The aim of this study was to evaluate the performance characteristics of an automated immunoassay for canine insulin-like growth factor 1 (IGF-1) measurement and to investigate the possible effects of some sources of variation, such as diurnal variations, feeding/fasting cycles, and glucocorticoid administration, in dogs. The immunoassay evaluated had an adequate analytical performance with intra- and inter-assay coefficients of variation (CVs) lower than 10%, linear regression equations with correlation coefficients of 0.9993 and 0.9988 after serial dilutions, and a limit of quantification of 7.1 ng/mL that was even lower than that reported by the manufacturer. The assay was significantly affected by hemolysis and lipemia producing a significant decrease in IGF-1 concentrations, but not by bilirubinemia. Serum IGF-1 concentrations did not show significant diurnal changes in fed or fasted dogs and were not affected by glucocorticoid administration.

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Objective—To evaluate serum haptoglobin concentration at feedlot arrival and subsequent performance and morbidity and mortality rates of calves that developed bovine respiratory disease. Animals—360 heifer calves and 416 steer and bull calves. Procedures—Serum samples were obtained from cattle at the time of arrival to a feedlot (day −1) and analyzed for haptoglobin concentration. In experiment 1, calves were classified into groups with a low (< 1.0 μg/mL), medium (1.0 to 3.0 μg/mL), or high (> 3.0 μg/mL) serum haptoglobin concentration and allotted into pens on the basis of group. In experiment 2, calves were classified as having or not having detectable serum haptoglobin concentrations. Results—In experiment 1, average daily gain from days 1 to 7 decreased as haptoglobin concentration increased. Dry-matter intake (DMI) from days 1 to 21 decreased with increasing haptoglobin concentration, and DMI typically decreased from days 1 to 63. Total bovine respiratory disease morbidity rate typically increased with increasing haptoglobin concentration. At harvest, no differences in carcass characteristics were observed on the basis of haptoglobin concentration. In experiment 2, cattle with measureable serum haptoglobin concentrations at arrival weighed less throughout the experiment, gained less from days 1 to 7, and had lower DMI from days 1 to 42. Overall morbidity rate was not different between groups, but cattle with detectable serum haptoglobin concentrations had higher odds of being treated 3 times. Conclusions and Clinical Relevance—Serum haptoglobin concentration in cattle at the time of feedlot arrival was not associated with overall performance but may have limited merit for making decisions regarding targeted prophylactic treatment.

Objective—To evaluate enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus in milk obtained from cows with subclinical mastitis. Sample—Milk samples obtained from 350 cows (1,354 mammary glands) on 11 Wisconsin dairy farms. Procedures—Of 252 S aureus isolates obtained from 146 cows, 83 isolates (from 66 cows with subclinical mastitis) were compared genotypically by use of pulsed-field gel electrophoresis and via PCR identification of toxic shock syndrome toxin 1 (TSST-1) and classical S aureus enterotoxin genes (sea, seb, sec, sed, and see). Results—Among the 83 S aureus isolates, ≥ 1 enterotoxin genes were identified in 8 (9.6%). Enterotoxin gene distribution was as follows: TSST-1, 7 isolates (8.4%); sec, 5 isolates (6.0%); and sed, 2 isolates (2.4%). Enterotoxin genes sea, seb, and see were not identified. Twelve pulsotypes and 5 subtypes were identified among the 83 isolates; 5 of the 12 pulsotypes were represented by only 1 isolate. In cows of 1 herd, only a single S aureus pulsotype was detected; in cows on most other farms, a variety of pulsotypes were identified. One pulsotype was recovered from 4 farms (n = 23 cows) and another from 5 other farms (16). Isolates with an enterotoxin gene were represented by 6 pulsotypes. Conclusions and Clinical Relevance—S aureus classical enterotoxins and TSST-1 were rarely recovered from milk samples obtained from cows with subclinical mastitis in Wisconsin. Diverse pulsotypes of S aureus were detected within and among farms, indicating that different strains of S aureus cause subclinical mastitis in dairy cows.

Objective—To assess expression pattern and subcellular compartmentalization of 5-lipoxygenase in cutaneous, UV radiation–induced, and oral squamous cell carcinomas (SCCs) in cats and determine the effects of cyclooxygenase or 5-lipoxygenase inhibition on proliferation or apoptosis in a feline oral squamous cell carcinoma (SCCF1) cell line. Sample—60 archived paraffin-embedded samples of SCCs from 60 cats and SCCF1 cells. Procedures—Retrospective immunohistochemical analysis of the archived samples of SCCs (20 cutaneous, 20 UV radiation–induced, and 20 oral tumors) was performed. Cell culture proliferation assays involving SCCF1 cells were performed, and tepoxalin-induced apoptosis and signaling were examined via western blotting and annexin V staining. Results—Immunohistochemically, staining for 5-lipoxygenase was most frequently of greatest intensity in oral SCCs, whereas staining of cutaneous and UV radiation–induced lesions had less consistent 5-lipoxygenase expression. Exposure of SCCF1 cells to the 5-lipoxygenase inhibitor tepoxalin resulted in apoptosis; the effect appeared to be mediated via alteration of cell signaling rather than via suppression of lipid mediators that are typically produced as a result of 5-lipoxygenase activity. Conclusions and Clinical Relevance—In cats, expression of 5-lipoxygenase in SCCs appeared to differ depending on tumor location. The influence of tepoxalin-induced 5-lipoxygenase inhibition on a poxygenase–expressing cell line coupled with the notable expression of 5-lipoxygenase in oral SCCs suggested that 5-lipoxygenase inhibition may have therapeutic benefits in affected cats. Although the safety of tepoxalin in cats has yet to be investigated, 5-lipoxygenase inhibitors should be evaluated for use as a potential treatment for SCCs in that species.

Objective-To determine variations in cytologic counts of bronchoalveolar lavage (BAL) fluid attributable to month of collection, first and second aliquots, and left and right lung sites in horses with recurrent airway obstruction (RAO). Animals-5 horses with RAO and 5 healthy horses without respiratory tract disease. Procedures-Horses were housed in a stable for 5 months prior to and throughout the study. Bronchoalveolar lavage fluid was collected from the right and left lung of each horse 3 times at monthly intervals (February, March, and April). Each BAL fluid collection was performed by use of 2 incremental instillations of 250 mL of isotonic saline (0.9% NaCl) solution in the same bronchial site. Analysis of BAL fluid included volume of BAL fluid recovered, a CBC, and differential cytologic counts. Results-Volume of BAL fluid recovered and cytologic counts did not differ in horses with RAO across time or between right and left lungs, except for the number of mast cells. Horses with RAO had significantly lower volumes of BAL fluid recovered, significantly lower percentages of macrophages and lymphocytes, and significantly higher percentages of neutrophils than did healthy horses. Despite individual variation, all horses with RAO had > 25% neutrophils throughout the study period. Conclusions and Clinical Relevance-Despite variation among horses, BAL fluid cytologic counts were repeatable over short and long periods and samples can be used for longitudinal studies as a diagnostic tool of pulmonary inflammation in horses with RAO.

Little is known about the sources and kinetics of enterotoxigenic Escherichia coli colonization in pigs during the pre-and post-weaning period. In this study, farrowing pens, sows, and piglets were tested for the presence of E. coli O149 by real-time polymerase chain reaction (PCR) after bacterial culture pre-enrichment on 2 farms, one with a history of post-weaning diarrhea (problem farm — PF) and the other without such a history (non-problem farm — NPF). Unlike those on the PF, the sows from the NPF did not carry E. coli O149 before parturition, although they were colonized to frequencies similar to animals on the PF soon afterwards. Most piglets from the NPF were colonized within a week after birth, whereas only a small proportion of those on the PF were colonized during that period. No difference was observed in the frequency of piglet colonization at the 2 farms either at weaning or during the following week. Post-weaning diarrhea (PWD), which is caused by enterotoxigenic E. coli (ETEC), is a multifactorial disease. The presence of ETEC alone is not always sufficient for the disease to develop. Many other factors are considered to be associated with the occurrence of PWD, including feed type (1,2), feeding regimen (1,3,4), the presence of other infectious agents (3,5), weaning age, and weight (6). Weaning, which is considered to be a major physiological and psychological stress factor, is critical for the disease to occur (7). Although piglets are already colonized with ETEC before weaning (4,8), on many farms, clinical disease occurs only after weaning (1). Both sows (9,10) and the environment (6) could be possible sources of infection for piglets, but results from previous studies have not resolved this issue because of the low sensitivity of ETEC detection methods. This study provides preliminary data based on a sensitive detection method for E. coli O149 in pigs and their environment. The results demonstrate the potential of real-time PCR for future studies on this topic.

We investigated vascular access ports for feline blood donation. Eight cats were anesthetized for conventional blood collection by jugular venipuncture at the beginning and end of the study. In-between conventional collections, vascular access ports were used for collection with or without sedation every 6 to 8 wk for 6 mo. Ports remained functional except for one catheter breakage, but intermittent occlusions occurred. Systolic blood pressure was lower during conventional collection. Behavioral abnormalities occurred during 3 port collections. Packed red cells prepared from collected blood were stored at 4°C for 25 d and assessed for quality pre- and post-storage. With both collection methods, pH and glucose level declined, and potassium level, lactate dehydrogenase activity and osmotic fragility increased. There were no differences between methods in pre-storage albumin and HCO3− levels, and pre and post-storage hematocrit, lactate dehydrogenase activity, and glucose and potassium levels. Pre-storage pH and pCO2 were higher with conventional collection, and pre- and post-storage osmotic fragility were greater with port collection. One port became infected, but all cultures of packed red cells were negative. Tissue inflammation was evident at port removal. In a second study of conventional collection in 6 cats, use of acepromazine in premedication did not exacerbate hypotension. The use of vascular access ports for feline blood donation is feasible, is associated with less hypotension, and may simplify donation, but red cell quality may decrease, and effects on donors must be considered.

Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.