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.

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.

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.

Evaluation of binding of fibrinogen and annexin V to equine platelets in response to supramaximal treadmill exercise.

There is evidence that equine platelet reactivity is altered by strenuous exercise. Changes in platelet reactivity could impact haemostasis following exercise-induced injury and may play a role in the pathophysiology of exercise-induced pulmonary haemorrhage. Interpretation of results of previous studies is hindered by potential in vitro-induced changes in platelet activity through the choice of anticoagulant and the use of platelet inhibitors. The present study was undertaken to re-evaluate the effect of exercise on equine platelets using methodologies that minimise in vitro-induced changes in platelet activation. The percentage of platelet-neutrophil aggregates increased significantly (P = 0.01) from mean +/- s.e. 3.5 +/- 0.6% at rest to 7.2 +/- 13% during exercise. There were no significant changes in binding of anti-fibrinogen antibody or annexin V to platelets in response to exercise. An inability to detect increased binding of fibrinogen or annexin V may be a result of poor test sensitivity or low statistical power. Alternatively, activated platelets may be quickly removed from the circulation and miss detection. The significance of increased numbers of platelet-neutrophil aggregates in association with exercise is currently unknown and warrants further investigation.

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.

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.

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.

Comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of Rhodococcus equi pneumonia in foals.

Recently, a technique was described for amplification of Rhodococcus equi-specific chromosomal and vapA DNA from blood and tracheal wash fluids. It was hypothesized that this technique would be more sensitive than standard culture techniques or serology for diagnosis of R. equi pneumonia in foals. Tracheal wash fluid, nasal swabs, whole blood samples, and serum samples from 56 foals with pneumonia were analyzed. Final clinical diagnosis was determined by the attending clinician on the basis of final interpretation of all available information about each foal, including clinical presentation, diagnostic test results, response to therapy, and outcome. Clinical diagnosis was used as a final reference standard for calculation of sensitivity, specificity, and predictive values for PCR, serology using an agar gel immunodiffusion test, and tracheal wash fluid culture. PCR of tracheal wash fluid using primers that recognized the vapA virulence plasmid of R. equi had a diagnostic sensitivity of 100% and specificity of 90.6%. Sensitivity and specificity were 57.1 and 93.8%, respectively, for standard microbiologic culture of tracheal wash fluid and 62.5 and 75.9%, respectively, for serology. PCR of tracheal wash fluid is more sensitive and specific for diagnosis of R. equi pneumonia than are other available diagnostic tests.

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.

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.

Clinical application of a polymerase chain reaction assay in the diagnosis of pneumonia caused by Rhodococcus equi in a horse.

Diagnosis of pneumonia caused by Rhodococcus equi can be made more rapidly by use of a polymerase chain reaction (PCR) assay than by use of conventional bacteriologic culture techniques. Use of a PCR assay aids in the differentiation between virulent and avirulent strains of R equi, and the assay may be used to identify R equi in feces and soil of breeding farms.

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.

Secondary immunodeficiencies of horses.

FPT of immunoglobulin in foals is the commonest form of acquired immunodeficiency in horses. FPT predisposes foals to bacterial infections and septicemia and easily is preventable and treatable if breeding farms and veterinarians are attentive to optimum foaling management practices. Other forms of acquired immunodeficiencies are uncommon in horses, although immune function may be transiently suppressed by a wide variety of drugs, infections, or other conditions. As immunologic testing becomes more sophisticated and more readily available to equine practitioners, acquired immunodeficiencies are likely to be characterized more frequently in horses.

Hepatic abscesses in three horses.

Hepatic abscesses were diagnosed in 3 adult horses. Two were < 4 years old and had evidence of concurrent immune-mediated conditions, including aseptic arthritis, immune-mediated thrombocytopenia, and immune-mediated anemia. Predisposing factors for hepatic abscess formation in these horses included prior abdominal surgery, proximal duodenitis/jejunitis, inflammatory bowel disease, and a penetrating foreign body in the large colon. Serum hepatic enzyme activities were within or slightly greater then reference limits in all 3 horses. The most pronounced and consistent abnormalities on CBC and serum biochemical analyses were hyperproteinemia, hyperglobulinemia, and a decreased albumin-to-globulin concentration ratio. Hepatic ultrasonography identified hepatic abscesses in all 3 horses. A variety of bacteria were isolated from these abscesses, including Staphylococus aureus and Bacteroides fragilis. One horse developed septic tibiotarsal arthritis, presumably as a result of intermittent bacteremia. Despite aggressive medical treatment, all horses were euthanatized because of a worsening condition and poor prognosis.

Platelets from thrombocytopenic ponies acutely infected with equine infectious anemia virus are activated in vivo and hypofunctional.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.

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.

The effect of supramaximal exercise on equine platelet function.

When blood is collected into sodium citrate in the proportion of 9 parts blood:1 part sodium citrate, the concentration of plasma sodium citrate in the sample will depend on the packed cell volume (PCV) of the blood sample. This difference in plasma sodium citrate concentration secondary to alterations in PCV significantly affects human platelet aggregation responses. Since horses attain a high PCV in response to high-intensity exercise we investigated the effect of differences in sample plasma sodium citrate concentration on equine platelet aggregability. In addition, low molecular weight heparin (LMWH) was evaluated as an alternative anticoagulant for assessment of platelet aggregability during strenuous exercise in horses. Blood samples were collected pre-exercise and at fatigue after supramaximal treadmill exercise into either 3.8% sodium citrate (9 parts blood:1 part sodium citrate) or 20 u LMWH/ml of blood. Platelet aggregation responses to 1.25 mumol/l adenosine diphosphate (ADP) were measured via optical aggregometry. For samples collected into sodium citrate, aggregability was significantly less than pre-exercise values in samples collected at fatigue and in pre-exercise samples in which sodium citrate concentrations were adjusted to equal those in fatigue samples. However, samples collected into LMWH showed significantly increased platelet aggregability in samples collected at fatigue when compared to pre-exercise samples. In conclusion, higher plasma sodium citrate concentration had a marked inhibitory effect on equine platelet aggregation responses. Low molecular weight heparin was a good alternative anticoagulant for assessment of equine platelet function and results indicate that equine platelet aggregability was enhanced in response to supramaximal exercise.