A retrospective analysis of renal carcinoma in the horse.

BACKGROUND: Renal carcinoma is a rare tumor of horses. HYPOTHESIS: Presenting complaints and clinical signs of this disease are vague and early diagnosis increases survival time. ANIMALS: Data were collected from the medical records of 4 horses presented to Washington State University as well as the 23 previously published case reports of horses with renal carcinoma. METHODS: Retrospective study. RESULTS: Renal carcinoma affects horses of all ages with most cases observed in geldings and Thoroughbreds. The most common presenting complaints are nonspecific and usually do not occur until late in the course of the disease. Routine laboratory results generally are unremarkable with no evidence of renal dysfunction. Urine and peritoneal fluid analyses are consistently abnormal, but the changes usually are nonspecific. Rectal palpation often allows detection of an abnormal kidney or a mass in the area of the kidney. Renal ultrasound examination is the most rewarding imaging procedure, and when combined with renal biopsy, antemortem diagnosis can be achieved. Renal carcinoma is both locally invasive and metastatic, necessitating careful staging for metastasis using thoracic radiography and abdominal ultrasound examination. If the tumor is localized to 1 kidney, nephrectomy is the treatment of choice. No chemotherapy or radiation treatment for renal carcinoma has been reported in the horse. Median survival for this series of cases was 11 days (0 days-1 year). CONCLUSIONS AND CLINICAL IMPORTANCE: Prognosis is poor to grave.

Pharmacokinetics of butorphanol in horses after intramuscular injection.

A two-way cross-over study of the pharmacokinetics of butorphanol after intravenous and intramuscular administration at 0.08 mg/kg in six adult horses was performed. Heparinized venous blood samples were obtained prior to drug administration and at 10, 20, 30, 45, 60, 120, 180, 240, and 360 min after IV injection. Samples were obtained at the same time points and at 6 h and 12 h after IM injection. Physical examination parameters were recorded at each time point. Plasma butorphanol concentrations were determined by high performance liquid chromatography. No significant differences in any physical parameters were observed after butorphanol administration except for an increase in respiratory rate at 60 and 180 min after IV administration. Absorption of butorphanol after IM administration was very rapid (half life of absorption of 6 min) but systemic availability after IM injection was low (37%). Terminal half-life after IV administration was much longer than half-life after IM administration (0.57 h and 7.7 h, respectively). This difference was attributed to detection of a deep compartment after IV administration that was not detectable after IM administration. To maintain targeted plasma butorphanol concentrations above 10 ng/mL, administration of 0.08 mg/kg IM every 3 h may be necessary.

Pharmacokinetics of butorphanol and evaluation of physiologic and behavioral effects after intravenous and intramuscular administration to neonatal foals.

BACKGROUND: Despite frequent clinical use, information about the pharmacokinetics (PK), clinical effects, and safety of butorphanol in foals is not available. OBJECTIVES: The purpose of this study was to determine the PK of butorphanol in neonatal foals after IV and IM administration; to determine whether administration of butorphanol results in physiologic or behavioral changes in neonatal foals; and to describe adverse effects associated with its use in neonatal foals. ANIMALS: Six healthy mixed breed pony foals between 3 and 12 days of age were used. METHODS: In a 3-way crossover design, foals received butorphanol (IV and IM, at 0.05 mg/kg) and IV saline (control group). Butorphanol concentrations were determined by high-performance liquid chromatography and analyzed using a noncompartmental PK model. Physiologic data were obtained at specified intervals after drug administration. Pedometers were used to evaluate locomotor activity. Behavioral data were obtained using a 2-hour real-time video recording. RESULTS: The terminal half-life of butorphanol was 2.1 hours and C0 was 33.2 +/- 12.1 ng/mL after IV injection. For IM injection, Cmax and Tmax were 20.1 +/- 3.5 ng/mL and 5.9 +/- 2.1 minutes, respectively. Bioavailability was 66.1 +/- 11.9%. There were minimal effects on vital signs. Foals that received butorphanol spent significantly more time nursing than control foals and appeared sedated. CONCLUSIONS AND CLINICAL IMPORTANCE: The disposition of butorphanol in neonatal foals differs from that in adult horses. The main behavioral effects after butorphanol administration to neonatal foals were sedation and increased feeding behavior.

Parasitemia in an immunocompetent horse experimentally challenged with Sarcocystis neurona sporocysts.

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 28 days, followed by 1000/day for 56 days. On day 98 of the study, six yearling colts were selected for attempted culture of S. neurona from blood, two testing positive, two testing suspect and two testing negative for antibodies against S. neurona on day 84 of the study. Two 10 ml tubes with EDTA were filled from each horse by jugular venipuncture and the plasma fraction rich in mononuclear cells was pipetted onto confluent equine dermal cell cultures. The cultures were monitored weekly for parasite growth for 12 weeks. Merozoites grown from cultures were harvested and tested using S. neurona-specific PCR with RFLP to confirm species identity. PCR products were sequenced and compared to known strains of S. neurona. After 38 days of in vitro incubation, one cell culture from a horse testing positive for antibodies against S. neurona was positive for parasite growth while the five remaining cultures remained negative for parasite growth for all 12 weeks. The Sarcocystis isolate recovered from cell culture was confirmed to be S. neurona by PCR with RFLP. Gene sequence analysis revealed that the isolate was identical to the challenge strain SN-37R and differed from two known strains UCD1 and MIH1. To our knowledge this is the first report of parasitemia with S. neurona in an immunocompetent horse.

Ulcerative dermatitis, thrombocytopenia, and neutropenia in neonatal foals.

This report describes transient ulcerative dermatitis, severe thrombocytopenia, and mild neutropenia in 6 foals from 4 mares from geographically diverse regions of the United States. The foals presented at <4 days of age with oral and lingual ulcers, and crusting and erythema around the eyes, muzzle, and perineal, inguinal, axillary, trunk, and neck regions. There was a severe thrombocytopenia (0-30,000 platelets/microL), leukopenia (1900-3200 white blood cells/microL), and mild neutropenia (500-1800 neutrophils/microL). Four of the 6 foals had petechiae and ecchymotic hemorrhages and 3 had bleeding tendencies. Results of examination of a bone marrow biopsy from 1 foal were normal and results of a platelet surface immunoglobulin test in another were negative. Histopathology of the skin in all foals showed subepidermal clefting with subjacent vascular dilation, dermal hemorrhage, and superficial papillary necrosis. The foals were treated supportively with broad-spectrum antibiotics (5/6), corticosteroids (3/6), gastric ulcer prophylaxis (6/6), whole-blood transfusion (4/6), and platelet-rich plasma (1/6). The skin lesions and thrombocytopenia (>50,000 platelets/microL) improved in 2 weeks (4/6). Two foals had a decline in their platelet counts when the steroids were decreased and needed protracted treatment. All foals survived and were healthy as yearlings. Two mares that had 2 affected foals each, upon subsequent pregnancies to different stallions, had healthy foals when an alternate source of colostrum was given. The findings in the cases in this report suggest a possible relationship between colostral antibodies or some other factor in the colostrum and the thrombocytopenia and skin lesions, although further investigation is warranted to confirm or refute this hypothesis.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona.

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus.

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor alpha (TNF alpha). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.

Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes.

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

Intravitreal transforming growth factor-beta 2 decreases cellular infiltration in endotoxin-induced ocular inflammation in rabbits.

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which has been identified in normal and inflamed ocular fluids, may play a role in the evolution of inflammatory ocular lesions. In this study we utilized a rabbit model of LPS-induced uveitis to determine if exogenous TGF-beta 2 could alter its course. Recombinant TGF-beta 2 (1-2000 ng), LPS (10 or 20 ng), or TGF-beta 2 (100 ng) plus LPS (10 ng) were injected intravitreally in one eye of a New Zealand white rabbit and the contralateral eye served as a paired control which received an equal volume of vehicle. The uveitic response was assessed by biomicroscopic examination of the anterior uvea and analysis of protein and cells in the aqueous humor. Ocular tissues were processed for histologic, immunohistochemical and in situ hybridization analyses. Rabbits injected with doses of TGF-beta 2 > or = 500 ng developed a mild uveitic response, compared to LPS alone, accompanied by expression of IL-1 beta mRNA and protein in the anterior uvea. Interestingly, rabbits coinjected with LPS (10 ng) and a nonuveitic dose (100 ng) of TGF-beta 2 exhibited a similar increase in ocular vascular permeability, but a decrease in inflammatory cell infiltration into the anterior uvea and aqueous humor (1185 +/- 117 versus 2465 +/- 176; p < 0.05). No evidence of inflammation was observed in eyes injected with 100 ng TGF-beta 2 alone. Similar to other models of inflammation, TGF-beta may interrupt the cascade of events leading to ocular inflammation, thereby suggesting therapeutic potential.

Increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia virus.

Seven ponies were infected with the virulent wild-type Wyoming strain of equine infectious anaemia virus (EIAV). Infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. Preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (IL-6) activity. Postinfection IL-6 activity was significantly increased as compared to preinfection values. The magnitude of increase in IL-6 was positively correlated with reverse transcriptase activity (an indirect measure of viraemia) but was not correlated with rectal temperature. IL-6 production in response to EIAV infection may play a role in pathogenesis of disease, especially the hyperglobulinaemia and apparent polyclonal B cell activation in these horses.

Thrombocytosis in 24 horses (1989-1994).

The records of horses presented to the Veterinary Teaching Hospital of North Carolina State University College of Veterinary Medicine between January 1, 1989 and April 30, 1994 were evaluated to determine risk factors associated with thrombocytosis. Of the 2,346 horses for which a CBC was performed, 24 (1.0%) had a platelet count > 400,000/microL. Demographic, diagnostic, physical examination, and clinicopathologic variables from these cases were compared with a reference population consisting of 189 horses with a normal platelet count presenting during the same period. Infectious/ inflammatory disorders were observed more commonly in horses with high platelet counts than in horses with normal platelet counts. Initial independent evaluation of demographic variables revealed that horses more than 3 years of age, females, and geldings were less likely to have thrombocytosis than were younger horses or stallions. Independent analysis of clinicopathologic variables revealed that horses with thrombocytosis were more likely to have hyperfibrinogenemia, leukocytosis, hypoproteinemia, and anemia than were horses with normal platelet counts. Physical examination parameters associated with thrombocytosis included tachycardia and pyrexia. In the final multivariable model, the variables with the strongest association with thrombocytosis included leukocytosis, anemia, and hyperfibrinogenemia. Thrombocytosis rarely causes clinical problems in horses and is not likely to require specific antiplatelet therapy. The strong association of thrombocytosis with infectious/inflammatory disorders, however, should lead clinicians to suspect these types of conditions in horses with high platelet counts.

Thrombocytopenia in horses: 35 cases (1989-1994).

The records of 3,952 equine patients presenting to the Veterinary Teaching Hospital at North Carolina State University College of Veterinary Medicine were evaluated to determine risk factors associated with thrombocytopenia. Of 2,346 horses from which a CBC was obtained, 35 (1.49%) were thrombocytopenic (platelet count < 75,000/microL). A reference population of 189 horses with normal platelet counts (75,000 to 300,000/microL) was also studied. Standardbred horses were at increased risk for thrombocytopenia, but age and gender were not identified as significant risk factors. Horses with infectious or inflammatory diseases were at increased risk for thrombocytopenia. The potential association of clinical and clinicopathologic factors with thrombocytopenia were assessed by reviewing a series of multiple logistic regression models. Clinical and clinicopathologic variables significantly associated with thrombocytopenia in the final model included increased PCV, increased band neutrophil count, increased total WBC, and decreased plasma protein concentration. Increased mature neutrophil count was associated with normal platelet counts. Thrombocytopenic horses were significantly more likely to die to be euthanized than were horses with normal platelet counts.

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses.

OBJECTIVE: To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. ANIMALS: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). PROCEDURE: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. RESULTS: Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.

Nucleic acid amplification for rapid detection of Rhodococcus equi in equine blood and tracheal wash fluids.

OBJECTIVE: To evaluate the ability of nucleic acid amplification techniques to detect Rhodococcus equi in equine buffy coat, blood, and tracheal wash fluid and to differentiate between virulent and avirulent strains of the bacteria. SAMPLE POPULATION: Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses. PROCEDURE: Logarithmic dilutions of virulent and avirulent strains of R equi were added to equine buffy coat and tracheal wash fluid samples. The DNA was extracted and amplified by polymerase chain reaction (PCR), using primers specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi. RESULTS: PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasmid primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacteria. Virulent strains of R equi could be identified by PCR and differentiated from avirulent strains within 12 to 24 hours after sample collection, with as few as 10 to 100 organisms present. CONCLUSIONS: PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and can differentiate between virulent and avirulent strains of the organism. CLINICAL RELEVANCE: Because PCR can confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination.

Pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses.

OBJECTIVE: To determine an infusion rate of butorphanol tartrate in horses that would maintain therapeutic plasma drug concentrations while minimizing development of adverse behavioral and gastrointestinal tract effects. ANIMALS: 10 healthy adult horses. PROCEDURE: Plasma butorphanol concentrations were determined by use of high-performance liquid chromatography following administration of butorphanol by single IV injection (0.1 to 0.13 mg/kg of body weight) or continuous IV infusion (loading dose, 17.8 microg/kg; infusion dosage, 23.7 microg/kg/h for 24 hours). Pharmacokinetic variables were calculated, and changes in physical examination data, gastrointestinal tract transit time, and behavior were determined over time. RESULTS: A single IV injection of butorphanol was associated with adverse behavioral and gastrointestinal tract effects including ataxia, decreased borborygmi, and decreased defecation. Elimination half-life of butorphanol was brief (44.37 minutes). Adverse gastrointestinal tract effects were less apparent during continuous 24-hour infusion of butorphanol at a dosage that resulted in a mean plasma concentration of 29 ng/ml, compared with effects after a single IV injection. No adverse behavioral effects were observed during or after continuous infusion. CONCLUSIONS AND CLINICAL RELEVANCE: Continuous IV infusion of butorphanol for 24 hours maintained plasma butorphanol concentrations within a range associated with analgesia. Adverse behavioral and gastrointestinal tract effects were minimized during infusion, compared with a single injection of butorphanol. Continuous infusion of butorphanol may be a useful treatment to induce analgesia in horses.

Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets.

OBJECTIVE: To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. SAMPLE POPULATION: Blood samples obtained from 4 Thoroughbreds. PROCEDURE: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein-conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode. RESULTS: Number of platelets (mean +/- SEM) in samples containing LMWH (109.5+/-11.3 x 10(3) cells/microl) was significantly less than the number in samples containing sodium citrate (187.3+/-30.3 x 10(3) cells/microl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding. CONCLUSIONS AND CLINICAL RELEVANCE: A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation.

Effects of intravenous administration of formaldehyde on platelet and coagulation variables in healthy horses.

OBJECTIVES: To assess safety and determine effects of IV administration of formaldehyde on hemostatic variables in healthy horses. ANIMALS: 7 healthy adult horses. PROCEDURE: Clinical signs and results of CBC, serum biochemical analyses, and coagulation testing including template bleeding time (TBT) and activated clotting time (ACT) were compared in horses given a dose of 0.37% formaldehyde or lactated Ringer's solution (LRS), IV, in a 2-way crossover design. In a subsequent experiment, horses received an infusion of 0.74% formaldehyde or LRS. In another experiment, horses were treated with aspirin to impair platelet responses prior to infusion of formaldehyde or LRS. RESULTS: Significant differences were not detected in any variable measured between horses when given formaldehyde or any other treatment. Infusion of higher doses of formaldehyde resulted in adverse effects including muscle fasciculations, tachycardia, tachypnea, serous ocular and nasal discharge, agitation, and restlessness. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous infusion of formaldehyde at doses that do not induce adverse reactions did not have a detectable effect on measured hemostatic variables in healthy horses.

Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses.

OBJECTIVE: To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. ANIMALS: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. PROCEDURE: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. RESULTS: Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). CONCLUSION: Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. CLINICAL RELEVANCE: Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.