systolic blood pressure did not change significantly in either group, 5 dogs developed systolic blood pressures > or = 180 mm Hg during the study. Appetite and energy level improved in most group 1 dogs with increases in Hct. Recombinant cEPO stimulated erythrocyte production in dogs with nonregenerative anemia secondary to chronic renal failure without causing the profound erythroid hypoplasia that can occur in rhEPO-treated dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs that had previously developed rhEPO-induced red cell aplasia.     

Comparison of biological activity and safety of recombinant canine erythropoietin with that of recombinant human erythropoietin in clinically normal dogs.

OBJECTIVE: To determine whether recombinant canine erythropoietin (rcEPO) stimulates erythropoiesis in dogs without causing the immunogenicity problem (ie, erythroid hypoplasia) associated with recombinant human erythropoietin (rhEPO). ANIMALS: 13 clinically normal dogs. PROCEDURE: Dogs were randomly assigned to 2 groups; 1 group (n = 6) received rhEPO, whereas the other group (7) received rcEPO. Both groups received SC injections of diluent for 4 weeks before initiating treatment with erythropoietin (100 U/kg of body weight, SC, 3 times/wk). Hematocrit and absolute reticulocyte count were monitored weekly, CBC were done monthly, and bone marrow aspirates for cytologic evaluation were obtained before and at 4, 8, 16, and 24 weeks during treatment. RESULTS: Weekly mean Hct and absolute reticulocyte count increased in both groups of dogs during the first 2 weeks of treatment. For dogs receiving rhEPO, precipitous decreases in reticulocyte number and more gradual decreases in Hct were associated with development of erythroid hypoplasia. Dogs receiving rhEPO developed erythroid hypoplasia by week 4 (n = 4), 8 (1), or 16 (1). With cessation of rhEPO treatment after diagnosis of erythroid hypoplasia, RBC production recovered 5 to 11 weeks (median, 7 weeks) later. In contrast, rcEPO treatment caused sustained increases in Hct and reticulocytosis. None of the dogs receiving rcEPO developed erythroid hypoplasia. CONCLUSIONS: rcEPO stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the erythroid hypoplasia encountered in rhEPO-treated dogs. CLINICAL RELEVANCE: Because rcEPO did not cause erythroid hypoplasia, rcEPO may represent an improved option, compared with rhEPO, for treatment of erythropoietin-dependent anemia in dogs.     

Use of human immunoglobulin in addition to glucocorticoids for the initial treatment of dogs with immune-mediated hemolytic anemia

Objective – To determine the utility of human intravenous immunoglobulin (hIVIG) for the initial treatment of canine immune-mediated hemolytic anemia (IMHA).
Design – Blinded, randomized, clinical trial.
Setting – Veterinary teaching hospital.
Animals – Twenty-eight, client-owned dogs with primary IMHA.
Interventions – At enrollment, after diagnosis of IMHA, dogs were randomly assigned to receive either hIVIG or placebo, in a blinded fashion. For the next 14 days, all dogs received glucocorticoids as the sole immunosuppressant agent. All dogs received low-molecular-weight heparin as an anticoagulant. D-dimer concentrations were evaluated at the beginning and end of the study protocol to monitor for thromboembolic complications.
Measurements and Main Results – Twenty-five of 28 dogs (89%) were discharged from the hospital. Thirteen of those received hIVIG and 12 received placebo. Twenty-four dogs (86%) were alive 14 days after enrollment, and of these 13 received hIVIG and 11 received placebo. D-dimer concentrations were elevated in 86% of all dogs at the time of diagnosis.
Conclusions – For initial treatment of dogs with IMHA, the addition of hIVIG to corticosteroid treatment did not improve initial response, nor did it shorten hospitalization.     

Non-purulent meningoencephalomyelitis of a Pacific striped dolphin (Lagenorhynchus obliquidens). The first evidence of morbillivirus infection in a dolphin at the Pacific Ocean around Japan

On March 22, 1998, a mature, male, hyposthenic Pacific striped dolphin (Lagenorhynchus obliquidens) was stranded at Aoshima Beach in Miyazaki prefecture, Japan. A necropsy performed 14 hr after death revealed mild diffuse congestion and edema of the leptomeninges and mild pulmonary atelectasis. Histopathologically, non-purulent inflammatory were observed throughout the cerebrum, thalamus, midbrain, pons, medulla oblongata, and spinal cord. Hematoxylin and eosin stain revealed no viral inclusion bodies. Immunohistochemistry using a monoclonal antibody against nucleoprotein of canine distemper virus (CDV-NP) revealed a number of CDV-NP-positive granular deposits in the cytoplasm and cell processes of the degenerating or intact neurons. The present paper is a first report of spontaneously occurred morbillivirus infection in a dolphin at the Pacific Ocean around Japan.     

Proliferative dermatitis associated with a novel alphaherpesvirus in an Atlantic bottlenose dolphin (Tursiops truncatus).

Herpesviruses and herpes-like viruses have been reported in only a small number of species of cetaceans, and, to date, clinical manifestations have been either as a life-threatening, disseminated infection or as a non-life-threatening dermatitis. A stranded juvenile Atlantic bottlenose dolphin, Tursiops truncatus, was admitted to the Dolphin and Whale Hospital for rehabilitation. On initial physical examination, the rostral skin had multifocal regions of hyperplasia, and the skin of the dorsum contained a large number of small papules. Histologically, epithelial hyperplasia was evident, and clusters of epithelial cells contained 5-15-microm intranuclear inclusion bodies. Transmission electron microscopic investigation revealed numerous 170-190-nm enveloped virions in both the intracellular spaces and the cytoplasm of epithelial cells, with numerous nucleocapsids noted in epithelial cell nuclei. Consensus primer polymerase chain reaction identified the presence of a novel herpesvirus associated with the lesions. Phylogenetic analysis of the deduced amino acid sequences of the herpesvirus DNA polymerase gene fragment showed it to align with alphaherpesvirus sequences from humans and domestic animals. Although clearly distinct, it was most closely related to two previously described alphaherpesviruses of dolphins. This case represents the first documentation of herpesvirus dermatitis in the Atlantic bottlenose dolphin.     

Use of famciclovir for the treatment of endotheliotrophic herpesvirus infections in Asian elephants (Elephas maximus).

Two juvenile Asian elephants (Elephas maximus) presented with an acute onset of facial edema and lethargy. Examination of the oral cavity of each animal revealed cyanosis of the tip and distal margins of the tongue suggestive of endothelial inclusion body disease (EIBD) of elephants. Whole-blood samples were obtained, and polymerase chain reaction tests confirmed the presence of elephant herpesvirus. The animals were administered famciclovir (Famvir, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19101, USA), a potent human anti-herpesvirus drug, in the course of their disease, and recovery followed a treatment regime of 3-4 wk. These are the first known cases of elephants surviving EIBD.     

Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose dolphin (Tursiops truncatus).

A 4-yr-old male bottlenose dolphin (Tursiops truncatus) developed an Aspergillus fumigatus pneumonia. Fungal elements were identified by cytology and microbiology from endoscopic bronchoalveolar lavage and brushings of a raised yellow endobronchial lesion. The results of qualitative immunodiffusion serology, a technique that identifies specific circulating antibodies to Aspergillus fumigatus, were suggestive of an active infection. The dolphin was treated with itraconazole for over 2 yr, which resulted in remission of clinical signs. Pneumonia caused by Aspergillus sp. accounts for the large majority of pulmonary mycoses in marine mammals. Bronchoscopy facilitated an early definitive diagnosis, accurate treatment, and remission.     

Effects of in vitro hemolysis on serum biochemistry values of the bottlenose dolphin (Tursiops truncatus).

The effects of in vitro hemolysis on 23 biochemical analytes were assessed in sera from 14 clinically healthy Atlantic bottlenose dolphins (Tursiops truncatus). Each serum sample was divided into three portions for analysis: 1) nonhemolyzed control; 2) moderate hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 150 mg/dl Hb; and 3) severe hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 500 mg/dl Hb. Moderate hemolysis resulted in statistically significant increases in the mean values of iron, lactate dehydrogenase, potassium, and uric acid and a decrease in creatinine (P < 0.001). Severe hemolysis resulted in statistically significant changes in the mean values of the above analytes in addition to the following increases: alanine aminotransferase, calcium, and serum globulins (P < 0.001) and albumin and total protein (P < 0.01). Total bilirubin and gamma glutamyl transferase levels were lower in the severely hemolyzed sample (P < 0.001). Differences in mean values for alkaline phosphatase between nonhemolyzed and hemolyzed serum were not significant but did show a downward trend in the hemolyzed sera. The presence and severity of hemolysis must be considered in the interpretation of the serum chemistry values.     

Use of recombinant human interferon alpha-2a in the management of a dog with epitheliotropic lymphoma

An 8-year-old, mixed-breed dog with preputial epitheliotropic lymphoma was initially treated with cyclophosphamide, vincristine, and prednisolone. A short-term partial response was followed by disease progression after 4 weeks. Recombinant human interferon alpha-2a was administered starting at week 7. The interferon therapy resulted in rapid resolution of clinical signs and a 10-week disease-free interval. The lymphoma recurred at 17 weeks and did not respond to rescue chemotherapy. Additional oral lesions were treated with localized radiotherapy followed by increased dosages of interferon. This additional interferon treatment resulted in another 12 weeks of stable disease.     

Use of recombinant human thyroid-stimulating hormone for thyrotropin stimulation test in euthyroid dogs

The purpose of this study was to evaluate the effects of the recombinant human thyroid-stimulating hormone (rhTSH) on serum total thyroxine (TT4) concentration in euthyroid dogs. Six healthy beagle dogs were used in each of the 3 phases of this study. Phase I: thyroid-stimulating hormone response tests were performed by using a total dose of 25 micrograms, 50 micrograms, and 100 micrograms of rhTSH, administered intravenously. Phases II and III: thyroid-stimulating hormone response tests were performed by using 50 micrograms of rhTSH administered by intramuscular and subcutaneous routes, respectively. In each phase and following all the administered doses of rhTSH, an increase in the serum TT4 concentration was noted, although it was not always significant. For phase I, there was a significant increase in serum TT4 concentrations. Based on this study, 50 micrograms was judged to be the optimal intravenous dose of rhTSH. For phases II and III, there was no significant increase in serum TT4 after the administration of rhTSH. Results of this study suggest that rhTSH could be a good substitute for bovine TSH, when used by the intravenous route, for the TSH stimulation test in dogs. Further studies are required to confirm its clinical usefulness.     

Expression of erythropoietin in cats treated with a recombinant adeno-associated viral vector

OBJECTIVE: To characterize the biological effects of IM administration of a recombinant adeno-associated virus serotype 2 (rAAV2) vector containing feline erythropoietin (fEPO) cDNA and determine whether readministration of the vector or removal of muscle tissue at the injection sites alters those effects. ANIMALS: 10 healthy 7-week-old specific pathogen-free cats. PROCEDURE: Cats received 1 X 10(7) infective units (iU; n = 3), 1 X 10(8) iU (3), or 1 X 10(9) iU (2) of rAAV2-fEPO vector IM (day 0). Two control cats received an rAAV2 vector containing the LacZ gene (1 X 10(9) iU, IM). In all cats, hematologic variables and serum fEPO concentration were measured at intervals; anti-rAAV2 antibody titer was measured on day 227. In cats that did not respond to treatment, the rAAV2-fEPO vector was readministered. Injection sites were subsequently surgically removed. RESULTS: Compared with control cats, cats treated with 1 X 10(9) iU of rAAV2-fEPO vector had increased Hct and serum fEPO concentrations. One of these cats developed pure RBC aplasia; its Hct normalized following injection site excision. Cats receiving lower doses of vector had no response; on retreatment, 1 of those cats developed sustained erythrocytosis that persisted despite injection site removal and the others did not respond or responded transiently. Antibodies against rAAV2 were detected in all vector-treated cats. CONCLUSIONS AND CLINICAL RELEVANCE: Gene therapy may be an effective treatment for cats with hypoproliferative anemia. However, rAAV2-fEPO vector administration may result in pure RBC aplasia or pathologic erythrocytosis, and injection site removal does not consistently abolish the biological response.     

Use of human immunoglobulin for treatment of severe erythema multiforme in a cat

A 5-month old female domestic shorthair cat developed lethargy and severe ulcerative skin lesions that covered more than half of its body after routine administration of rabies vaccine, anthelmintic, and ear medication. Clinical and histologic findings were consistent with a severe cutaneous drug reaction or erythema multiforme. The cat's condition continued to deteriorate despite drug withdrawal and supportive care. Administration of human intravenous immunoglobulin was well tolerated by the cat and led to rapid resolution of ulcerative cutaneous lesions, accompanied by substantial improvement in the cat's demeanor within 8 days. Human intravenous immunoglobulin appears to be a novel promising treatment for life-threatening cutaneous drug reactions.     

Use of recombinant human thyroid-stimulating hormone for thyrotropin-stimulation testing of euthyroid cats

OBJECTIVE: To evaluate response of euthyroid cats to administration of recombinant human thyroid-stimulating hormone (rhTSH). ANIMALS: 7 healthy cats. PROCEDURE: Each cat received each of 5 doses of rhTSH (0, 0.025, 0.050, 0.100, and 0.200 mg), IV, at 1-week intervals. Serum concentration of total thyroxine (TT4) and free thyroxine (fT4) was measured immediately before each injection (time 0) and 2, 4, 6, and 8 hours after administration of each dose. RESULTS: Overall TT4 response did not differ significantly among cats when administered doses were > or = 0.025 mg. Serum TT4 concentrations peaked 6 to 8 hours after administration for all doses > or = 0.025 mg. For all doses > or = 0.025 mg, mean +/- SEM TT4 concentration at 0, 6, and 8 hours was 33.9 +/- 1.7, 101.8 +/- 5.9, and 101.5 +/- 5.7 nmol/L, respectively. For all doses > or = 0.025 mg, mean fT4 concentration at 0, 6, and 8 hours was 38.7 +/- 2.9, 104.5 +/- 7.6, and 100.4 +/- 8.0 pmol/L, respectively. At 8 hours, the fT4 response to 0.025 and 0.050 mg was less than the response to 0.100 and 0.200 mg. Adverse reactions after rhTSH administration were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: The TSH stimulation test can be performed in cats by IV administration of 0.025 to 0.200 mg of rhTSH and measurement of serum TT4 concentrations at time of injection and 6 or 8 hours later. Clinical validation of the TSH stimulation test would facilitate development of additional tests of thyroid gland function, such as a TSH assay.