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Monensin has been approved by the FDA for incorporation into lactating dairy cow rations. It is widely utilized for its beneficial impacts on energy metabolism, milk production, and cow health, particularly in transition cows that are at increased risk of ketosis, displaced abomasums, parturient paresis, and clinical mastitis. Although its safety and efficacy at the FDA-approved levels are well-documented, overexposures are likely to occur frequently without established diagnostic criteria for confirming such excess doses. Potentially toxic feed mixing errors on large livestock production facilities can impact hundreds to thousands of sheep, feeder cattle, or dairy cows within a matter of hours.<P> California is the largest dairy state in the United States, home to 1.8 million lactating cows producing approximately 24% of the nation's milk supply (1.6 billion kg/yr). Studies indicate that monensin is not detectable in edible tissues or milk from cows experimentally fed approved or sub-lethal doses. However large-scale feed mixing errors clearly have the potential to create public health concerns for food safety (meat and milk). Each such episode requires toxicological analysis using validated analytical methods. <P>Establishment of solid diagnostic criteria, combining monensin concentrations in biological specimens with other diagnostic parameters are needed for evidence-based determinations of withholding time when the recommended monensin dosage in feed is exceeded. This study aims to establish reference ranges for monensin concentrations in various biological specimens using validated analytical methods so that veterinary diagnosticians can provide rapid and accurate results and interpretations to food animal practitioners. <P>This project addresses multiple High Priority Issues including: Sustainable Production Systems, Feed and Feed Systems Security, Rapid and Accurate Diagnostics and Biodefense of Food Systems; and Animal Well-Being. Enhancing the veterinary diagnostician's ability to utilize both clinical and post-mortem specimens to diagnose monensin toxicosis will greatly improve the accuracy and timeliness of suspect monensin case investigations. The results of this study will provide essential data to veterinary diagnosticians, and may lead to a novel way to screen dairy cattle for appropriate supplementation with monensin.<P> RESEARCH HYPOTHESES: Quantitative serum and tissue monensin concentrations will differ between cattle with monensin toxicosis and cattle exposed to approved dietary levels of monensin. The veterinary diagnostician's ability to diagnose monensin toxicosis will improve with the addition of quantitative tissue monensin analysis to histopathological examination versus histopathological data alone. <P>OBJECTIVES: <BR> Aim 1: To determine serum, liver, skeletal muscle and myocardial monensin concentrations in cattle fed different concentrations of monensin.<BR> Aim 2: To determine the biological specimen that correlates best with dietary monensin level <BR>Aim 3: To explore the correlation between the severity of histopathological findings with tissue monensin concentrations.
Non-Technical Summary: California is the largest dairy state in the United States, home to 1.8 million lactating cows producing approximately 24% of the nation's milk supply (1 .6 billion kg/yr). Monensin is routinely added to dairy cattle rations for its beneficial impacts on energy metabolism, milk production, and health, particularly in transition cows at increased risk of ketosis, displaced abomasums, parturient paresis, and clinical mastitis. Potentially toxic feed mixing errors on large livestock production facilities, or continuous administration of levels above the FDA approved level can impact hundreds to thousands of dairy cows within a matter of hours; and thus have the potential to create public health (food safety) concerns which need to be addressed without delay. A recent literature search found few validated reference values or accepted analytic procedures for monitoring levels of monensin in serum, milk, liver, and meat to address these concerns in urgent situations. The proposed study aims to establish clear guidelines in suspect cases of overexposure to monensin by establishing reference values for monensin concentrations in a variety of biological matrices, including serum, urine, milk and edible tissues. If a correlation of serum, milk, and tissue monensin concentrations in cattle with adequate and toxic dietary levels of monensin can be established, these reference values can be used by veterinary diagnostic laboratories across the US [and other countries where monensin is approved for dairy cows, such as Canada] to improve the accuracy and timeliness of the diagnostic work-up of suspect monensin intoxications. Data from this study can also provide useful to develop new screening tools of cattle for adequate monensin supplementation. The potential of monensin intoxications is present as almost all dairies add monensin to their rations. Multiple episodes of both acute and chronic ionophore toxicoses in cattle have been reported in the scientific literature. Intoxication most commonly results from feed mixing errors that either result in inclusion of monensin into the diets of more sensitive non-target species (especially horses) or in excessive concentrations in the diets of target species (sheep and cattle) for which monensin is an approved feed additive. Monensin is relatively safe in cattle, which has allowed it to be approved for use in beef and dairy cattle. However, mixing errors or monensin levels well above the recommended use level have the potential to affect many cattle within a very short time period. These events have the potential to create widespread public health concerns, whether those concerns are a matter of perception only, or constitute an actual food safety concern. Veterinarians, along with producers and veterinary diagnostic laboratories, will need to establish a definite diagnosis whether an overexposure has occurred or not, and provide solid assessment of potential food safety concerns. The proposed study will generate critical data. <P> Approach: Animal Groups: Animal studies will be conducted in compliance with the Animal Welfare Act, U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Guide for Care and Use of Agriculture Animals in Agriculture Research and Teaching. We propose to use a minimal number of animals to establish pilot data. Two groups of 5 late-lactation Holstein dairy cows each will be randomly assigned to two treatment groups. Cows will be fed properly balanced rations that meet or exceed the nutrient requirements of dairy cattle based on both the 1989 and 2001 National Research Council's recommendations. Cows are fed twice daily after milking. Dosing: A bolus of monensin will be administered orally once daily for 10 days. Group A will receive the FDA-approved monensin dose of 1 mg/kg p.o. for 10 days. Group B will receive 30 mg/kg p.o. for 10 days. Sample collection: Blood, milk and urine samples will be collected immediately prior to the first dose, and 30 min, 1 hr, 2 hr, 4 hr, 8 hr, and 24 hr (just prior to second dose) after the first dose; one day 5 prior to dosing; and on day 10 immediately prior to the last dose, and 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 24 hr after the last dose. Blood will be spun down and serum will be stored frozen until analysis. Collection times will be recorded. Endpoints: Milk production, feed consumption, cardiac troponin I, clinical chemistry evaluations, and clinical signs of weakness, cardiac failure and any other abnormalities identified on daily physical examination will be assessed. Complete post-mortem evaluations and monensin measurements in various tissues will be performed. The following tissues will be collected and stored frozen for monensin analysis: myocardium, skeletal muscle, liver, and kidney. Histopathological evaluation will be conducted [blind] by a board certified veterinary pathologist of all major tissues, such as portions of brain, liver, kidneys, lungs, spleen, rumen, reticulum, omasum, abomasum, intestines, adrenal glands, skeletal muscle, trachea, thyroid, diaphragm, esophagus, heart, great vessels, will be collected, immersed in 10% buffered neutral formalin for 24 hours, sectioned, stained with hematoxylin and eosin and examined by light microscopy. Lesions will be graded according to severity and tissue distribution. Monensin analysis: A sensitive and selective liquid chromatography-mass spectrometry method for the detection of monensin in various biological matrices was recently developed in the CAHFS Toxicology Laboratory. The LC-MS/MS method is suitable for rapid analysis for monensin at a detection limit of 1 ng/g. Serum, urine, milk, liver and muscle tissues will be analyzed quantitatively for monensin. Data analysis: If data is distributed normally, a one-way or two-way Analysis of Variance (ANOVA) will be performed. Otherwise, the parametric the Kruskal Wallis (one-way) or Friedman (two-way) analyses will be done. Anticipated results: We expect to find a significant difference in tissue monensin concentrations between both study groups. We also expect a correlation between the histopathological findings and the detected monensin concentrations.