Introduction

Monensin is an antibiotic produced as a byproduct of fermentation by Streptomyces cinnamonensis and belongs to a family of drugs known as polyether antibiotics or ionospheres. Monensin was the first antibiotic that showed an effect at practical concentrations for incorporation in feed as an anticoccidial agent.¹ However, in recent years it has been widely used as a feed additive to improve performance in livestock production systems. Monensin has also been used in the treatment and prevention of ketosis, lactic acidosis, bloat and acute pulmonary edema and emphysema.²

Monensin has a low therapeutic index and may be fatal in certain species when used in excessive doses. Accidental monensin intoxication, usually occur after mixing errors that result from its inclusion in feeds of non-target species or in excessive concentration in the diets of target species.³ Monensin intoxication has been reported in cattle^{4, 5} water buffaloes,⁶ sheep,^7,8 horses,⁹ swine,¹⁰ chickens,¹¹ ostriches,¹² deer,¹³ and dogs.^14,15 Monensin has been approved for use in non-lactating goats.¹⁶ However, there are no published reports of field cases of monensin toxicosis in goats. Experimental data indicated that the single oral LD₅₀ dose for monensin in goats is 26.4 mg kg^-1 of body weight.¹⁷

Susceptibility to monensin toxicity varies considerably between species. Horses are unusually sensitive to monensin and other ionophores intoxication and fish are the most tolerant to high levels of ionophores. The LD₅₀ of monensin in horses is as low as 1.4 mg kg^-1, while its LD₅₀ for chicken, the least sensitive species, is 214.0 mg kg^-1.³ Monensin toxicity may also be potentiated by concurrent use of various antibiotics, including tiamulin, oleandomycin chloramphenicol and macrolides, and sulfa drugs.^3,4

In this study, clinical, laboratory and pathological features of sub-acute monensin intoxication have been investigated in goats.

Materials and Methods

Animals and treatments. Seven clinically healthy local-breed, female, non-lactating and non-pregnant goats weighing 35-40 kg and aged 2-4 years were purchased from a local market. Prior to commencement of the experiment, goats received 0.2 mg kg^-1 of subcutaneous ivermectin (Razak Co., Tehran, Iran,) and 7.5 mg kg^-1 of oral rafoxanide (Daru, Tehran, Iran) and were kept in indoor pen and fed for 14 days to ensure proper acclimation. Fresh water was available all the time. Monensin (Razak Co., Tehran, Iran) was administered orally via orogastric tube at the dose of 13.0 mg kg^-1 body weight daily for five days.

Blood collection and serum biochemistry. Venous blood samples with and without anticoagulant were collected for hematology and measurement of total plasma protein and serum concentrations of troponin I, albumin, blood urea nitrogen (BUN), creatinine, total antioxidant capacity (TAC), activities of creatine kinase (CK), aspartate amio transferase (AST) and lactate dehydrogenase (LDH) on day 0 and afterward daily until day 10. The total protein was determined by refractometry and the fibrinogen concentration was measured by the heat-precipitation refractometry method. Other biochemical parameters were measured by an automated biochemical analyzer (Model Targa 3000, Biotecnica, Rome, Italy) using commercial kits for assessment of iron, CK, AST, LDH, BUN, creatinine, bilirubin and albumin (Pars Azmoon, Tehran, Iran) and for TAC (Randox, Antrim, UK). Serum troponin I concentration was determined using an ELISA kit (Monobind Inc, Lake Forest, USA).

The concentration of malondialdehyde (MDA) was estimated in serum according to the method of Placer et al.¹⁸ The reaction mixture consisted of 0.20 mL of serum, 1.30 mL of 0.20 M Tris, 0.16 M KCl of buffer (pH 7.4) and 1.50 mL of thiobarbituric acid reagent. The mixture was heated in a boiling water bath for 10 min. After cooling, 3 mL of pyridine/n-butanol (3:1, v/v) and 1.00 mL of 1M sodium hydroxide were added and mixed by vigorous shaking. A blank was run simultaneously by incorporating 0.20 mL distilled water instead of the serum. The absorbance of the test sample was read at 548 nm. The nmol of MDA per ml of serum was calculated using 1.56 × 10⁵ as extinction coefficient.

The activities of erythrocyte glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined in erythrocyte hemolysate obtained immediately after sampling from the blood with anticoagulant. Activity of GPx was measured using commercially available kits (Ransel test kit; Randox Laboratories Ltd., London, UK). This method is based on that of Pagalia and Valentine.¹⁹

Activity of SOD was measured based on a modified method of iodophenyl nitrophenol phenyl tetrazolium chloride (INT; Randox Laboratories Ltd., London, UK). One unit of SOD causes a 50% inhibition of the rate of reduction of INT under the assay conditions.

Pathological examinations. The goats were immediately necropsied after sacrificing on day 10 and all macroscopic changes of different organs were recorded. To examine microscopic lesions, tissue samples of heart, liver, kidneys and skeletal muscles (quadriceps muscles) were collected, fixed in 10% neutral buffered formalin and processed for routine histology. Tissue sections were stained with hematoxylin and eosin (H & E) for light microscopic examination.

Statistical analysis. Statistical analysis was conducted using SPSS for windows (Version 16; SPSS Inc., Chicago, USA). Based on Kolmogorov–Smirnov normality test, non - parametric Friedman and Wilcoxon Signed tests were used to compare significant differences within group for measured parameters. For all comparisons, p < 0.05 was considered as significant. For controlling the rate of type I error, the P value of each pairwise comparison was multiplied by the number of comparisons.

Results

 Clinical findings of monensin toxicosis in goats. Clinical signs of toxicity such as diarrhea and tachycardia were observed in monensin exposed goats. Monensin caused significant elevation of heart rate and a significant decrease in ruminal contraction. Body temperature was significantly decreased during the first four days of the experiment and then returned to baseline values. No significant change was observed in the respiratory rate.

Effect of monensin on serum troponin I, CK, AST and LDH. Significant increase in serum troponin I concentration was observed on days 3, 4, 7 and 8 of the study with a median peak concentration of 2.03 ng mL^-1 on day 8. The CK and LDH activities were significantly elevated in all timepoints compared to baseline values. Significant increase in activity of serum AST was observed on days 8, 9 and 10 (Table 1).

Effect of monensin on serum MDA, TAC and erythrocyte SOD and GPx. Significant elevation of erythrocyte SOD activity was observed on all days compared to baseline value. Serum MDA levels were significantly increased on days 1 and 8 of the experiment. Significant decrease of erythrocyte GPx activity was observed on days 2 and 5. No significant change was observed in serum TAC level (Tables 2 and 3).

Effect of monensin on serum albumin, bilirubin and plasma total protein. Serum bilirubin concentrations were significantly elevated on all days after monensin administration when compared with baseline levels. Monensin administration had no significant effect on plasma total protein, fibrinogen and serum albumin concentration during the study period in comparison with baseline values (Table 4).

Effect of monensin on serum levels of BUN, creatinine and serum troponin I. Significant increase of serum BUN concentration was observed on days 1 to 4. The concentration of serum creatinine was significantly increased on days 1, 6 and 7. Monensin exposure ended up significant elevation of serum troponin I concentration on days 3, 8 and 9 in goats, (Table 5).

Pathological findings. At necropsy, the goats showed pulmonary edema, mild pleural, pericardial and peritoneal effusions, congestion of the liver and kidneys, pale area on the heart and skeletal muscles and sub-endocardial and sub-epicardial hemorrhages. Histopathology revealed severe coagulative necrosis in skeletal muscles which was associated with infiltration of mononuclear inflammatory cells, (Fig. 1). Vacuolar degeneration and severe multifocal necrosis were seen in cardiac muscle, (Figs. 2 and 3). The kidneys showed congestion and renal tubular necrosis, (Fig. 4). Congestion and hemosiderin–laden cells were observed in the liver.