Objective: To evaluate platelet-neutrophil aggregate (PNA) formation and neutrophil shape as indicators of neutrophil activation in dogs with systemic inflammatory diseases and after blood sample incubation with various platelet and neutrophil agonists. Animals: 20 dogs with systemic inflammatory response syndrome (SIRS) and 10 healthy Beagles. Procedures: Neutrophils were isolated from blood samples directly after blood sample collection and after incubation of blood samples with phorbol myristate acetate, collagen, adenosine diphosphate, epinephrine, or various concentrations of lipopolysaccharide or arachidonic acid. CD61+ neutrophils as an indicator of PNA formation were evaluated, and neutrophil size and granularity were assessed via flow cytometry. Results: Dogs with SIRS had more PNA formation, larger neutrophil size, and less granularity relative to control dogs, but no differences were evident when these dogs were grouped by whether they had sepsis (n = 6) or disseminated intravascular coagulation (12). A significant increase in PNA formation occurred after neutrophil incubation with all agonists, and incubation with phorbol myristate acetate elicited the strongest response. Neutrophils increased in size and decreased in granularity after incubation with all agonists except epinephrine. Incubation with lipopolysaccharide or arachidonic acid resulted in a dose-dependent effect on PNA formation and neutrophil shape. Conclusions and Clinical Relevance: SIRS appeared to increase the degree of PNA formation and neutrophil shape change. Similar changes after neutrophil incubation with platelet agonists suggested that platelet activation has a role in PNA formation. Additional studies are necessary to determine the clinical importance and diagnostic value of PNA formation in dogs with SIRS and sepsis.

Objective: To evaluate the B-mode and Doppler ultrasonographic appearance of presumptively normal main axillary and large superficial cervical lymph nodes (MALNs and SCLNs, respectively) in adult dogs. Animals: 51 healthy adult dogs (data from 1 dog were not analyzed). Procedures: For each dog, weight, distance from the cranial aspect of the first sternebra to the caudal aspect of the left ischiatic tuberosity, and thoracic height and width at the level of the xiphoid process were recorded. Via B-mode and Doppler ultrasonography, echogenic characteristics, size in relation to body size and weight, and vascular supply of the MALNs and the SCLNs were evaluated (1 SCLN in 1 dog was not ultrasonographically visible). Results: Most MALNs were clearly margined, solitary, and ovoid; echopatterns were homogenous or cortical and hypo- to isoechoic, compared with surrounding soft tissues. Size measurements of MALNs correlated with dogs' body length, thoracic width and height, and body weight. Most SCLNs were clearly margined, fusiform, and hypoechoic (compared with surrounding soft tissues) with a cortical or homogenous echopattern. Size measurements of SCLNs correlated with dogs' body length, thoracic width and height, and body weight. In 50 of the 100 MALNs, an intranodal vascular supply was detected; in contrast, an intranodal vascular supply in SCLNs was detected infrequently. Conclusions and Clinical Relevance: Results indicated that, in dogs, anatomically separate lymph nodes have different echogenic and vascular characteristics; body size (skeletal length, height, and width), along with body weight, were correlated with sizes of presumptively normal MALNs and SCLNs.

Objective: To determine effects of leukotriene (LT) C4 on ion transport across equine tracheal epithelium. Sample: Tracheal epithelium from cadavers of 24 horses considered free of respiratory tract disease. Procedures: Mucosae were mounted into Ussing chambers, and short-circuit current (Isc) was monitored over time. Effects of LTC4 were examined for various conditions, including addition of amiloride (10μM) to the mucosal bath solution, addition of bumetanide (10μM) to the serosal bath solution, addition of barium (1mM) to the serosal bath solution, and substitution of gluconate for chloride and HEPES for bicarbonate in bath solutions. Electrolyte transport was assessed via 22Na and 36Cl isotope fluxes. Results: Addition of LTC4 (50nM) to the serosal bath solution caused an increase in Isc for basal conditions and a larger increase after pretreatment with amiloride. The increase was negated in part by the addition of bumetanide to the serosal bath solution and further reduced by substitution of HEPES for bicarbonate in bath solutions. Remaining current was reduced to values less than those before treatment with LTC4 by the addition of barium to the serosal solution. There was a small increase in Isc after the addition of amiloride and substitution of gluconate for chloride. Radioisotope flux indicated that addition of LTC4 to the serosal bath solution increased chloride secretion and reduced sodium absorption. Conclusions and Clinical Relevance: LTC4 stimulated chloride secretion through a predominately bumetanide-sensitive pathway, with a smaller contribution from a bicarbonate-dependent pathway. Thus, LTC4 appears to be a potential mediator of airway hypersecretion in horses.

Objective: To determine the prevalence of Mycoplasma bovis infection in the lungs of cattle at various times after arrival at a feedlot, to measure the relationship between clinical disease status and the concentration and genotype of M bovis within the lungs, and to investigate changes in the genotype of M bovis over time. Sample: Bronchoalveolar lavage fluid (BALF) from 328 healthy or pneumonic beef cattle and 20 M bovis isolates obtained from postmortem samples. Procedures: The concentration of M bovis in BALF was determined via real-time PCR assays, and M bovis isolates from BALF were genotyped via amplified fragment length polymorphism (AFLP) analysis. Results: Prevalence of M bovis in BALF was 1 of 60 (1.7%) at arrival to a feedlot and 26 of 36 (72.2%) and 36 of 42 (85.7%) at ≤ 15 days and 55 days after arrival, respectively. Neither the concentration nor the AFLP type of M bovis in BALF was correlated with clinical disease status. The M bovis AFLP type differed between early and later sampling periods in 14 of 17 cattle. Conclusions and Clinical Relevance: The findings implied spread of M bovis among calves and suggested that host factors and copathogens may determine disease outcomes in infected calves. Chronic pulmonary infection with M bovis may represent a dynamic situation of bacterial clearance and reinfection with strains of different AFLP type, rather than continuous infection with a single clone. These findings impact our understanding of why cattle with chronic pneumonia and polyarthritis syndrome inadequately respond to antimicrobial treatment.

Objective: To examine the effect of endotoxins on metabolism and histopathologic changes of isolated perfused equine forelimbs. Sample: Forelimbs (comprising the metacarpus and digit) were collected from cadavers of 12 healthy adult horses after slaughter at an abattoir (14 limbs; 1 forelimb of 10 horses and both forelimbs of 2 horses). Procedures: Forelimbs were perfused for 10 hours with autologous blood, with and without the addition of endotoxin (80 ng of lipopolysaccharide [LPS]/L). Two limbs of the endotoxin exposure group and 2 nonperfused limbs were loaded to failure of the suspensory apparatus of the pedal bone to evaluate the effect of body weight. Metabolic and histologic variables were evaluated. Results: Blood pressure increased during the first hour and did not differ between groups. Lactate dehydrogenase activity was similar in both groups and increased significantly during the 10-hour period; glucose consumption at 5 hours and lactate concentration at 8 hours were significantly higher in limbs exposed to endotoxin. The width of secondary epidermal lamellae was greater in LPS limbs. In the primary dermal lamellae of LPS limbs, there were significantly more vessels with an open lumen and aggregates of intravascular neutrophils. Conclusions and Clinical Relevance: In the blood-perfused isolated forelimbs of equine cadavers, exposure to LPS led to significant changes in the laminar tissue as well as to metabolic changes. Therefore, endotoxin should be considered as a causative factor for laminitis and not merely as a risk factor.

Objective: To determine the pharmacokinetics of a long-acting formulation of ceftiofur, ceftiofur crystalline-free acid (CCFA), following SC injection to Asian elephants (Elephas maxim us). Animals: 11 adult Asian elephants. Procedures: Each elephant received CCFA (6.6 mg/kg, SC) in the area caudoventral to the base of an ear. Blood samples were collected from an ear vein immediately prior to and at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours after CCFA administration. Plasma concentrations of desfuroylceftiofur acetamide (the acetamide derivative of ceftiofur) were measured via ultrahigh-pressure liquid chromatography–tandem mass spectrometry. Data were analyzed via a noncompartmental pharmacokinetics approach. Results: The mean ± SD maximum plasma concentration of desfuroylceftiofur acetamide was 1.36 ± 0.74 μg/mL and was detected at 4718 ± 31.30 hours. The mean ± SD area under the curve from time 0 to infinity was 2278 ± 55.8 μg•h/mL, and the mean residence time from time 0 to infinity was 158.2 ± 90.2 hours. The terminal elimination half-life associated with the slope of the terminal phase had a harmonic mean ± pseudo-SD of 83.36 ± 30.01 hours. Conclusions and Clinical Relevance: Elephants tolerated CCFA at a dose of 6.6 mg/kg, SC, well. Dosing recommendations will depend on the mean inhibitory concentration of ceftiofur for each bacterial pathogen. Desfuroylceftiofur acetamide concentrations remained > 0.25 μg/mL for the entire 168-hour study period, which suggested CCFA would provide clinically relevant antimicrobial activity against certain pathogens for 7 to 10 days.

Objective: To investigate the effect of exercise during yearling sales preparation on the risk of interruptions during training in Thoroughbred racehorses. Animals: 114 Thoroughbred racehorses. Procedures: Information regarding the daily exercise of yearlings during sales preparation was obtained prospectively from a convenience sample of stud farms. Yearlings were followed to entry into race training, and subsequently, daily training information was recorded until the end of the racing season. Competing-risks survival analysis was used to model time from entry into race training to voluntary training interruption (no known condition or disease identified) and time from entry into race training to involuntary training interruption (due to presence of a condition or disease) occurring before the first trial (practice race for education). Total hand walking time and mechanical walker time accumulated during sales preparation were the main exposures of interest. Results: 82 of 114 (71.9%) horses had an interruption before the first trial; 65 (79%) interruptions were voluntary, and 17 (21 %) interruptions were involuntary. Increased total hand walking time was significantly associated with decreased risk of voluntary interruptions, whereas longer cumulative distances at a canter were significantly associated with decreased risk of involuntary interruptions. Conclusions and Clinical Relevance: Results identified an association between early exercise during sales preparation and decreased risk of voluntary interruption and increased risk of involuntary interruption during training of 2-year-old Thoroughbred racehorses. Further investigation into the effects of early exercise on racing performance is needed, but results have indicated that there may be an opportunity to modify early exercise programs.

Objective: To determine whether the method of lipopolysaccharide (LPS) administration (intermittent vs continuous) affects the magnitude and duration of the systemic inflammatory response in horses and whether prolonged (48 hours) endotoxemia induces laminitis. Animals: 12 healthy adult horses (10 mares and 2 geldings). Procedures: Horses were randomly assigned to receive LPS (total dose, 80 μg; n = 4) or saline (0.9% NaCl) solution (80 mL/h; 4) via constant rate infusion or 8 bolus IV injections of LPS (10 μg, q 6 h;4) during a 48-hour period. Physical examinations were performed every 4 hours, inflammatory cytokine gene expression was determined for blood samples obtained every 8 hours, and IV glucose tolerance tests were performed. Results: All LPS-treated horses had signs of depression and mild colic; those signs abated as the study progressed. Administration of LPS increased expression of interleukin-1β, interleukin-6, and interleukin-8, but results were not significantly different between LPS treatment groups. Cytokine expression was significantly higher on the first day versus the second day of LPS treatment. Interleukin-1β expression was positively correlated with rectal temperature and expression of other cytokines. Glucose and insulin dynamics for both LPS groups combined did not differ significantly from those of the saline solution group. Signs of laminitis were not detected in any of the horses. Conclusions and Clinical Relevance: Horses developed LPS tolerance within approximately 24 hours after administration was started, and the method of LPS administration did not affect the magnitude or duration of systemic inflammation. Laminitis was not induced in horses.

Objective-To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs. Animals-22 healthy adult dogs and 10 dogs with eccentrocytosis. Procedures-2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method. Results-Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results. Conclusions and Clinical Relevance-The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.