OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl2, and concentrations of platelet-derived growth factor-BB and transforming growth factor-β1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System’s (NAHMS) 2007‐2008 beef study, producers from 24 states were offered the opportunity to evaluate their animals for internal parasites and for overall responses to treatment with anthelmintics. A lapse of 45 d was required between initial sampling and any previous treatments. Choice of anthelmintic (oral benzimidazoles, and both injectable and pour-on endectocides) was at the discretion of the producer so as not to alter the local control programs. Fresh fecal samples were collected from 20 animals, or from the entire group if less than 20, then randomly assigned to 1 of 3 participating laboratories for examination. Analyses consisted of double centrifugation flotation followed by enumeration of strongyle, Nematodirus, and Trichuris eggs (the presence of coccidian oocysts and tapeworm eggs was also noted). Where strongyle eggs per gram (epg) exceeded 30, aliquots from 2 to 6 animals were pooled for egg isolation and polymerase chain reaction (PCR) analysis for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. Results from 72 producers (19 States) indicated that fecal egg count reductions were < 90% in 1/3 of the operations. All operations exhibiting less than a 90% reduction had used pour-on macrocyclic lactones as the anthelmintic treatment. While some of these less than expected reductions could have been the result of improper drug application, PCR analyses of the parasite populations surviving treatment, coupled with follow-up studies at a limited number of sites, indicated that less than expected reductions were most likely due to anthelmintic resistance in Cooperia spp. and possibly Haemonchus spp.

Objective—To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Sample—Citrate-anticoagulated whole blood from 30 healthy adult horses. Procedures—Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead–calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples. Results—Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP. Conclusions and Clinical Relevance—Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.

Objective: To measure platelet membrane–derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions. Animals: 31 clinically normal dogs (19 males and 12 females). Procedures: Citrate-anticoagulated blood samples obtained from each dog were centrifuged at 2,500 × g to isolate platelet-poor plasma (PPP), then PPP was centrifuged at 21,000 × g to isolate microparticle-free plasma (MPF) and microparticle-enriched plasma (MPEP). Whole blood and paired samples of fresh and frozen-thawed PPP, MPF, and MPEP were dual labeled for flow cytometric detection of membrane CD61 (constitutive platelet antigen) and annexin V (indicating phosphatidylserine externalization). Platelets and PMPs were enumerated with fluorescent, size-calibrated beads. Thrombin generation in fresh and frozen-thawed PPP, MPF, and MPEP was measured via kinetic fluorometric assays configured with low tissue factor and low phospholipid concentrations. Results: Initial centrifugation yielded PPP with < 0.5% the platelets of whole blood, with median counts of 413 PMPs/μL for males and 711 PMPs/μL for females. Sequential centrifugation resulted in a 10-fold concentration of PMPs in MPEP and virtually depleted PMPs from MPF. Thrombin generation depended on PMP content, with median endogenous thrombin potential of 0, 893, and 3,650 nmol•min for MPF, PPP, and MPEP, respectively. Freeze-thaw cycling caused significant increases in PMP counts and phosphatidylserine externalization. Conclusions and Clinical Relevance: Canine PMPs were major determinants of thrombin-generating capacity; preanalytic variables influenced plasma PMP content. Processing conditions described here may provide a basis for characterization of PMPs in clinical studies of thrombosis in dogs.

improved method for recovery of ascarid and other nematode eggs from soil, emphasis on Baylisascaris procyonis and Toxocara cati

High-intensity exercise can be associated with the occurrence of muscle injury, as well as the induction of an acute-phase response (APR). The present study aims to investigate the synthesis and profile of serum proteins in horses before and after participating in 2 different exercise protocols and to relate this profile to the presence or absence of muscular injury caused by exercise. Ten purebred Arabian (n = 5) and Criollo (n = 5) horses were subjected to 2 different tests on a treadmill, one consisting of short-duration and rapid-acceleration training (TRA) that was mostly anerobic and the other of long-duration and slow-acceleration training (TLD) that was predominantly aerobic. Blood samples were obtained before the beginning of exercise (T0) and at 6 post-exercise time points: immediately after (T1) and 30 min (T2), 3 h (T3), 12 h (T4), 24 h (T5), and 48 h (T6) after exercise. Hematocrit was determined by the microhematocrit method. Plasma and serum samples were prepared by centrifugation (1500 × g for 5 min) for plasma concentrations of fibrinogen, total serum proteins (TP), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and creatine-kinase (CK) serum activity. Total protein concentration and CK serum activity were determined in an automated biochemistry analyzer. Fibrinogen was determined by the heat precipitation method in microhematocrit capillary tubes. Estimated concentrations of haptoglobin (Hp) significantly decreased after TRA and estimated concentrations of alpha-1 acid glycoprotein (AGP) significantly increased after both protocols at T2. Albumin increased after the TLD exercise protocol. Changes in hematocrit, haptoglobin, and albumin concentrations in horses subjected to different treadmill exercise protocols are related to a physiological response to hemoconcentration and hemolysis. Increases of AGP in the TLD protocol suggest the release of catecholamines as a response to avoid oxidative damage to tissue.

Unbound or free cortisol constitutes a small fraction of total plasma cortisol, but is believed to represent the biologically active portion of this circulating glucocorticoid. We tested the hypothesis that the percentage free cortisol was altered in plasma from dogs with hyperadrenocorticism, which could account for a greater target tissue response to this circulating hormone. The percentage free cortisol in plasma samples from human beings, healthy dogs, and dogs with hyperadrenocorticism was estimated, using centrifugal ultrafiltration-dialysis. Total cortisol concentrations were determined by use of radioimmunoassay. Total cortisol concentrations appeared greater in plasma from human beings than in plasma from either group of dogs. However, the percentage free cortisol was lower in plasma from human beings, resulting in a calculated concentration of free cortisol that was quite similar between plasma from human beings and healthy dogs. Total plasma cortisol concentrations were greater (P < 0.01) in samples from dogs with hyperadrenocorticism (190 +/- 113 nmol/L; mean +/- SD) than in healthy dogs (102 +/-85 nmol/L), but the percentage free cortisol was not different between these 2 groups (dogs with hyperadrenocorticism, 16 +/- 9%; healthy dogs, 13 +/- 6%). However, plasma free cortisol concentrations (product of total and the percentage of free cortisol) were greater (P < 0.01) in samples from dogs with hyperadrenocorticism (36 +/- 41 nmol/L) than in those from healthy dogs (16 +/- 9 nmol/L). Significant (P < 0.001) positive linear relationships were found between total cortisol concentrations and percentage free cortisol in plasma samples from healthy dogs and dogs with hyperadrenocorticism. Furthermore, the slope of these lines was not different between the 2 groups, providing no evidence for alterations in cortisol binding associated with hyperadrenocorticism. The higher total cortisol concentrations in dogs affected with this disease do, however, result in greater concentrations of free cortisol in circulation, contributing to the development of clinical signs observed in this disease.

Veterinary diagnostic endocrinology laboratories frequently receive hemolyzed plasma, serum, or blood samples for hormone analyses. However, except for the previously reported harm done by hemolysis to canine insulin, effects of hemolysis on quantification of other clinically important hormones are unknown. Therefore, these studies were designed to evaluate effects of hemolysis on radioimmunoassay of thyroxine, 3,5,3'-triiodothyronine, progesterone, testosterone, estradiol, cortisol, and insulin in equine, bovine, and canine plasma. In the first experiment, hormones were measured in plasma obtained from hemolyzed blood that had been stored for 18 hours. Blood samples were drawn from pregnant cows, male and diestrous female dogs, and male and pregnant female horses. Each sample was divided into 2 equal portions. One portion was ejected 4 times with a syringe through a 20-gauge (dogs, horses) or 22-gauge (cows) hypodermic needle to induce variable degrees of hemolysis. Two subsamples of the blood were taken before the first and after the first, second, and fourth ejections. One subsample of each pair was stored at 2 to 4 C and the other was stored at 20 to 22 C for 18 to 22 hours before plasma was recovered and stored at -20 C. The second portion of blood from each animal was centrifuged after collection; plasma was recovered and treated similarly as was blood. Concentrations of thyroxine in equine plasma, of 3,5,3'-triiodothyronine, estradiol, and testosterone in equine and canine plasma, and of cortisol in equine plasma were not affected by hemolysis. Storage of bovine blood at either temperature and equine blood at 20 to 22 C caused progesterone concentrations to decrease (P < 0.05); the effect was not enhanced or diminished by hemolysis. Insulin concentration in equine blood decreased (P < 0.05) at both temperatures; this effect was exacerbated by hemolysis. In the second experiment, blood samples from horses and dogs were hemolyzed and plasma was immediately recovered and stored for 18 to 22 hours at 2 to 4 C or 20 to 22 C. Storage of hemolyzed equine plasma did not affect concentrations of progesterone, insulin, or thyroxine at either temperature. Whereas progesterone concentration was not affected in hemolyzed canine plasma, hemolysis decreased (P < 0.05) insulin concentration when plasma was stored at 20 to 22 C. These results emphasize the importance of examining effects of sample collection and handling procedures on hormone stability and the danger of extrapolating results of such studies from one species to another and from one hormone to another.

OBJECTIVE To evaluate the in vitro effect of 20% N-acetylcysteine (NAC) on the viscosity of normal canine bile. ANIMALS Bile samples obtained from 10 adult dogs euthanized for reasons unrelated to biliary disease. PROCEDURES Each sample was centrifuged to remove particulates, then divided into 3 aliquots. One aliquot remained untreated (control). Each of the other aliquots was diluted 1:4 with 20% NAC or sterile water. The viscosity of all samples was measured with a rotational viscometer at 25°C. Viscosity of control samples was measured immediately after centrifugation and at 1 and 24 hours after treatment application to the diluted samples. Viscosity of diluted samples was measured at 1 and 24 hours after treatment application. RESULTS Mean viscosity differed significantly among the 3 groups at both 1 and 24 hours after treatment application. Relative to control samples, the addition of NAC and sterile water decreased the viscosity by approximately 3.35 mPa·s (95% confidence interval [CI], 1.58 to 5.12 mPa·s) and 2.74 mPa·s (95% CI, 1.33 to 4.14 mPa·s), respectively. Mean viscosity of the NAC-treated samples was approximately 0.61 mPa·s (95% CI, 0.21 to 1.01 mPa·s) less than that for the sterile water–treated samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that in vitro dilution of canine bile 1:4 with 20% NAC significantly decreased the viscosity of the resulting mixture. Further research is necessary to determine whether NAC is a safe and effective noninvasive treatment for dogs with persistent biliary sludge or gallbladder mucoceles.