Estimation of the probability for exceeding thresholds of urine specific gravity and plasma concentration of furosemide at various intervals after intravenous administration of furosemide in horses

OBJECTIVE: To estimate the probability of concurrently exceeding thresholds for plasma concentration of furosemide and urine specific gravity after IV administration of furosemide in horses. ANIMALS: 12 mature healthy Thoroughbred (n = 6) or Quarter Horse (6) mares. PROCEDURE: Venous blood was collected from each horse prior to and 0.25, 0.5, 0.75, 1, 2, 3, 4, 4.5, 5, and 6 hours after IV administration of 250 mg (first experiment) or 500 mg (second experiment) of furosemide. Urine was collected hourly between 1 and 6 hours after administration of furosemide at both doses. Concentrations of furosemide were determined by use of an ELISA. Concentration of furosemide and urine specific gravity was modeled as a function of time, accounting for inter- and intrahorse variabilities. On the basis of pharmacokinetic and specific gravity data, the probability of exceeding a concentration of 100 ng of furosemide/ml as a function of time was determined, using a semiparametric smooth functional averaging method. A bootstrap approach was used to assess the inherent variation in this estimated probability. RESULTS: The estimated probability of exceeding the threshold of 100 ng of furosemide/ml and urine specific gravity < 1.012 was approximately 0% between 4.0 and 5.5 hours after IV administration of 250 mg of furosemide/horse, and ranged from 0 to 1% between 4 and 5.5 hours after IV administration of 500 mg of furosemide/horse. The probability of a horse being falsely identified as in violation of regulatory concentrations was inversely associated with time. CONCLUSIONS AND CLINICAL RELEVANCE: Coupling plasma furosemide concentration with urine specific gravity testing will greatly reduce the chance that some horses are misclassified as being in violation of regulatory concentrations.     

Estimation of the probability for exceeding a threshold concentration of furosemide at various intervals after intravenous administration in horses

OBJECTIVE: To estimate the probability for exceeding a threshold concentration of furosemide commonly used for regulatory purposes after IV administration of furosemide in horses. ANIMALS: 12 mature healthy horses (6 Thoroughbreds and 6 Quarter Horses). PROCEDURE: Venous blood was collected from each horse prior to and 0.25, 0.5, 0.75, 1, 2, 3, 4, 4.5, 5, and 6 hours after administration of 250 or 500 mg of furosemide. Concentrations of furosemide were determined, using an ELISA. Concentration of furosemide was modeled as a function of time, accounting for inter- and intrahorse variabilities. On the basis of pharmacokinetic data, the probability for exceeding a concentration of 100 ng/ml as a function of time was determined, using a semiparametric smooth functional averaging method. A bootstrap approach was used to assess inherent variation in this estimated probability. RESULTS: The estimated probability of exceeding the threshold of 100 ng of furosemide/ml ranged from 11.6% at 4 hours to 2.2% at 5.5 hours after IV administration of 250 mg of furosemide/horse and 34.2% at 4 hours to 12.3% at 5.5 hours after IV administration of 500 mg of furosemide/horse. The probability of a horse being falsely identified in violation of regulatory concentrations was inversely associated with time and positively associated with dose. CONCLUSIONS AND CLINICAL RELEVANCE: Interhorse variability with respect to pharmacokinetics of furosemide will result in misclassification of some horses as being in violation of regulatory concentrations.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia antibody to purified P26 viral protein

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-gel immunodiffusion.     

Equine Infectious Anemia: Active Surveillance in Central Italy 2007-2009

Equine infectious anemia (EIA) is an infectious and potentially fatal viral disease of equids. EIA virus is usually transmitted from horse to horse by large biting insects, such as horseflies. The aim of this study was to evaluate the results of a national surveillance plan from 2007 to 2009 and evaluate the potential risk factors of EIA in horse populations in central Italy. In 2007, 18 of 6,773; in 2008, 30 of 7,940; and in 2009, 21 of 11,666 equines tested were seropositive for EIA. No statistical association was found between location or sex and the diagnosis of EIA. The seroprevalence rate (2007-2008-2009) was higher among older equids (older than 6 years) than among young (3 months to 5 years old) (P < .05). Likewise, the seroprevalence rate (2007-2008-2009) was higher among mules than among other horses (P < .05). Until 2007, the national equine register did not exist in Italy; therefore, it was difficult to measure the percentage of untested horses that presented a real but unquantified risk for continued EIA virus transmission. By introducing new laws governing the control and conducting active surveillance for EIA, it has been possible, in Italy, to develop a firm foundation of knowledge concerning the persistence and transmission of EIA and the risk factors and to better control the spread of this infection in horses.     

Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection

The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected with 10(6)TCID50 of the Wyoming strain of EIA virus and in 50% of the HLC from febrile acute or subacute horses. The direct fluorescent antibody testing of HLC derived from chronic and inapparent carrier horses did not detect cell-associated EIA virus. The pony inoculation test proved to be the most reliable and accurate method for detecting infective EIA virus in horses in various stages of EIA infection and accurately correlated with the AGID test.     

Evaluation of hemorheologic variables as implications for exercise-induced pulmonary hemorrhage in racing thoroughbreds.

Hematologic and rheologic changes were examined in 49 Thoroughbreds before and after competitive racing. Mean postrace values for RBC count, hemoglobin concentration, and PCV increased by 58 to 61%, whereas blood viscosity increased 2 to 3 times. Postrace echinocyte numbers were 162% greater than prerace values. Smaller, but statistically significant, changes were found for mean corpuscular hemoglobin concentration, red cell distribution width, plasma total protein concentration, total WBC count, neutrophil count, and lymphocyte count. Variables measured did not predict whether a horse was a bleeder not treated with furosemide, a bleeder treated with furosemide, or a nonbleeder.     

Direct enzyme immunoassay of progesterone in bovine plasma

The present study was undertaken to develop a novel, practical and simple procedure for enzyme immunoassay (EIA) of plasma progesterone in cows. Diluted plasma was heated for 70°C for 30 min and applied directly to wells of a microtitre plate without extraction. Then plasma was incubated with antiprogesterone antibody and horseradish peroxidase‐labeled progesterone. The sensitivity of the assay was estimated as 4.4 pg/mL (0.11 pg/well). The intra‐assay and interassay coefficients of variation were 5.7–19.1% and 6.6–19.3%, respectively. When 0.3, 1 and 3 ng of progesterone were added to plasma, the recovery rates ranged between 79.9 and 108.4%. Only 4 h were needed to complete an assay to measure progesterone concentration. To apply the present direct EIA, progesterone concentration in plasma was assayed in crossbred cows used for the embryo transfer program. During insertion of controlled‐internal drug release (CIDR), progesterone concentrations were kept at a high level, although the removal of CIDR with treatment of dinoprost trometamine reduced progesterone concentration drastically. These results suggest that the present direct EIA is a practical and suitable method for measuring the plasma concentration of progesterone.     

Network simulation modeling of equine infectious anemia in the non-racehorse population in Japan

An equine infectious anemia (EIA) transmission model was developed by constructing a network structure of horse movement patterns in a non-racehorse population. This model was then used to evaluate the effectiveness and efficiency of several EIA surveillance strategies. Because EIA had not been detected in Japan since 1993, it was appropriate to review the current surveillance strategy, which aims to eradicate EIA by intensive testing, and to consider alternative strategies suitable for the current EIA status in Japan.The non-racehorse population was divided into four sectors based on horse usage: the equestrian sector, private owner sector, exhibition sector, and fattening sector. To evaluate the risk of disease spread within and between sectors accompanied by horse movements, a stochastic individual-based network model was developed based on a previous survey of horse movement patterns. Surveillance parameters such as targeting sectors and frequency of testing were added into the model to compare surveillance strategies.The disease spread heterogeneously among sectors. Infection occurred mainly in the equestrian sector; the infection was less disseminated in other sectors. Therefore, we considered that the equestrian sector posed a higher risk of disease dissemination within and between sectors through horse movements. However, surveillance strategies targeting only the equestrian sector were not effective enough for early detection of the disease. Alternatively, targeting horses that moved permanently and those in the private owner sector in addition to the equestrian sector is recommended to achieve effectiveness equivalent to that of the current surveillance. In terms of surveillance efficacy, by increasing the testing interval (once yearly to once every 3 years), this testing scheme could reduce the number of tested horses to 44% of the current surveillance, while maintaining almost equivalent effectiveness. Intensive strategies targeting high-risk populations are considered to enhance effectiveness and efficiency of surveillance. The approach in this study may be helpful in the decision-making process that is involved in setting up strategies for risk-based surveillance.     

Distribution of equine infectious anemia in horses in the north of Minas Gerais State, Brazil.

The paper examines the prevalence of equine infectious anemia (EIA) in horse populations in the northern part (comprising 89 cities) of Minas Gerais State, Brazil, from January 2002 to December 2004. Data on 8,981 agar gel immunodiffusion test results from the region were used as input for a statistical and autoregressive analysis model to construct a city-level map of the distribution of EIA prevalence. The following EIA prevalence (P) levels were found: 49 cities with 0 < P < or = 0.5%, 26 with 0.5% < P < or = 1.5%, 10 with 1.5% < P < or = 5%, and 4 with 5% < P < or = 25%.     

Simplified method to measure glucocorticoid metabolites in faeces of horses

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3α,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3α,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.     

The development of a sensitive enzyme immunoassay for the determination of estrone and estradiol-17beta in bovine blood plasma based on the same homologous combination with antiserum and steroid-enzyme conjugate

The development of a sensitive enzymeimmunoassay (EIA) for the determination of estrone (E1) and estradiol-17beta (E2 beta) in bovine plasma is described. The assay is a homologous double-antibody EIA with E2beta 17hemisuccinate (HS) as hapten for the immunoreactive reagent. The antiserum was raised against E2beta 17HS bovine serum albumin conjugate in the rabbit, and E2beta 17HS-horseradish peroxidase was used as steroid-enzyme conjugate. Each estrogen EIA was distinguished only by using the each working standard and sample for the EIA. Bovine plasma E1 and E2beta were extracted and purified before EIA. The antiserum was used at 1:1,750,000 dilutions for EIA. Estrone and E2beta showed high cross-reactivity with the antiserum (E1: 350.7%, E2beta:100%). The sensitivities were <0.03 pg/well for E1 and <0.12 pg/well for E2beta. Recovery rates of E1 and E2beta added to bovine blood plasma were 94.5% and 93.9%, respectively. The precision for EIA of estrogens was below 9.7%. The profiles of either estrogen as determined by EIA corresponded closely well with follicle dynamics in the cow during the estrous cycles and with placental function in pregnant animals. In conclusion, our new EIA can be applied with sufficient sensitivities, recovery and precision for the routine analysis of E1 and E2beta concentrations in bovine plasma.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Serological diagnosis of influenza A infections in the horse by enzyme immunoassay. Comparison with the complement fixation test

An enzyme immunoassay (EIA) using horseradish peroxidase and a type-specific antigen is described for the detection and quantitation of anti-influenza antibodies in the horse. Compared with the complement fixation (CF) test (using the same antigen), EIA proved to be superior with respect to sensitivity and reliability. The internal variation of EIA was low and thus small titres in EIA can be considered of diagnostic significance. However, no strict correlation with CF was observed. The use of an immunoconjugate against equine IgM in parallel with IgG would certainly improve the sensitivity of the test, especially in early stages of infection.     

A western blot test for the serological diagnosis of equine infectious anemia

After electrophoretic separation in SDS-PAGE structural proteins of the virus of Equine Infectious Anemia (EIA) were easily blotted by the semi-dry-blotting method onto nitrocellulose filters. Strips of these filters were used for antibody demonstration, and positive reactions thereof were intensified by a biotin-avidin-peroxidase system. Sensitivity of this system was so high as to allow readable interpretation of bands up to the dilution of 1:6,400 of a strongly positive serum. Frequently this procedure allowed to make a firm diagnostic Western-Blot diagnosis on far weaker equine sera. Interpretation of results proved, however, difficult with sera, which formed one single band only. This observation, although of weak grade, had to be made in some 5% of sera stemming from horses with a certainly negative history of EIA. Consequently, we conclude, that a policy followed in the serodiagnostic Western-Blot of human AIDS should also be adopted for the interpretation of the EIA Western-Blot, namely to declare as positive merely horse sera which evidence more than one single band, whereof at least one band should represent a viral glycoprotein.     

Furosemide-induced changes in plasma and blood volume of horses

The effect of furosemide administration (1 mg/kg body weight, i.v.) on plasma and blood volumes in 6 intact and 4 splenectomized horses was measured using Evans blue dye dilution, hematocrit, and hemoglobin and plasma total solids concentrations. Body weight decreased by 33.6 +/- 3.3 and 33.7 +/- 0.8 g/kg 4 h after furosemide administration to intact and splenectomized mares, respectively. Plasma volume, estimated by Evans blue dye dilution, was reduced by 8.3 +/- 3.3% (mean +/- SE) 4 h after furosemide administration. The reduction in plasma volume was first detectable 5-10 min after furosemide administration and was greatest 15-30 min (13.0 +/- 0.8%) after dosing. This study demonstrates that furosemide produces significant and rapid reductions in plasma volume in horses. These decreases in plasma volume only partially resolve 4 h after furosemide administration.     

An enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of equine infectious anemia in Brazil

An enzyme-linked immunosorbent assay (ELISA) test was developed for the detection of specific antibodies against the unique infectious anemia (EIA) virus in equine sera. The ELISA test was faster and more sensitive when compared with the classic test of agar gel immunodiffusion (AGID). A total of 200 sera were tested: 100 from negative horses and 100 from positive horses by AGID. The ELISA test showed 92 horse sera negative and 100 horse sera positive by AGID with values of optical density (OD) less than 0.139 and higher than 0.139, respectively. Eight horse sera were negative by AGID and higher than 0.139 by ELISA. Six of these became AGID positive also when re-tested 30 days later, and two were of the horses that showed clinical signs of EIA and died before re-testing.     

Review of furosemide in horse racing: its effects and regulation

Furosemide has been used empirically and has been legally approved for many years by the US racing industry for the control of exercise-induced pulmonary haemorrhage (EIPH) or bleeding. Its use in horses for this purpose is highly controversial and has been criticized by organizations outside and inside of the racing industry. This review concentrates on its renal and extra-renal actions and the possible relationship of these actions to the modification of EIPH and changes in performance of horses. The existing literature references suggest that furosemide has the potential of increasing performance in horses without significantly changing the bleeding status. The pulmonary capillary transmural pressure in the exercising horse is estimated to be over 100 mmHg. The pressure reduction produced by the administration of furosemide is not of sufficient magnitude to reduce transmural pressures within the capillaries to a level where pressures resulting in rupture of the capillaries, and thus haemorrhage, would be completely prevented. This is substantiated by clinical observations that the administration of furosemide to horses with EIPH may reduce haemorrhage but does not completely stop it. The unanswered question is whether the improvement of racing times which have been shown in a number of studies are due to the reduction in bleeding or to other actions of furosemide. This review also discusses the difficulties encountered in furosemide regulation, in view of its diuretic actions and potential for the reduction in the ability of forensic laboratories to detect drugs and medications administered to a horse within days or hours before a race. Interactions between nonsteroidal anti-inflammatory drugs (NSAIDs) and furosemide have also been examined, and the results suggest that the effects of prior administration of NSAID may partially mitigate the renal and extra-renal effects which may contribute to the effects of furosemide on EIPH.     

Effect of dose of furosemide on plasma tCO2 changes in standardbred horses

Nine Standardbred horses of similar athletic fitness (six mares, three geldings), ranging from 4 to 11 years of age, were used to determine the effects of 0, 250, or 500 mg intravenously administered furosemide on plasma tCO₂ changes over time. All horses were either currently racing or in advanced stages of race training before entering a qualifying race. Horses were randomly allotted to one of the three treatment levels of furosemide during 3 consecutive weeks. Jugular venous samples were obtained from horses at rest in box stalls before and hourly for 6 hours after administration of furosemide. Body weights of horses ranged from 356 to 456 kg, and the mean was 417 kg. Thus, the dose of furosemide received by each horse ranged from 0.55 to 0.70 mg/kg body weight for the 250-mg injections and from 1.1 to 1.4 mg/kg body weight for the 500-mg injections. Furosemide caused metabolic alkalosis in the horses. Least square means (±SEM) were determined and horses had adjusted plasma tCO₂ of 32.2, 33.9, and 34.7 ± 0.41 for the 0-, 5-, and 10-mL doses of furosemide, respectively. The type 3 tests of hypotheses found that there was a difference (P < .0001) across time, a difference (P = .0016) according to furosemide dose, and a difference (P < .0001) according to treatment × hour. There was no difference (P > .05) according to week or treatment × week. These data suggest that either 250 or 500 mg furosemide given to Standardbred race horses induces statistically similar metabolic alkalosis.     

The influence of furosemide on plasma elimination and urinary excretion of drugs in standardbred horses

A study of the effects of intravenous administration of either 150 mg or 250 mg of furosemide to standardbred mares pre-treated with other drugs was undertaken to determine whether a unique pattern of drug elimination into urine and from plasma for each compound occurred. Furosemide significantly reduced the plasma concentrations of codeine compared to control 2-6 h after furosemide administration. In contrast, the plasma concentrations of theophylline, phenylbutazone, pentazocine, guaifenesin and flunixin were not markedly altered by furosemide. In the case of acepromazine, clenbuterol and fentanyl, the data generated were insufficient to state with certainty whether or not furosemide affected the plasma concentrations of these three drugs. A significant reduction was noted in the urinary concentrations of guaifenesin, acepromazine, clenbuterol, phenylbutazone, flunixin, fentanyl and pentazocine within 1-4 h of furosemide administration. The urinary concentrations of theophylline remained reduced as long as 8 h after furosemide injection. Furosemide administration to horses pre-treated with codeine resulted in depression of urinary morphine concentrations 2-4 h and 9-12 h after furosemide injection. A lower furosemide dose (150 mg) produced changes in drug urinary excretion and plasma elimination equivalent to the higher dose (250 mg). It is evident that furosemide affects the urinary and plasma concentrations of other co-administered drugs but not in a predictable fashion, which limits the extrapolation of these results to as yet untested drugs.     

Effects of extrathoracic airway obstruction on intrathoracic pressure and pulmonary artery pressure in exercising horses

OBJECTIVE: To determine whether dorsal displacement of the soft palate (DDSP) results in pulmonary artery hypertension and leads to increases in transmural pulmonary artery pressure (TPAP); to determine whether pulmonary hypertension can be prevented by prior administration of furosemide; and to determine whether tracheostomy reduces pulmonary hypertension. ANIMALS: 7 healthy horses. PROCEDURE: Horses were subjected to 3 conditions (control conditions, conditions after induction of DDSP, and conditions after tracheostomy). Horses were evaluated during exercise after being given saline (0.9% NaCl) solution or furosemide. RESULTS: Controlling for drug, horse, and speed of treadmill, DDSP-induced increase in intrathoracic pressure was associated with a significant increase in minimum (36 mm Hg), mean (82 mm Hg), and maximum (141 mm Hg) pulmonary artery pressure, compared with values for control horses (30, 75, and 132 mm Hg, respectively). Increases in pulmonary artery pressure did not induce concomitant increases in TPAP. Tracheostomy led to a significant reduction of minimum (53 mm Hg), and mean (79 mm Hg) TPAP pressure, compared with values for control horses (56 and 83 mm Hg, respectively). When adjusted for horse, speed of treadmill, and type of obstruction, all aspects of the pulmonary artery and TPAP curves were significantly decreased after administration of furosemide, compared with those for horses given saline (0.9% NaCl) solution. CONCLUSIONS: DDSP was associated with increases in pulmonary artery pressure but not with increases in TPAP. CLINICAL RELEVANCE: Expiratory obstructions such as DDSP are likely to result in pulmonary hypertension during strenuous exercise, but may not have a role in the pathogenesis of exercise-induced pulmonary hemorrhage.