M. Elena Gorman

Selenium Supplementation Restores Innate and Humoral Immune Responses in Footrot-Affected Sheep

Dietary selenium (Se) alters whole-blood Se concentrations in sheep, dependent upon Se source and dosage administered, but little is known about effects on immune function. We used footrot (FR) as a disease model to test the effects of supranutritional Se supplementation on immune function. To determine the effect of Se-source (organic Se-yeast, inorganic Na-selenite or Na-selenate) and Se-dosage (1, 3, 5 times FDA-permitted level) on FR severity, 120 ewes with and 120 ewes without FR were drenched weekly for 62 weeks with different Se sources and dosages (30 ewes/treatment group). Innate immunity was evaluated after 62 weeks of supplementation by measuring neutrophil bacterial killing ability. Adaptive immune function was evaluated by immunizing sheep with keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. At baseline, FR-affected ewes had lower whole-blood and serum-Se concentrations; this difference was not observed after Se supplementation. Se supplementation increased neutrophil bacterial killing percentages in FR-affected sheep to percentages observed in supplemented and non-supplemented healthy sheep. Similarly, Se supplementation increased KLH antibody titers in FR-affected sheep to titers observed in healthy sheep. FR-affected sheep demonstrated suppressed cell-mediated immunity at 24 hours after intradermal KLH challenge, although there was no improvement with Se supplementation. We did not consistently prevent nor improve recovery from FR over the 62 week Se-treatment period. In conclusion, Se supplementation does not prevent FR, but does restore innate and humoral immune functions negatively affected by FR.

Aged Beagle dogs have decreased neutrophil phagocytosis and neutrophil-related gene expression compared to younger dogs.

Information on aging and the innate immune response in dogs is needed in order to understand the impact of age on the innate immune system and to design diets that might improve age-associated changes in innate immunity. The purpose of this study was to investigate the effects of age on phagocytosis and antibacterial activity of peripheral blood neutrophils, and on expression levels of selected mRNA specific for neutrophil migration or killing functions. Three groups of Beagle dogs were evaluated in a cross-sectional study design: puppies less than 1 year of age (n=15), adults 1-7 years of age (n=37) and senior adults over 8 years of age (n=25). Neutrophil phagocytosis and killing of Lactococcus lactis were assessed, and the relative abundance of mRNAs for myeloperoxidase (MPO), l-selectin, interleukin-8 receptor (IL-8R) and interleukin-1beta-converting enzyme (ICE) was determined. Results showed that in Beagle dogs there is a significant effect of age on percent bacterial killing by neutrophils. Average decline in phagocytic ability at 4 and 10 years of age (compared to 1 year of age) was 25% and 39%, respectively. In addition, younger dogs have significantly higher levels of mRNA for IL-8R, l-selectin and ICE. These results suggest that older dogs have depressed innate immune responses compared with younger dogs, which may contribute to increased morbidity and mortality with aging.

Influence of dietary antioxidants and fatty acids on neutrophil mediated bacterial killing and gene expression in healthy Beagles

Dietary (n-3) fatty acids from fish oil have been used to modulate immune function in many mammalian species. Together, dietary antioxidants and behavioral enrichment have been shown to enhance neutrophil phagocytosis in geriatric Beagle dogs. The purpose of this study was to further investigate the effects of vitamins E and C, in combination with dietary fish oil, on neutrophil mediated bacterial killing, and on transcript levels of selected neutrophil mRNA. Fifty adult Beagle dogs were randomized into five dietary treatment groups for 60 days. All foods were complete and balanced and met the nutrient profiles of AAFCO for adult dogs. For 60 days before study initiation, dogs consumed a pretrial food that contained 74 IU/kg vitamin E and 0 mg/kg vitamin C. The five experimental foods were confirmed by analytical methods to contain ≥640 IU/kg vitamin E and 130 mg/kg vitamin C (as fed). Experimental foods ranged from low levels of EPA and DHA (pretrial food and lowest experimental food had 0.01% EPA and no detectable DHA) to the highest experimental food with 0.25% EPA and 0.17% DHA. Ex vivo bactericidal activity of activated, peripheral-blood neutrophils against Lactococcus lactis was determined after 1 h incubation. Bactericidal activity was calculated as a percentage of control values (bacteria incubated in media without neutrophils). Transcript levels of genes involved in neutrophil-mediated immune functions were determined by real-time qPCR. Dogs in all treatment groups had increased serum vitamin E concentration (P<0.01). After consuming experimental food for 60 days, neutrophils from dogs in all 5 treatment groups also had increased bactericidal activity (P<0.01). Dietary fish oil however, had no effect on bactericidal activity. Stepwise multiple regression analysis demonstrated that the change in neutrophil mediated bacterial killing was significantly correlated to changes in gene expression of interleukin-8 receptor (IL-8R), interleukin converting enzyme (ICE), and myeloperoxidase (MPO; r(2)=0.33; P=0.003). When stepwise multiple regression analysis was performed considering each mRNA as a dependent variable and change in selected individual and summed fatty acid concentrations as independent variables, change in the ratio of saturated fatty acids (SFA) to polyunsaturated fatty acids (PUFA) was significant (P≤0.05) in the mRNA regression analyses for IL-8R, ICE, MPO, and cyclooxygenase-2. In summary, circulating neutrophils from dogs fed diets enriched in vitamins E and C had significantly increased bactericidal activity as well as altered gene expression. Change in SFA to PUFA ratio also altered neutrophil gene expression.

Comparison between manual and automated total nucleated cell counts using the ADVIA 120 for pleural and peritoneal fluid samples from dogs, cats, horses, and alpacas

BACKGROUND Analysis of body fluids includes an estimate of total nucleated cell count (TNCC). Automated methods may enhance the accuracy and timeliness of TNCC results. OBJECTIVE The purpose of this report was to assess the ability of the ADVIA 120 hematology analyzer to accurately count nucleated cells in pleural and peritoneal fluids from animals, compared with manual counts. METHODS Pleural and peritoneal fluids submitted in EDTA tubes to our laboratory over a 17-month period were used in the study. TNCC/microL was determined by a manual method, using a hemocytometer, and by an automated method, using the ADVIA 120. Correlation of results was determined by Passing-Bablok regression, Bland-Altman plots, and Pearson correlation analysis. RESULTS Samples from dogs (n=36), cats (n=36), horses (n=59), and alpacas (n=11) were analyzed. High correlation in TNCC between methods was found for peritoneal fluid (n=93, r=.959), pleural fluid (n=49, r=.966), and all fluids combined (n=142, r=.960) (P<.001). Variation between methods was greater in samples with TNCCs<1000/microL (r=.62, P<.001). The ADVIA systematically overestimated the number of cells in all fluid samples by 95 cells/microL (confidence interval=19.2-190.5/muL). CONCLUSION The ADVIA 120 reliably determines TNCC in pleural and peritoneal effusions and can be recommended for routine veterinary laboratory analysis.

Hematologic effects of subcutaneous administration of recombinant human granulocyte colony-stimulating factor (filgrastim) in healthy alpacas

OBJECTIVE To determine the effects of SC administration of filgrastim on cell counts in venous blood and bone marrow of healthy adult alpacas. ANIMALS 10 healthy alpacas. PROCEDURES Alpacas were randomly assigned to receive treatment with filgrastim (5 microg/kg, SC; n=5) or an equivalent volume of physiologic saline (0.9% NaCl) solution (5) once a day for 3 days. Blood samples were obtained via jugular venipuncture 1 day prior to treatment and once a day for 5 days commencing 24 hours after the first dose was administered. Complete blood counts were performed for each blood sample. Bone marrow aspirates were obtained from the sternum of each alpaca 48 hours before the first treatment was administered and 72 hours after the third treatment was administered. Myeloid-to-erythroid cell (M:E) ratio was determined via cytologic evaluation of bone marrow aspirates. RESULTS In filgrastim-treated alpacas, substantial increases in counts of WBCs and neutrophils were detected within 24 hours after the first dose was administered. Band cell count and percentage significantly increased 24 hours after the second dose. Counts of WBCs, neutrophils, and band cells remained high 48 hours after the third dose. Red blood cell counts and PCV were unaffected. The M:E ratio also increased significantly after treatment with filgrastim. CONCLUSIONS AND CLINICAL RELEVANCE Filgrastim induced rapid and substantial increases in numbers of circulating neutrophils and M:E ratios of bone marrow in healthy alpacas. Therefore, filgrastim may be useful in the treatment of camelids with impaired bone marrow function.

Serum biochemistry panels in African buffalo: Defining reference intervals and assessing variability across season, age and sex

Serum biochemical parameters can be utilized to evaluate the physiological status of an animal, and relate it to the animal’s health. In order to accurately interpret individual animal biochemical results, species-specific reference intervals (RI) must be established. Reference intervals for biochemical parameters differ between species, and physiological differences including reproductive status, nutritional resource availability, disease status, and age affect parameters within the same species. The objectives of this study were to (1) establish RI for biochemical parameters in managed African buffalo (Syncerus caffer), (2) assess the effects of age, sex, pregnancy, and season on serum biochemistry values, and (3) compare serum biochemistry values from a managed herd to a free-ranging buffalo herd and to values previously published for captive (zoo) buffalo. Season profoundly affected all biochemistry parameters, possibly due to changes in nutrition and disease exposure. Age also affected all biochemical parameters except gamma glutamyl transferase and magnesium, consistent with patterns seen in cattle. Sex and reproductive status had no detectable effects on the parameters that were measured. The biochemical profiles of managed buffalo were distinct from those observed in the free-ranging herd and captive buffalo. Biochemical differences between buffalo from captive, managed, and free-ranging populations may be related to nutritional restriction or lack of predation in the context of management or captivity. The reference intervals provided in this study, in addition to the seasonal and age-related patterns observed, provide a foundation for health investigations that may inform management strategies in this ecologically and economically important species.

Primary ureteral giant cell sarcoma in a Pomeranian

An 8-year-old male neutered Pomeranian dog was presented to the Veterinary Teaching Hospital at Oregon State University for surgical treatment of hydronephrosis of the left kidney and a left cranial abdominal mass. A primary ureteral mass was found during exploratory surgery, and the mass was resected and ureteral anastomosis was performed. Cytologic evaluation of the mass revealed 3 distinct cell populations, including a large number of multinucleated giant cells, a moderate number of thin spindle-shaped cells, and cohesive clusters of transitional epithelial cells. The cytologic diagnosis was giant cell sarcoma. The diagnosis was confirmed by histologic examination, and immunohistochemical staining was performed. The spindle-shaped cells and multinucleated giant cells were both immunoreactive for vimentin and spindle-shaped cells for S-100. Tumor cells did not express wide-spectrum cytokeratin, broad-spectrum muscle actin, smooth muscle actin, sarcomeric actin, desmin, BLA36, Mac 387, synaptophysin, neuron-specific enolase, glial fibrillary acid protein, or von Willebrand factor. These findings are most consistent with an anaplastic sarcoma with giant cells. This is the first case report of a primary ureteral giant cell sarcoma in a dog.

What is your diagnosis? Peritoneal fluid from a 1-year-old female German Shepherd dog: Peritoneal fluid from a dog

A 1-year-old intact female German Shepherd dog was presented to the Oregon State University Small Animal Veterinary Teaching Hospital for progressive inappetance, weight loss, and lethargy. Within the past 2 weeks the dog’s abdomen appeared progressively bloated, and episodes of labored breathing and whimpering were noted. The dog was not currently on heartworm preventative or a deworming protocol, and the owner had been administering 325 mg of aspirin twice daily for 2 weeks due to signs of discomfort. Recent blood work revealed a normocytic hypochromic regenerative anemia, neutrophilia with a left shift, monocytosis, eosinophilia, moderate hypoglycemia (glucose 52 mg/dL, reference interval 70–117 mg/dL), and moderate hypoalbuminemia (albumin 1.8 mg/dL, reference interval 2.5–4.0 mg/dL). On physical examination the dog appeared emaciated with a 1.5/5 body condition score and had a visibly distended abdomen. Abdominal palpation identified ascites with the bulk of the distention localized to the cranial abdomen. Abdominal ultrasonographic examination revealed a large hyperechoic structure extending from the xiphoid process to the mid-abdomen. On lateral radiographs the structure was filled with mineral to bone-dense material. Aspiration of the ascitic fluid was performed and the fluid was submitted for analysis. The fluid contained 96,870 nucleated cells/mL and 130,000 RBCs/mL and had a total protein concentration of 3.0 g/dL. A direct smear and a smear of cytocentrifuged fluid were prepared and examined (Figure 1). Figure 1. Cytocentrifuged preparation of peritoneal fluid from a 1-yearold female German Shepherd dog. Modified Wright’s. (A) 10 objective. (B) 100 objective.