Abstract
Six adult horses were administered sodium monensin, 1.0–1.5 mg/kg, via gastric gavage. Anorexia and/or diarrhea occurred within 24 hr after monensin administration in all 6 horses. Cardiac disease and dysfunction were evaluated by both elevations in heart rate, echocardiography, and an increase in serum concentrations of cardiac troponin I (cTnI), occurred in 4 horses. The development and severity of cardiac disease was likely affected by the monensin dose, vehicle (water or corn oil) mixed with monensin, and/or whether the monensin was administered to fed or fasted horses. Initial increases in cTnI concentrations occurred between 24 and 72 hr after monensin administration. The 2 horses with the highest cTnI concentrations died or were euthanized within 5 days after monensin administration and had severe cardiac disease. One horse had increased cTnI concentrations from day 2 to day 16, but no apparent change in ventricular contractile function was evident on echocardiography. The fourth diseased horse did not return to cTnI reference intervals until day 27 after monensin administration, and the ventricular function was still abnormal just before euthanasia 9 months later. Cardiac troponin I measurements could be useful in managing farm outbreaks of accidental monensin feeding by the early identification of horses with cardiac disease.
Introduction
Monensin is an ionophore antibiotic used in the dairy and beef industries to decrease the incidence of ketosis, improve feed efficiency (growth promotion), and help control ruminal acidosis, and, in the poultry industry, it is used as a coccidiostatic agent. 8,12,16,17 In the horse, however, it is highly toxic, and naturally occurring outbreaks of monensin toxicosis are well documented. 4,6,11,26 The proposed median lethal dose (LD50) of monensin for horses is reported to be 2–3 mg/kg based on a single dose study in nonfasted horses. 22
The toxic effect of monensin is caused by abnormal movement of sodium, potassium, and calcium ions into muscle cells, with myocardial necrosis believed to be the result of myocyte osmolar imbalance and mitochondrial dysfunction. 23,28 Cardiac disease and heart failure are the most common and serious consequences of monensin ingestion in horses. 4,6,11 Specific biochemical detection of cardiac disease such as caused by monensin poisoning in horses has previously relied upon measurement of creatine kinase myocardial band (CKMB), but few veterinary laboratories assay CKMB, and the specificity of this test for detecting cardiac disease is unknown in horses and is poor in other species. 1,31 Cardiac troponin I (cTnI) is a highly sensitive and specific biomarker of myocardial injury in human medicine. 18 In mice, rats, dogs, and humans, cTnI is considered the most effective biomarker for myocardial injury in cardio-toxicity. 25 Evaluation of cTnI in the horse is limited, except for reports that documented cTnI values in horses in training or at rest and for isolated case reports of acute ventricular tachycardia. 3,9,13,19,27,30 Recently, the present authors reported that cTnI, measured by either the access immunoassay a or the point-of-care analyzer i-STAT 1, b is an accurate method of detecting cardiac disease in the horse (MS Kraus, SA Jesty, AR Gelzer, et al.: 2007, Characterization of cardiac troponin I as an indicator of cardiac damage in horses utilizing an i-STAT-1. Abstract in proceedings of the Annual American College of Veterinary Internal Medicine Forum. J Vet Med Intern Med 21:604–605). The purpose of the current study was to describe the clinical signs and cTnI changes in concentration over time after administration of monensin at different doses, by using doses below the reported LD50. The monensin was mixed either in water or corn oil and was administered to either fasted or fed horses.
Materials and methods
Six healthy adult horses, age 5–12 years, were used in the present study. The horses were housed in individual 4.27 m × 4.27 m stalls and fed free-choice grass hay, in addition to 1 kg of an equine sweet feed twice daily. At baseline, all 6 horses had cardiac evaluations performed, which included a clinical examination, a normal resting heart rate (36–44 bpm), and an echocardiography at rest and immediately after treadmill exercise. Treadmill exercise was an initial 2 min at 4 m/sec. At 2 min, the treadmill was inclined to a 6.3% slope. At 4 min, the treadmill was accelerated to 6 m/sec and kept at that speed for 1 min. At each subsequent minute, the treadmill was accelerated by 1 m/sec until each horse began to have difficulty in maintaining its position near the front of the treadmill. Fractional shortening of the left ventricle, the percentage difference between M-mode measurements of the left ventricular lumen diameter at end diastole and end systole, was recorded for each horse at rest and immediately after the treadmill exercise. Horses were administered sodium monensin c via a nasogastric tube as a single treatment, with dosages varying between 1–1.5 mg/kg. The dosages of 1–1.5 mg/kg were chosen because they are 50% less than the published LD50 (2–3 mg/kg) for the horse. 22 Monensin was mixed in either 1 liter of water (1 horse) or 227 g of corn oil d (5 horses) for gastric gavage and administered to 2 horses on feed and to 4 horses after withholding feed for 6 hr (see Table 1). Blood for cTnI serum measurements was collected just before and at 1 and 3 hr after baseline treadmill examination and at the following times after monensin administration: 0, 0.5, 1, 3, 6, 12, 24, 36, 48, 72, and 96 hr. Further daily sampling was performed in horses that had elevated heart rates at 96 hr after receiving monensin. Blood was immediately centrifuged after collection, and serum was frozen at −70°C for storage before cTnI measurement. The frozen samples were analyzed within 1 month after completion of the study. The cTnI concentrations were determined by a commercial immunoassay a that uses 2 mouse monoclonal antibodies directed against human cTnI, with a lower limit of detection of 0.01 ng/ml; cTnI values for normal horses were not available from the testing laboratory, but, in a previous study of normal adult horses in training, cTnI tested by another 2-site immunoassay was <0.11 ng/ml, 27 consistent with values obtained with the assay used in the current study in horses before monensin dosing. The horse cTnI amino acid sequence is nearly identical to human cTnI, and differences are outside the epitope region used in human commercial analyzers. 29 After administration of monensin, horses were monitored hourly for any abnormal clinical signs (e.g., colic, diarrhea, anorexia, trembling, and ataxia), and heart rates were determined at least every 6 hr. By study design, any horse that developed signs of fulminant heart failure (i.e., coughing, jugular pulses, edema, and dyspnea) was euthanized with a concentrated pentobarbital solution, e and a complete necropsy was performed on each animal. Postmortem examination was performed within 6 hr of euthanasia, and tissue samples were fixed in 10% neutral buffered formalin. Formalin-fixed samples were further processed for sectioning, embedded in paraffin wax, sectioned at 4 μm, and stained with hematoxylin and eosin. Horses with tachycardia that persisted for 1 week after monensin administration were monitored for at least 4 additional weeks by a continuous electrocardiogram (ECG) loop recorder f in an effort to capture any tachyarrhythmias that might occur. The device was implanted subcutaneously over the left heart apex with the animal under local anesthesia of the area with a subcutaneous 2% lidocaine infusion. The ECG device was programmed to record heart rates greater than 110 bpm. The device was interrogated weekly for any arrhythmias that might have occurred. Horses that did not develop tachycardia after monensin administration were exercised on the treadmill at 10 m/sec for 2 min on day 4. After the second treadmill test, serum samples were collected again for cTnI measurements at 1, 3, 6, 12, and 24 hr after exercise. The study protocol and care of the horses were approved by the Cornell University College of Veterinary Medicine Animal Care and Use Committee.
Results
All 6 horses were healthy at the start of the trial and had a mean fractional shortening percentage of 36.4% (range: 32.7–45.7%) at rest and 51.8% (range: 43–63.35%) immediately after the treadmill exercise.
Dose of sodium monensin, vehicle mixture, and feed status of 6 horses administered sodium monensin via a nasogastric tube and the outcome.*
cTnI = cardiac troponin I; N = normal; S = survived; I = increased; D = died; E = euthanized.
No disease.
Died or euthanized because of cardiac failure.
Cardiac disease without cardiac failure.
All hay and grain withheld for 6 hr before monensin administration. Feed was offered 1 hr after administration.
Serum cardiac troponin I (cTnI) concentrations for each horse 0–12 hr after monensin administration.
Serum cardiac troponin I (cTnI) concentrations for each horse 1–27 days after monensin administration. Horse nos. 1 and 3 that had no elevation in cTnI concentration do not show up on the Fig. 2 graph because of the scale needed to reflect the magnitude of increase in cTnI in diseased horses.
Within 6 hr after administration of the monensin, all 6 horses developed complete or partial anorexia. The anorexia resolved in 4 of the horses (nos. 1, 2, 5, and 6) within 48 hr. Horse no. 3 regained a normal appetite on day 4, and horse no. 4 remained anorexic until euthanasia on day 5. Five of the 6 horses developed diarrhea between 12 and 24 hr after the monensin administration. The diarrhea was not severe and resolved within 12–48 hr in all horses, except for horse no. 4, which had severe diarrhea from day 2 to day 5, when signs of heart failure were noted and euthanasia was performed. Clostridium difficile toxin was found in the stool of that horse. Horse no. 2, who received the highest dose of monensin (1.5 mg/kg) of any horse in the study, had grade 2 of 5 ataxia on day 2. Four horses had elevations in heart rate (an increase of 8–20 bpm) within 24 hr of monensin administration. Horse nos. 1 and 3 did not show any elevation in heart rate during the study and did not have increased cTnI concentrations during the 96 hr post-monensin observation period, nor after the post-monensin treadmill exercise (the highest cTnI at any of the collection times after the treadmill examination was 0.08 ng/ml). The highest heart rates in the 4 horses with tachycardia were 66 bpm (horse no. 2 at 72 hr), 60 bpm (horse no. 4 at 96 hr), 70 bpm (horse no. 5 at 48 hr), and 64 bpm (horse no. 6 at 72 hr).
Serum cTnI concentrations for all 6 horses are reported in Figure 1 (the first 12 hr after monensin administration with the X-axis in hours) and Figure 2 (beginning at 24 hr after monensin with the X-axis in days). Only horses that developed tachycardia had increased cTnI concentrations. Horse no. 2 died acutely between 83 and 84 hr after monensin administration, although appetite, attitude, and manure consistency had returned to normal, and there was no clinical evidence of cardiac failure preceding the death. Horse no. 5 was monitored for an additional 35 days. The horse had no detected tachyarrhythmias, and ventricular contractility decreased from 45% (before monensin) to 33% at 48 hr after monensin but was 41% at day 4. The horse appeared healthy 1 year later. Horse no. 6 was monitored for 7 months, during which time appetite, attitude, and body weight remained normal; no arrhythmias were detected by the continuous ECG loop recorder, and there were no outward clinical signs of heart failure. The horse was euthanized for other reasons 7 months after monensin administration, and, at that time, fractional shortening had only improved from a low of 9% on day 15 after monensin to 23%.
Gross findings after necropsy of horse nos. 2, 4, and 6 were primarily limited to the heart. In 2 of the horses, there was a mild pericardial effusion, and, in all 3 horses, there were numerous, variably sized foci of pallor and hemorrhage streaked throughout the myocardium. Histologic examination in all 3 horses revealed that the myocardium was replaced by numerous dissecting bands of fibrosis that entrapped degenerate and necrotic cardiac myofibers. The myofibers were in variable stages of degeneration and necrosis characterized by loss of cross striations, sarcoplasmic floccular vacuolization and fragmentation, sarcoplasmic hypereosinophilia, sarcoplasmic mineralization, and myofiber atrophy (Fig. 3). The myocardial interstitium was expanded by small to moderate numbers of lymphocytes, macrophages, and rare neutrophils. In some areas, there was marked interstitial edema. One of the 3 horses (horse no. 2) necropsied had similar changes present within skeletal muscle. All horses had varying degrees of chronic passive congestion in the liver typified by centrilobular hepatocyte atrophy and accumulation of hemo-siderin-laden macrophages. Other histologic findings believed to be unrelated to cardiac dysfunction included chronic parasite tracts in the liver (1 horse), adrenocortical adenoma (1 horse), nephritis (2 horses), and fibrinonecrotic colitis in the horse (no. 4) with the C. difficile infection.
Discussion
The present experimental study, although based on a small number of horses, demonstrated that the LD50 of monensin may vary considerably, depending upon a fed or fasted state, and by vehicle of administration (corn oil vs. water). The 2 fasted horses that received the highest doses of monensin (1.2 and 1.5 mg/kg) mixed in corn oil died or were euthanized with severe cardiac disease. These dosages were 40% and 20% less, respectively, than the minimal reported LD50 (2.0–3.0 mg/kg) for orally administered sodium monensin in horses. 22 In a previous study, only 1 of 5 horses that received 2.0 mg/kg died, but monensin was mixed in mineral oil for gastric gavage, and the horses were not held off feed before administration. Both factors may have caused differences in bioavailability in comparison with the present study. Intestinal absorption of monensin, a lipid soluble antibiotic, was likely greater in the current study, because a highly absorbed oil, corn oil, was used in contrast to poorly absorbed mineral oil (used as the vehicle in the previous study). 22 In rats, absorption of dichloro-diphenyl-trichloroethane (DDT), another highly lipid-soluble product, was estimated to be 2 to 3 times greater when the DDT was mixed with vegetable oil in comparison with a mineral-oil mixture, which was similar to DDT mixed in water. 20 In addition, withholding feed is known to increase absorption of some antibiotics in the horse. 7,21 From an experimental point of view, the current study may be important for a researcher who desires to cause nonlethal cardiac disease in the horse with monensin. Based upon the limited number of horses, the authors recommend that for future studies intended to produce cardiac disease without failure, would be to administer no more than 1.0 mg/kg monensin mixed in corn oil and administered after a 6-hr fast.
Myocardium; horse. Cardiac myofibers are variably degenerate with loss of sarcoplasmic detail and karyorrhexis (arrow). The interstitium is mildly edematous and expanded by small numbers of lymphocytes and macrophages (asterisk). Hematoxylin and eosin. Bar = 200 μm.
The clinical signs of monensin toxicity observed in the current study were similar to those reported in naturally occurring outbreaks and in 4 reported experimental studies. 4–6,11,22,23,26 Anorexia was the first and most consistent clinical sign observed in the horses in the current study. Anorexia has not been described as an initial clinical finding in some natural outbreaks of monensin toxicity, but this might be because, in some reports, the horses were group fed and anorexia might not have been noticed. In a subacute feeding experiment, all horses that received 125 or 279 ppm of monensin mixed in concentrate developed anorexia within 24 hr after monensin was given. 22 The 2 horses in that study that received the highest dose (279 ppm in feed) had progressive ataxia within 12–36 hr after monensin ingestion. Neurologic signs were the predominant clinical signs associated with monensin poisoning in 1 outbreak in which draft horses were accidentally given feed that contained 516 ppm. 26 Ataxia was also noticeable within 35 min in a pony experimentally given monensin intravenously, but the pony was normal within 60 min. 10 Ataxia was observed in only 1 horse in the current study. In addition, microscopic lesions were not observed in the spinal cord of the draft horses involved in the naturally occurring outbreak 26 or in the horse with ataxia (no. 2) in the current study. The pathophysiology of the neurologic disease is unknown but appears to be a relatively early finding in some horses that receive very high doses of monensin and may precede clinical, microscopic, and biochemical evidence of cardiac disease.
Signs of abdominal discomfort were noted in 2 horses in the present study, and diarrhea occurred in 5 horses. Abdominal pain was previously reported in horses that were poisoned with monensin. 26 Diarrhea is occasionally reported in horses and is a common finding in cattle with monensin toxicosis. 16 Abdominal pain and diarrhea may have been caused by changes in intestinal flora caused by the antimicrobial properties of the ionophore. In fact, horse no. 4, the only one with severe diarrhea, had C. difficile toxins in the feces and was found to have fibrinonecrotizing colitis on necropsy that was consistent with clostridial enteritis. Clostridium difficile diarrhea in adult horses is usually associated with oral administration of antibiotics. 14
Cardiac troponins (both cTnI and cTnT) are highly sensitive and specific biomarker of myocardial injury in humans. 25 Cardiac troponin I is the preferred biomarker for cardiotoxicity in laboratory animals. 25 Cardiac troponin I appears to be more sensitive than cTnT or CKMB in humans in the early phase of myocardial infarction. 18 Cardiac troponin is highly stable for up to 12 months when samples are stored at −70°C. 2 In the horse, properly frozen plasma or serum samples can be assayed by either the immunoassay method used in the current study or a point of care analyzer (Kraus MS et al.: 2007, Characterization of cardiac troponin I). Cardiac troponin was measured in 5 horses involved in a natural outbreak of monensin poisoning, and 2 of the 5 horses had mild increases in cTnI, above the reference interval. This outbreak was described as an atypical monensin toxicosis, with most of the affected horses having neurologic signs without biochemical or postmortem evidence of cardiac muscle disease. 26
All horses in the current study had serum cTnI concentrations within the reported reference interval for horses in training 27 before monensin administration, both at rest and after high-speed treadmill exercise. The 2 horses that were not fasted and were administered 1 mg/kg of monensin did not have increases in cTnI concentration during the 96-hr post–monensin administration testing period or after a second treadmill exercise. The absence of any troponin increase during these study times would suggest that there was no cardiac muscle damage in these horses. Cardiac damage from monensin ingestion is dose-dependent, and horses absorbing smaller doses might have no cardiac disease. All 4 fasted horses that received monensin in vegetable oil had elevations in cTnI concentration. Elevations were first noted at 24 hr in horse no. 2 (which received 1.5 mg/kg) and at 48 hr in 2 horses and 72 hr in another horse. The 2 horses that were euthanized or died within the first week of the study because of severe cardiac disease had the highest cTnI concentrations in the study. The second cTnI spike, at 120 hr in horse no. 4, may have been associated with systemic illness from the C. difficile enteritis. Cardiac troponin I is known to be increased in some critically ill humans, presumably because of the adverse effects of the primary disease on the heart. 15 The presumed fatal arrhythmia (acute death without any signs of heart failure) in horse no. 2 was the reason a rhythmmonitoring device was placed in horse nos. 5 and 6 in the study, but no tachyarrhythmias were found in these horses during the monitoring period. Fatal arrhythmias were previously reported as a cause of death in horses poisoned by monensin. 11,24 The 2 horses with cardiac disease (increased heart rates and cTnI values), but without physical signs of heart failure, had a gradual decline in heart rate and cTnI over time. In these horses, 17 (horse no. 5) and 27 (horse no. 6) days were required before the value returned to <0.11 ng/mg, the interval obtained before dosing in the present study and reported as the reference interval for horses with a similar cTnI assay. 27 In horses 5 and 6, cTnI (0.05 and 0.03 ng/ml, respectively) concentrations were within the reference interval at these times, but in both horses, cTnI was still 0.02 ng/ml above their pre-monensin concentrations. The information on changes in cTnI values in the present study is clinically important because measuring cTnI, which can now be performed on site in 10 min, at 24–72 hr after monensin exposure could be helpful in separating diseased versus horses without disease. Similarly, horses with marked elevations at 48 hr or less might be predictive of severe disease.
In summary, from a clinical standpoint, the current report is noteworthy because, to the authors' knowledge, it is the first to describe temporal changes in cTnI in horses exposed to monensin. Measurement of cTnI concentration could be used to separate diseased versus horses without disease in a natural monensin exposure outbreak. The availability of a stall-side cTnI assay offers an additional practical advantage of measuring cTnI when investigating horses exposed to monensin. In addition, the magnitude of cTnI elevations may provide prognostic information during naturally occurring monensin outbreaks.
Footnotes
a.
Beckman Coulter Inc., Fullerton, CA.
b.
Heska Corp., Loveland, CO.
c.
Sodium monensin, Sigma-Aldrich, St. Louis, MO.
d.
Mazola corn oil, ACH Food Companies Inc., Memphis, TN.
e.
Fatal-Plus Solution, Vortech Pharmaceutical Ltd., Dearborn, MI.
f.
Reveal®, Medtronic Inc., Minneapolis, MN.