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Equine toxicoses: Investigative strategies and approaches for performance horses
EQUINE TOXICOSES: INVESTIGATIVE STRATEGIES AND APPROACHES FOR PERFORMANCE HORSES Mark Wickstrom, MSc, DVM, PhD, and Barry Blakley, MSc, DVM, PhD The investigation of suspect toxicosis is one of the most complex diagnostic problems faced by equine practitioners, requiring a wide range of clinical skills. When poisoning occurs, there is often no clear evidence of exposure to a toxic agent. Establishing an accurate diagnosis of poisoning requires a systematic approach, beginning with a comprehensive history, followed by clinical examination of affected horses, clinical pathologic testing, postmortem examinations, and analytic toxicology testing. The diagnosis of potential toxicosis in performance horses especially in the racetrack environment presents unique challenges to the veterinarian. Economic concerns including wagering, competition, inflated animal values, and insurance issues combine to create a situation in which intentional poisonings using obscure toxicants and unusual routes of exposure may occur. Collection of an accurate history is frequently confounded under these circumstances. This problem, coupled with the wide range of potential malicious toxic agents available, reinforces the requirement to approach these cases in a thorough and systematic fashion.
From the Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Address reprint requests to Mark Wickstrom, MSc, DVM, PhD, Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4 Canada. Reprinted with permission. Copyright 2002, Elsevier Science (USA). All rights reserved. 0737-0806/02/2209-0003$35.00/0 doi:10.1053/jevs.2002.34474
Volume 22, Number 9, 2002
Throughout the investigative process, potential medico-legal issues should be kept in the forefront. Care must be taken to maintain complete and accurate medical records and to maintain chain of custody on all antemortem and postmortem samples submitted to the diagnostic laboratory, because suspicious or unexplained deaths frequently end up in court. The investigation of suspect toxicosis is one of the most complex diagnostic problems faced by equine practitioners, requiring a wide range of clinical skills. When poisoning occurs, there is often no clear evidence of exposure to a toxic agent. Animals are frequently found dead or displaying generalized clinical signs that may be attributable to any of hundreds of potential toxicants as well as nontoxic causes. Diagnosis based on clinical signs alone is often impossible. Toxicologic diseases may be acute or chronic, and the clinical presentation will vary depending on the route, extent, and frequency of exposure, the time elapsed since the most recent exposure, age and physiologic condition of the animal, and many other factors. The veterinarian observing these signs may see only one phase of the syndrome and can be easily mislead, especially if faced with a rapidly deteriorating situation and the pressure to “do something.” Identification of the nature, source, and route of exposure of the toxicant is essential to prevent further exposure and initiate decontamination and specific treatment of affected animals. Without this information, the veterinarian is limited to supportive and symptomatic therapeutic measures. These must of course be initiated, and severely affected horses must be stabilized to the extent possible. However, immediately after the clinical crisis is controlled, it is essential to begin the work of establishing an accurate diagnosis because the “window of oppor-
tunity” within which this may be done can be quite narrow. Establishing an accurate diagnosis of toxicosis requires a systematic approach, beginning with a comprehensive history1,2 and followed by clinical examination of affected horses and collection of appropriate samples for analytic chemistry and clinical pathologic testing.3,4 Complete necropsies, including gross and histopathologic examinations, should be conducted on any animals that died.5 Throughout the investigative process, potential medico-legal issues should be kept in the forefront. Care must be taken to maintain complete and accurate medical records and to maintain chain of custody on all antemortem and postmortem samples submitted to the diagnostic laboratory. Suspicious or unexplained deaths may end up in court, sometimes years after their original presentation. This article discusses a systematic approach to diagnosis of suspect toxicosis in the horse, with emphasis on performance horses and the unique environment of the racehorse industry.
PERFORMANCE HORSES The performance horse adds a unique dimension to equine practice. There may be many features involved in dealing with performance horses that are not part of a typical mixed practice or equine practice environment. Many of the standard criteria used to establish a diagnosis involving a potential poison in these cases may be absent or even intentionally misleading. Performance horses are usually valuable animals. With economic issues such as wagering and distorted animal values influencing the actions of owners, trainers, and competitors, the opportunity for the occurrence of unusual events, including toxicoses, is real.
383
HISTORY Investigative strategies need to reflect the unique circumstances that may occur at the racetrack. The relative importance of each step in the classical systematic investigative strategy outlined above may vary in some instances involving performance horses. Malicious poisoning and insurance fraud are infrequent occurrences in most types of practice, but in racetrack investigations, legal implications (both criminal and civil proceedings) are often important considerations. In most toxicologic investigations, circumstantial, historical, and epidemiologic information plays a key role.4,6,7 This type of information may have limited value or may, in fact, be misleading in suspected cases of poison-
384
ing in the performance horse, especially if the owner or trainer of the horse is involved in the poisoning incident. There may be definite attempts to provide a false, inadequate, or misleading history to hide ulterior motives. Other incidents of malicious poisoning may involve individuals with no direct association with the horse. In either case, there may be few clues to find, and even the most astute diagnostician may have difficulty sorting out fact from fiction. Obtaining a comprehensive history of the horse, its environment, and management practices at the facility where the exposure occurred is the first step in any case, but the interview process may be less informative as compared with many investigations. Because equine poisonings have a high probability of ending up in court, it is important to remember that the animal’s record is a medico-legal document, subject to subpoena, and it must be composed accordingly. The paper trail needs to be com-
plete and interpretable, even years after the event. Document, date, and sign it – do not rely on memory. Poor medical records are a frequent cause of professional and legal embarrassment in the courts and professional disciplinary hearings.
ENVIRONMENTAL ASSESSMENT Examination of the environment or the premises inhabited by the horse is an important step in most toxicologic investigations.7,8 Information relating to water supply, feed sources, supplements, and any recent changes in management or husbandry practices often provides valuable evidence of potential exposure sources. While this exercise is an essential part of the investigation, in the case of performance horses, it is less likely to generate positive leads. Unlike many pasture situations, horses at the racetrack tend to be under close supervision and are fed high-quality feed in a highly con-
JOURNAL OF EQUINE VETERINARY SCIENCE
Table
1. Environmental Samples for Diagnostic Toxicology* Sample
Amount†
Comments
Concentrate feeds, mineral supplements
2 kg
Forages (pasture, hay)
5 kg
Multiple representative samples should be collected; may be pooled to create a composite; seal in plastic bags or glass jars for shipping.‡ Collect from multiple locations in pasture or storage facility, or from multiple bales using forage core sampler; cut to 3-inch lengths and seal in plastic bags for shipping. Press and dry or freeze entire plant, including root. Allow water to run to clear pipes before collecting; store and ship refrigerated in glass jar.§ Entire suspect bait or unidentified material (eg, pesticide) and label should be submitted; seal in plastic bag or glass jar and keep frozen.
Suspect toxic plants
All
Water
1L
Baits or unidentified material
All
*These are general guidelines. More detailed information concerning sample collection, handling, storage, and shipping for specific suspect toxicants can be obtained from the diagnostic laboratory or a veterinary toxicologist. †Collect and submit more than the minimum amount required for the test. In some situations (with stable toxicants), it may be prudent to split valuable samples and retain half, in the event that a shipment is lost or damaged en route to the diagnostic laboratory. ‡Samples should be frozen for some suspect toxicants. §Some organic contaminants require special containers and preservation methods.
trolled and restricted physical environment. Consequently, opportunities for accidental poisonings of the sort encountered in horses managed under more natural conditions are uncommon. For example, plant poisonings, or exposure to pesticide sprays or other agrochemicals, which are commonly encountered in routine equine practice, have a low probability of occurrence. Common natural routes of exposure to toxic agents, including dermal, oral, or inhalation, are also less frequent in the racetrack setting. Exposure to toxicants, if it is malicious or intentional in nature, must occur during periods of minimal supervision. In most situations, the toxic agent would need to be delivered at a high dose over a short period of time. Consequently, the route of exposure often selected is by injection. Detection of a small injection site on a large animal may be virtually impossible. Various drugs or toxicants may be easily administered in this fashion with only a small probability of identifying the site of administration. Selected agents may also be administered by cutaneous routes, especially if they are applied in solvents such as dimethyl
Volume 22, Number 9, 2002
sulfoxide. One should assume that unnatural routes of exposure have a reasonable probability of occurrence until proven to the contrary. Although they are less likely to represent a problem with performance horses, feed or water sources of exposure may need to be ruled out by systematic collection of representative samples3,4,9 (Table 1). Hay and other feed, supplements, and bedding should be inspected for the presence of toxic plants and evidence of consumption. Horses housed on racetrack premises are often restricted to a single stall, unless training is in progress. If a horse is intentionally poisoned, there is a high probability that it will happen in the stall because the majority of the unsupervised time is spent in that location. Spatial relationships associated with the incident may be difficult to ascertain because only a single animal with restricted and controlled movement is involved. Spatial relationships normally influenced by environmental or physical factors such as fences or water sources may not be evident. However, if multiple horses are affected, the opportunity to identify spa-
tial relationships between animals and exposure sources will improve. The following incident at the racetrack illustrates an unusual spatial relationship. Two horses died approximately 1 week apart under similar mysterious circumstances. Investigation of the first death was limited because the horse was not considered valuable. The death of the second horse, a valuable animal, raised concerns and prompted a full investigation. A unique spatial relationship was identified. The horses occupied “mirror image” locations in the barn. The malicious poisoning was intended to kill the valuable horse, but the guilty individual, who was unfamiliar with the facility and following incomplete directions, entered the stall area from the opposite end of the premises. Consequently, the second horse on the right, rather than the second to last horse on the left, was poisoned. The intended horse was poisoned 1 week later, once the mistake was realized. The discovery of this unusual spatial pattern influenced the direction of the subsequent investigation.
CLINICAL SIGNS AND CLINICAL PATHOLOGIC OBSERVATIONS Clinical signs and clinical pathologic data often provide critical information to aid in the diagnosis of poisoning.6,7 Clinical examination of affected horses should include observation of the onset, duration, severity, and progression of signs. It should be noted that clinical signs alone are seldom sufficient to establish a diagnosis. In suspected toxicoses involving only a single animal, the manifestations of the syndrome may not be classical because even the most common signs associated with particular toxicants are exhibited only by a proportion of poisoned animals. There is always a range in clinical manifestations, even under controlled, experimental exposure scenarios. In addition, the duration or
385
level of exposure, drug interactions, and numerous environmental and physiologic factors may mask or alter vital parameters with considerable diagnostic significance. If several horses are poisoned, the probability of at least one animal exhibiting key clinical manifestations essential for a reliable diagnosis is increased. In cases involving sudden death with minimal supervision, clinical manifestations may not be documented. In these situations, examination of the immediate surrounding area and extremities of the horse may provide indirect evidence implicating violent seizures or intense physical activity before death, which may be supportive of a particular diagnosis. In the live animal, it is essential to collect a variety of samples for clinical chemistry and hematologic analyses, including serum electrolytes, serum chemistry panel, complete blood count, and urinalysis. Because many drugs in particular are excreted in the urine, it is the sample of choice for many potentially malicious poisonings. It is often important to resist the temptation to focus on any particular agent too early. If a hepatotoxin is suspected, one should not limit the evaluation to liver enzyme measurements. If initial impressions were incorrect and underlying liver disease was not present, the “window of opportunity” to collect alternate samples for additional testing may have passed. Monensin or other ionophore poisoning, which is a frequently encountered problem in the horse, is a classic example. Rapid elimination, a limited ability to quantify levels in tissues, and only transient changes in many clinical pathologic measurements severely compromise diagnostic options only a few days after the poisoning incident. Elevated levels of muscle enzymes in the blood and the presence of myoglobin in the urine represent crucial
386
information. These indicators of muscle damage are elevated early in the syndrome but return to normal levels after a few days. Functional changes in the muscle may develop, but the evidence of acute muscle damage in the blood or urine may be absent.10 Circumstances of exposure and the type of toxicant encountered in performance animals may deviate considerably from those usually observed in cases of equine poisonings. For example, toxicants, which under most circumstances involve oral ingestion, may be administered by unnatural routes, such as intravenous injection. As a consequence, the onset and nature of the clinical manifestations may be unusual or atypical, and anticipated necropsy findings may be absent or masked by the altered pathogenesis. In most practice situations, the most probable diagnosis in a case of equine poisoning may be associated with local agricultural or husbandry practices. In contrast, with performance horses, the unusual or unexpected may become the norm. Human or veterinary drugs, or even more obscure toxicants, such as thallium, may become the poison of choice. Intentional overdoses of compounds such as insecticides that may be readily applied to the skin in rapid fashion without detection should be placed high on the list of suspicious agents. Cholinesterase inhibitors, such as organophosphate insecticides, cause sudden death with few distinctive postmortem changes and require specific tissue collection and analytical procedures. Carbamate insecticides, which act in a fashion similar to the organophosphate compounds, readily disassociate from target enzyme receptors. Consequently, if the analysis is delayed, no physiologic abnormality may be evident. The sophisticated racetrack environment is not only a source of unusual toxicants but the accessibility of these toxic agents to unscrupulous individuals may also be greater. Human medications and drugs of abuse are prime examples. In one instance, a horse developed a strange clinical syndrome characterized by sudden onset of weakness, lethargy, and coma, followed rapidly by death. Routine drug analysis and necropsy findings provided no
leads. However, analysis of a blood sample for glucose implicated the involvement of a massive insulin overdose. A complete evaluation of serum biochemical variables, no matter how irrelevant it may seem early in the investigation, may provide essential information in the end.
NECROPSY AND POSTMORTEM EVALUATION Necropsy and postmortem histopathologic analysis often provide critical evidence to support a tentative diagnosis of poisoning.5 From a legal perspective, anatomic pathology is often considered more definitive than other types of evidence, such as circumstantial or historical information or clinical signs. Data derived from the necropsy often provide the most complete picture of the pathogenesis of events in cases of toxic exposure. Observed patterns of lesions may shed light on the duration and level of exposure and its relationship to historical and circumstantial evidence. It may be possible to alter or misrepresent circumstantial or clinical evidence, but the pathogenesis of the toxic syndrome as documented by necropsy findings cannot easily be modified. Hence, necropsy findings provide one of the best sources of unbiased evidence. Ideally, gross and histopathologic examinations should be conducted by a board-certified veterinary pathologist with the credentials to adequately represent the case in court, if necessary. Unfortunately, with many toxicants, findings at postmortem are unremarkable, and negative findings are not unusual. Acute death may allow little time for obvious anatomic lesions to develop. For example, monensin poisoning is associated with myopathy and congestive heart failure, but in cases of massive overdose, the horse frequently dies before distinct gross lesions develop, and necropsy findings may be nonspecific. Other toxicants, such as cholinesterase inhibitors, produce only biochemical lesions. Therefore, the reliance on tissue and fluid analysis for specific toxicants or biochemical changes bec omes important.
JOURNAL OF EQUINE VETERINARY SCIENCE
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ANALYTICAL TOXICOLOGY Analytic toxicology testing often provides the final piece of information that confirms or establishes the etiologic diagnosis with reasonable probability. There is a common perception that analytical evidence is legally sound and difficult to dispute in the courts. Those familiar with testing methodologies, however, are aware of the many pitfalls of chemical analysis. Proper tissue selection and collection and storage procedures are crucial3,4 (Table 2). Toxicants are not uniformly distributed. Often, there is a particular tissue that is considered optimal for the analysis, and that tissue may vary depending on whether the exposure circumstances were acute or chronic in nature. For example, insec-
ticide poisoning associated with organochlorine pesticides may be acute or chronic. In acute poisonings, stomach contents and liver may contain diagnostically relevant levels. In contrast, the lipid-soluble nature of these insecticides may limit the usefulness of these tissues in cases of chronic poisoning, making brain the tissue of choice. Many toxicants are eliminated in a predicable, time-dependent fashion. Relative concentrations in a variety of tissues may provide valuable information concerning the route of exposure, duration of exposure, and possibly the dose of the toxicant. This information when presented in association with other types of evidence may clearly add credibility to the etiologic diagnosis. The “fit” in the puzzle becomes increasingly tight. In some instances, analytic measurements may provide false-negative results. Postmortem breakdown of the toxicant, rapid elimination before death, inappropriate tissue selection, or im-
proper handling or storage of the sample may be contributing factors to the occurrence of false-negative results. Analysis of feed, water, or tissue samples may indicate normal background levels of the toxicant. Such results are disturbing, particularly in light of necropsy or clinical pathologic findings that are consistent with the suspected etiologic diagnosis. The puzzle appears to have a “missing piece” or a “poor fit.” Under these circumstances, it is important to evaluate other potential explanations for the discrepancy. The following scenario, which is frequently encountered in horses, illustrates other avenues for investigation.
CASE Monensin poisoning is suspected in the death of a horse. Clinical and pathologic findings are consistent with the diagnosis, but analysis of tissues and sus-
2. Tissue Selection and Handling for Analytic Toxicology Testing*
Table
Sample
Amount†
Storage Condition‡
Comments§
Blood
5-10 mL
EDTA tube, refrigerate
Serum
5-10 mL
Urine
100 mL
Feces
250 g
Tissue biopsies
Glass vial (eg, clean red-top tube), freeze Plastic screw-cap vial, refrigerate Plastic bag or glass jar, freeze Plastic bag, freeze
Most metals, cholinesterase inhibitors, pesticides, cyanide, some organics, etc. Remove clot within 2 h; avoid hemolysis; trace elements; use special tubes for zinc; drugs, alkaloids, etc. Drugs, alkaloids, some metals, paraquat, oxalates, etc.
Liver
All of biopsy sample 10 g Plastic bag, freeze, note area sampled 500 g Plastic bags or glass jars, freeze 200 g Plastic bag, freeze
Kidney
200 g
Plastic bag, freeze
Brain
Half
Plastic bag, freeze
Fat Ocular fluid Miscellaneous tissue
200 g 1 eye 100 g
Plastic bag, freeze Plastic bag, freeze Plastic bag, freeze
Hair Ingesta¶
Recent oral exposures or drugs or other toxicants excreted in bile Especially liver; for organochlorines, metals, pyrrolizidine alkaloids, etc. Dermal exposure to pesticides, chronic accumulation of some metals Confirm recent oral exposure; some metals, toxic plant identification, plant toxins, drugs, pesticides, etc. Metals, insecticides, alkaloids, some drugs, some plant and mycotoxins, anticoagulant rodenticides, etc. Major excretory organ for many drugs, some metals, pesticides, antifreeze, etc. Sagittal section, half frozen, half in formalin for pathology; organochlorine and anticholinesterase insecticides, sodium, mercury, pyrethrins, etc. Lipophilic toxicants; organochlorine pesticides, polychlorinated biphenyls Nitrate, ammonia, potassium, magnesium, other electrolytes Tissue-specific tests; eg, barbiturates (spleen), paraquat (lung), etc.
*These are general guidelines. More detailed information concerning sample collection, handling, storage, and shipping for specific suspect toxicants can be obtained from the diagnostic laboratory or a veterinary toxicologist. †Collect and submit more than the minimum amount required for the test. In some situations (with stable toxicants), it may be prudent to split valuable samples and retain half, in the event that a shipment is lost or damaged en route to the diagnostic laboratory. ‡Freeze or refrigerate as soon as possible after collection and maintain appropriate storage conditions during shipping. §These examples are not intended to be exhaustive. Consult a toxicologist or analytic chemist for additional information. ¶Collect separate samples from stomach, small intestines, and colon.
388
JOURNAL OF EQUINE VETERINARY SCIENCE
pect feed is negative for monensin. Rapid elimination of monensin before death and poor sensitivity associated with tissue analysis may explain the negative tissue analysis, but the negative feed analysis causes more uncertainty. Conflicting evidence of this nature creates considerable problems from both insurance and legal perspectives. Often, the inability to resolve this inconsistency results in prolonged and perhaps unnecessary legal proceedings. There are a number of potential explanations, which are, in most instances, difficult to prove or disprove for situations such as this in which monensin is not detected in suspect feed or is detected only in low concentrations, insufficient to cause sudden death. Mixing errors are often the source of monensin in the ration, but the toxicant may not be uniformly distributed in the feed. Analysis of a nonrepresentative sample may provide misleading information. Assuming the assay methods and results are valid, other explanations are possible. For example, horses deficient in vitamin E or selenium may be more susceptible to the toxic effects of ionophores.11 This enhanced susceptibility may trigger the development of clinical manifestations of toxicity at lower levels of exposure than normally required. Drug interactions may also be a contributing factor. Any agent that inhibits the metabolism of monensin may enhance its toxicity. Macrolide antibiotics and certain mycotoxins have been reported to act in this manner.10 Finally, in situations similar to this, it is important to test for all potential ionophores in the feed. Monensin, although the most frequently encountered ionophore, may not be the cause in every instance. Many laboratories offer screening methods for several ionophores that are routinely used as feed additives. In addition to false-negative results, certain assay procedures may generate
Volume 22, Number 9, 2002
false-positive results. These results may lead to unnecessary insurance or legal claims. Highly toxic substances detected at low levels in tissue or feed samples may be implicated as causes of equine toxicoses. Feed additives such as monensin or lincomycin, an antibiotic, which should not be present in horse rations, may be detected at low levels. It is extremely important to determine whether the result is, in fact, a true value and not a false-positive value. Discussion of the result with the analytical chemist should provide valuable information. Detection limits, specificity, and the type of methodology used in the analysis may provide insight concerning the probability of a false-positive result. If some doubt remains, it is recommended that a duplicate sample be sent to another certified laboratory that uses a different method of analysis. Failure to eliminate false-positive results will lead the investigation down inappropriate pathways and ultimately will hamper proper resolution of the case. This may become an expensive oversight if legal proceedings are well advanced before the error is discovered.
SUMMARY Toxicoses in the horse may be technically and medically complex. Expertise, including that of the attending veterinarian, veterinary toxicologist, pathologist, and the analytical chemist, plays a key role in arriving at an accurate diagnosis. If there are insurance issues or the potential for litigation, it is critical that full and open communication be maintained at all levels from the beginning of the case. Misleading or incomplete evidence may have a negative impact on the final outcome. It is important to realize that veterinary medicine, including veterinary toxicology, is an art. There is a probability, large or small, that a toxic agent played a role in the patho-
genesis of the event under consideration. This is an opinion provided by experts based on the balance of probability and medical certainty as it relates to the facts presented in the case.
REFERENCES 1. Carson TL: Taking and interpreting a toxicological history, in Kirk RW, Bonagura JD (eds): Current Veterinary Therapy XI. Philadelphia, PA, Saunders, 1992, pp 160-162 2. Fitzgerald KT: Taking a toxicologic history, in Peterson ME, Talcott PA (eds): Small Animal Toxicology. Philadelphia, PA, Saunders, 2001, pp 27-113 3. Buck WB, Osweiler GD: Diagnostic toxicology, in Van Gelder GA (ed): Clinical and Diagnostic Veterinary Toxicology (ed 3). Dubuque, IA, Kendall/Hunt, 1988, pp 44-51 4. Puschner B, Galey FD: Diagnosis and approach to poisoning in the horse. Vet Clin North Am (Equine Pract) 17:399-409, 2001 5. Johnson BJ: Handling forensic necropsy cases. Vet Clin North Am (Equine Pract) 17:411-418, 2001 6. Osweiler GD: Diagnostic toxicology, in Osweiler GD (ed): Toxicology. Philadelphia, PA, Williams & Wilkins, 1996, pp 37-44 7. Blodgett DJ: The investigation of outbreaks of toxicologic disease. Vet Clin North Am (Food Anim Pract) 4:145-158, 1988 8. Smith RD: Epidemiology for the equine practitioner. Vet Clin North Am (Equine Pract) 17:419-432, 2001 9. Hancock DD, Blodgett D, Gay CC: The collection and submission of samples for laboratory testing. Vet Clin North Am (Food Anim Pract) 4:33-60, 1988 10. Oehme FW, Pickrell JA: An analysis of chronic oral toxicity of polyether ionophore antibiotics in animals. Vet Human Toxicol 41:251-257, 1999 11. Novilla MN: The veterinary importance of the toxic syndrome induced by ionophores. Vet Human Toxicol 34:66-70, 1992
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From the Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Address reprint requests to Mark Wickstrom, MSc, DVM, PhD, Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4 Canada. Reprinted with permission. Copyright 2002, Elsevier Science (USA). All rights reserved. 0737-0806/02/2209-0003$35.00/0 doi:10.1053/jevs.2002.34474
Volume 22, Number 9, 2002
Throughout the investigative process, potential medico-legal issues should be kept in the forefront. Care must be taken to maintain complete and accurate medical records and to maintain chain of custody on all antemortem and postmortem samples submitted to the diagnostic laboratory, because suspicious or unexplained deaths frequently end up in court. The investigation of suspect toxicosis is one of the most complex diagnostic problems faced by equine practitioners, requiring a wide range of clinical skills. When poisoning occurs, there is often no clear evidence of exposure to a toxic agent. Animals are frequently found dead or displaying generalized clinical signs that may be attributable to any of hundreds of potential toxicants as well as nontoxic causes. Diagnosis based on clinical signs alone is often impossible. Toxicologic diseases may be acute or chronic, and the clinical presentation will vary depending on the route, extent, and frequency of exposure, the time elapsed since the most recent exposure, age and physiologic condition of the animal, and many other factors. The veterinarian observing these signs may see only one phase of the syndrome and can be easily mislead, especially if faced with a rapidly deteriorating situation and the pressure to “do something.” Identification of the nature, source, and route of exposure of the toxicant is essential to prevent further exposure and initiate decontamination and specific treatment of affected animals. Without this information, the veterinarian is limited to supportive and symptomatic therapeutic measures. These must of course be initiated, and severely affected horses must be stabilized to the extent possible. However, immediately after the clinical crisis is controlled, it is essential to begin the work of establishing an accurate diagnosis because the “window of oppor-
tunity” within which this may be done can be quite narrow. Establishing an accurate diagnosis of toxicosis requires a systematic approach, beginning with a comprehensive history1,2 and followed by clinical examination of affected horses and collection of appropriate samples for analytic chemistry and clinical pathologic testing.3,4 Complete necropsies, including gross and histopathologic examinations, should be conducted on any animals that died.5 Throughout the investigative process, potential medico-legal issues should be kept in the forefront. Care must be taken to maintain complete and accurate medical records and to maintain chain of custody on all antemortem and postmortem samples submitted to the diagnostic laboratory. Suspicious or unexplained deaths may end up in court, sometimes years after their original presentation. This article discusses a systematic approach to diagnosis of suspect toxicosis in the horse, with emphasis on performance horses and the unique environment of the racehorse industry.
PERFORMANCE HORSES The performance horse adds a unique dimension to equine practice. There may be many features involved in dealing with performance horses that are not part of a typical mixed practice or equine practice environment. Many of the standard criteria used to establish a diagnosis involving a potential poison in these cases may be absent or even intentionally misleading. Performance horses are usually valuable animals. With economic issues such as wagering and distorted animal values influencing the actions of owners, trainers, and competitors, the opportunity for the occurrence of unusual events, including toxicoses, is real.
383
HISTORY Investigative strategies need to reflect the unique circumstances that may occur at the racetrack. The relative importance of each step in the classical systematic investigative strategy outlined above may vary in some instances involving performance horses. Malicious poisoning and insurance fraud are infrequent occurrences in most types of practice, but in racetrack investigations, legal implications (both criminal and civil proceedings) are often important considerations. In most toxicologic investigations, circumstantial, historical, and epidemiologic information plays a key role.4,6,7 This type of information may have limited value or may, in fact, be misleading in suspected cases of poison-
384
ing in the performance horse, especially if the owner or trainer of the horse is involved in the poisoning incident. There may be definite attempts to provide a false, inadequate, or misleading history to hide ulterior motives. Other incidents of malicious poisoning may involve individuals with no direct association with the horse. In either case, there may be few clues to find, and even the most astute diagnostician may have difficulty sorting out fact from fiction. Obtaining a comprehensive history of the horse, its environment, and management practices at the facility where the exposure occurred is the first step in any case, but the interview process may be less informative as compared with many investigations. Because equine poisonings have a high probability of ending up in court, it is important to remember that the animal’s record is a medico-legal document, subject to subpoena, and it must be composed accordingly. The paper trail needs to be com-
plete and interpretable, even years after the event. Document, date, and sign it – do not rely on memory. Poor medical records are a frequent cause of professional and legal embarrassment in the courts and professional disciplinary hearings.
ENVIRONMENTAL ASSESSMENT Examination of the environment or the premises inhabited by the horse is an important step in most toxicologic investigations.7,8 Information relating to water supply, feed sources, supplements, and any recent changes in management or husbandry practices often provides valuable evidence of potential exposure sources. While this exercise is an essential part of the investigation, in the case of performance horses, it is less likely to generate positive leads. Unlike many pasture situations, horses at the racetrack tend to be under close supervision and are fed high-quality feed in a highly con-
JOURNAL OF EQUINE VETERINARY SCIENCE
Table
1. Environmental Samples for Diagnostic Toxicology* Sample
Amount†
Comments
Concentrate feeds, mineral supplements
2 kg
Forages (pasture, hay)
5 kg
Multiple representative samples should be collected; may be pooled to create a composite; seal in plastic bags or glass jars for shipping.‡ Collect from multiple locations in pasture or storage facility, or from multiple bales using forage core sampler; cut to 3-inch lengths and seal in plastic bags for shipping. Press and dry or freeze entire plant, including root. Allow water to run to clear pipes before collecting; store and ship refrigerated in glass jar.§ Entire suspect bait or unidentified material (eg, pesticide) and label should be submitted; seal in plastic bag or glass jar and keep frozen.
Suspect toxic plants
All
Water
1L
Baits or unidentified material
All
*These are general guidelines. More detailed information concerning sample collection, handling, storage, and shipping for specific suspect toxicants can be obtained from the diagnostic laboratory or a veterinary toxicologist. †Collect and submit more than the minimum amount required for the test. In some situations (with stable toxicants), it may be prudent to split valuable samples and retain half, in the event that a shipment is lost or damaged en route to the diagnostic laboratory. ‡Samples should be frozen for some suspect toxicants. §Some organic contaminants require special containers and preservation methods.
trolled and restricted physical environment. Consequently, opportunities for accidental poisonings of the sort encountered in horses managed under more natural conditions are uncommon. For example, plant poisonings, or exposure to pesticide sprays or other agrochemicals, which are commonly encountered in routine equine practice, have a low probability of occurrence. Common natural routes of exposure to toxic agents, including dermal, oral, or inhalation, are also less frequent in the racetrack setting. Exposure to toxicants, if it is malicious or intentional in nature, must occur during periods of minimal supervision. In most situations, the toxic agent would need to be delivered at a high dose over a short period of time. Consequently, the route of exposure often selected is by injection. Detection of a small injection site on a large animal may be virtually impossible. Various drugs or toxicants may be easily administered in this fashion with only a small probability of identifying the site of administration. Selected agents may also be administered by cutaneous routes, especially if they are applied in solvents such as dimethyl
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sulfoxide. One should assume that unnatural routes of exposure have a reasonable probability of occurrence until proven to the contrary. Although they are less likely to represent a problem with performance horses, feed or water sources of exposure may need to be ruled out by systematic collection of representative samples3,4,9 (Table 1). Hay and other feed, supplements, and bedding should be inspected for the presence of toxic plants and evidence of consumption. Horses housed on racetrack premises are often restricted to a single stall, unless training is in progress. If a horse is intentionally poisoned, there is a high probability that it will happen in the stall because the majority of the unsupervised time is spent in that location. Spatial relationships associated with the incident may be difficult to ascertain because only a single animal with restricted and controlled movement is involved. Spatial relationships normally influenced by environmental or physical factors such as fences or water sources may not be evident. However, if multiple horses are affected, the opportunity to identify spa-
tial relationships between animals and exposure sources will improve. The following incident at the racetrack illustrates an unusual spatial relationship. Two horses died approximately 1 week apart under similar mysterious circumstances. Investigation of the first death was limited because the horse was not considered valuable. The death of the second horse, a valuable animal, raised concerns and prompted a full investigation. A unique spatial relationship was identified. The horses occupied “mirror image” locations in the barn. The malicious poisoning was intended to kill the valuable horse, but the guilty individual, who was unfamiliar with the facility and following incomplete directions, entered the stall area from the opposite end of the premises. Consequently, the second horse on the right, rather than the second to last horse on the left, was poisoned. The intended horse was poisoned 1 week later, once the mistake was realized. The discovery of this unusual spatial pattern influenced the direction of the subsequent investigation.
CLINICAL SIGNS AND CLINICAL PATHOLOGIC OBSERVATIONS Clinical signs and clinical pathologic data often provide critical information to aid in the diagnosis of poisoning.6,7 Clinical examination of affected horses should include observation of the onset, duration, severity, and progression of signs. It should be noted that clinical signs alone are seldom sufficient to establish a diagnosis. In suspected toxicoses involving only a single animal, the manifestations of the syndrome may not be classical because even the most common signs associated with particular toxicants are exhibited only by a proportion of poisoned animals. There is always a range in clinical manifestations, even under controlled, experimental exposure scenarios. In addition, the duration or
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level of exposure, drug interactions, and numerous environmental and physiologic factors may mask or alter vital parameters with considerable diagnostic significance. If several horses are poisoned, the probability of at least one animal exhibiting key clinical manifestations essential for a reliable diagnosis is increased. In cases involving sudden death with minimal supervision, clinical manifestations may not be documented. In these situations, examination of the immediate surrounding area and extremities of the horse may provide indirect evidence implicating violent seizures or intense physical activity before death, which may be supportive of a particular diagnosis. In the live animal, it is essential to collect a variety of samples for clinical chemistry and hematologic analyses, including serum electrolytes, serum chemistry panel, complete blood count, and urinalysis. Because many drugs in particular are excreted in the urine, it is the sample of choice for many potentially malicious poisonings. It is often important to resist the temptation to focus on any particular agent too early. If a hepatotoxin is suspected, one should not limit the evaluation to liver enzyme measurements. If initial impressions were incorrect and underlying liver disease was not present, the “window of opportunity” to collect alternate samples for additional testing may have passed. Monensin or other ionophore poisoning, which is a frequently encountered problem in the horse, is a classic example. Rapid elimination, a limited ability to quantify levels in tissues, and only transient changes in many clinical pathologic measurements severely compromise diagnostic options only a few days after the poisoning incident. Elevated levels of muscle enzymes in the blood and the presence of myoglobin in the urine represent crucial
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information. These indicators of muscle damage are elevated early in the syndrome but return to normal levels after a few days. Functional changes in the muscle may develop, but the evidence of acute muscle damage in the blood or urine may be absent.10 Circumstances of exposure and the type of toxicant encountered in performance animals may deviate considerably from those usually observed in cases of equine poisonings. For example, toxicants, which under most circumstances involve oral ingestion, may be administered by unnatural routes, such as intravenous injection. As a consequence, the onset and nature of the clinical manifestations may be unusual or atypical, and anticipated necropsy findings may be absent or masked by the altered pathogenesis. In most practice situations, the most probable diagnosis in a case of equine poisoning may be associated with local agricultural or husbandry practices. In contrast, with performance horses, the unusual or unexpected may become the norm. Human or veterinary drugs, or even more obscure toxicants, such as thallium, may become the poison of choice. Intentional overdoses of compounds such as insecticides that may be readily applied to the skin in rapid fashion without detection should be placed high on the list of suspicious agents. Cholinesterase inhibitors, such as organophosphate insecticides, cause sudden death with few distinctive postmortem changes and require specific tissue collection and analytical procedures. Carbamate insecticides, which act in a fashion similar to the organophosphate compounds, readily disassociate from target enzyme receptors. Consequently, if the analysis is delayed, no physiologic abnormality may be evident. The sophisticated racetrack environment is not only a source of unusual toxicants but the accessibility of these toxic agents to unscrupulous individuals may also be greater. Human medications and drugs of abuse are prime examples. In one instance, a horse developed a strange clinical syndrome characterized by sudden onset of weakness, lethargy, and coma, followed rapidly by death. Routine drug analysis and necropsy findings provided no
leads. However, analysis of a blood sample for glucose implicated the involvement of a massive insulin overdose. A complete evaluation of serum biochemical variables, no matter how irrelevant it may seem early in the investigation, may provide essential information in the end.
NECROPSY AND POSTMORTEM EVALUATION Necropsy and postmortem histopathologic analysis often provide critical evidence to support a tentative diagnosis of poisoning.5 From a legal perspective, anatomic pathology is often considered more definitive than other types of evidence, such as circumstantial or historical information or clinical signs. Data derived from the necropsy often provide the most complete picture of the pathogenesis of events in cases of toxic exposure. Observed patterns of lesions may shed light on the duration and level of exposure and its relationship to historical and circumstantial evidence. It may be possible to alter or misrepresent circumstantial or clinical evidence, but the pathogenesis of the toxic syndrome as documented by necropsy findings cannot easily be modified. Hence, necropsy findings provide one of the best sources of unbiased evidence. Ideally, gross and histopathologic examinations should be conducted by a board-certified veterinary pathologist with the credentials to adequately represent the case in court, if necessary. Unfortunately, with many toxicants, findings at postmortem are unremarkable, and negative findings are not unusual. Acute death may allow little time for obvious anatomic lesions to develop. For example, monensin poisoning is associated with myopathy and congestive heart failure, but in cases of massive overdose, the horse frequently dies before distinct gross lesions develop, and necropsy findings may be nonspecific. Other toxicants, such as cholinesterase inhibitors, produce only biochemical lesions. Therefore, the reliance on tissue and fluid analysis for specific toxicants or biochemical changes bec omes important.
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ANALYTICAL TOXICOLOGY Analytic toxicology testing often provides the final piece of information that confirms or establishes the etiologic diagnosis with reasonable probability. There is a common perception that analytical evidence is legally sound and difficult to dispute in the courts. Those familiar with testing methodologies, however, are aware of the many pitfalls of chemical analysis. Proper tissue selection and collection and storage procedures are crucial3,4 (Table 2). Toxicants are not uniformly distributed. Often, there is a particular tissue that is considered optimal for the analysis, and that tissue may vary depending on whether the exposure circumstances were acute or chronic in nature. For example, insec-
ticide poisoning associated with organochlorine pesticides may be acute or chronic. In acute poisonings, stomach contents and liver may contain diagnostically relevant levels. In contrast, the lipid-soluble nature of these insecticides may limit the usefulness of these tissues in cases of chronic poisoning, making brain the tissue of choice. Many toxicants are eliminated in a predicable, time-dependent fashion. Relative concentrations in a variety of tissues may provide valuable information concerning the route of exposure, duration of exposure, and possibly the dose of the toxicant. This information when presented in association with other types of evidence may clearly add credibility to the etiologic diagnosis. The “fit” in the puzzle becomes increasingly tight. In some instances, analytic measurements may provide false-negative results. Postmortem breakdown of the toxicant, rapid elimination before death, inappropriate tissue selection, or im-
proper handling or storage of the sample may be contributing factors to the occurrence of false-negative results. Analysis of feed, water, or tissue samples may indicate normal background levels of the toxicant. Such results are disturbing, particularly in light of necropsy or clinical pathologic findings that are consistent with the suspected etiologic diagnosis. The puzzle appears to have a “missing piece” or a “poor fit.” Under these circumstances, it is important to evaluate other potential explanations for the discrepancy. The following scenario, which is frequently encountered in horses, illustrates other avenues for investigation.
CASE Monensin poisoning is suspected in the death of a horse. Clinical and pathologic findings are consistent with the diagnosis, but analysis of tissues and sus-
2. Tissue Selection and Handling for Analytic Toxicology Testing*
Table
Sample
Amount†
Storage Condition‡
Comments§
Blood
5-10 mL
EDTA tube, refrigerate
Serum
5-10 mL
Urine
100 mL
Feces
250 g
Tissue biopsies
Glass vial (eg, clean red-top tube), freeze Plastic screw-cap vial, refrigerate Plastic bag or glass jar, freeze Plastic bag, freeze
Most metals, cholinesterase inhibitors, pesticides, cyanide, some organics, etc. Remove clot within 2 h; avoid hemolysis; trace elements; use special tubes for zinc; drugs, alkaloids, etc. Drugs, alkaloids, some metals, paraquat, oxalates, etc.
Liver
All of biopsy sample 10 g Plastic bag, freeze, note area sampled 500 g Plastic bags or glass jars, freeze 200 g Plastic bag, freeze
Kidney
200 g
Plastic bag, freeze
Brain
Half
Plastic bag, freeze
Fat Ocular fluid Miscellaneous tissue
200 g 1 eye 100 g
Plastic bag, freeze Plastic bag, freeze Plastic bag, freeze
Hair Ingesta¶
Recent oral exposures or drugs or other toxicants excreted in bile Especially liver; for organochlorines, metals, pyrrolizidine alkaloids, etc. Dermal exposure to pesticides, chronic accumulation of some metals Confirm recent oral exposure; some metals, toxic plant identification, plant toxins, drugs, pesticides, etc. Metals, insecticides, alkaloids, some drugs, some plant and mycotoxins, anticoagulant rodenticides, etc. Major excretory organ for many drugs, some metals, pesticides, antifreeze, etc. Sagittal section, half frozen, half in formalin for pathology; organochlorine and anticholinesterase insecticides, sodium, mercury, pyrethrins, etc. Lipophilic toxicants; organochlorine pesticides, polychlorinated biphenyls Nitrate, ammonia, potassium, magnesium, other electrolytes Tissue-specific tests; eg, barbiturates (spleen), paraquat (lung), etc.
*These are general guidelines. More detailed information concerning sample collection, handling, storage, and shipping for specific suspect toxicants can be obtained from the diagnostic laboratory or a veterinary toxicologist. †Collect and submit more than the minimum amount required for the test. In some situations (with stable toxicants), it may be prudent to split valuable samples and retain half, in the event that a shipment is lost or damaged en route to the diagnostic laboratory. ‡Freeze or refrigerate as soon as possible after collection and maintain appropriate storage conditions during shipping. §These examples are not intended to be exhaustive. Consult a toxicologist or analytic chemist for additional information. ¶Collect separate samples from stomach, small intestines, and colon.
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pect feed is negative for monensin. Rapid elimination of monensin before death and poor sensitivity associated with tissue analysis may explain the negative tissue analysis, but the negative feed analysis causes more uncertainty. Conflicting evidence of this nature creates considerable problems from both insurance and legal perspectives. Often, the inability to resolve this inconsistency results in prolonged and perhaps unnecessary legal proceedings. There are a number of potential explanations, which are, in most instances, difficult to prove or disprove for situations such as this in which monensin is not detected in suspect feed or is detected only in low concentrations, insufficient to cause sudden death. Mixing errors are often the source of monensin in the ration, but the toxicant may not be uniformly distributed in the feed. Analysis of a nonrepresentative sample may provide misleading information. Assuming the assay methods and results are valid, other explanations are possible. For example, horses deficient in vitamin E or selenium may be more susceptible to the toxic effects of ionophores.11 This enhanced susceptibility may trigger the development of clinical manifestations of toxicity at lower levels of exposure than normally required. Drug interactions may also be a contributing factor. Any agent that inhibits the metabolism of monensin may enhance its toxicity. Macrolide antibiotics and certain mycotoxins have been reported to act in this manner.10 Finally, in situations similar to this, it is important to test for all potential ionophores in the feed. Monensin, although the most frequently encountered ionophore, may not be the cause in every instance. Many laboratories offer screening methods for several ionophores that are routinely used as feed additives. In addition to false-negative results, certain assay procedures may generate
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false-positive results. These results may lead to unnecessary insurance or legal claims. Highly toxic substances detected at low levels in tissue or feed samples may be implicated as causes of equine toxicoses. Feed additives such as monensin or lincomycin, an antibiotic, which should not be present in horse rations, may be detected at low levels. It is extremely important to determine whether the result is, in fact, a true value and not a false-positive value. Discussion of the result with the analytical chemist should provide valuable information. Detection limits, specificity, and the type of methodology used in the analysis may provide insight concerning the probability of a false-positive result. If some doubt remains, it is recommended that a duplicate sample be sent to another certified laboratory that uses a different method of analysis. Failure to eliminate false-positive results will lead the investigation down inappropriate pathways and ultimately will hamper proper resolution of the case. This may become an expensive oversight if legal proceedings are well advanced before the error is discovered.
SUMMARY Toxicoses in the horse may be technically and medically complex. Expertise, including that of the attending veterinarian, veterinary toxicologist, pathologist, and the analytical chemist, plays a key role in arriving at an accurate diagnosis. If there are insurance issues or the potential for litigation, it is critical that full and open communication be maintained at all levels from the beginning of the case. Misleading or incomplete evidence may have a negative impact on the final outcome. It is important to realize that veterinary medicine, including veterinary toxicology, is an art. There is a probability, large or small, that a toxic agent played a role in the patho-
genesis of the event under consideration. This is an opinion provided by experts based on the balance of probability and medical certainty as it relates to the facts presented in the case.
REFERENCES 1. Carson TL: Taking and interpreting a toxicological history, in Kirk RW, Bonagura JD (eds): Current Veterinary Therapy XI. Philadelphia, PA, Saunders, 1992, pp 160-162 2. Fitzgerald KT: Taking a toxicologic history, in Peterson ME, Talcott PA (eds): Small Animal Toxicology. Philadelphia, PA, Saunders, 2001, pp 27-113 3. Buck WB, Osweiler GD: Diagnostic toxicology, in Van Gelder GA (ed): Clinical and Diagnostic Veterinary Toxicology (ed 3). Dubuque, IA, Kendall/Hunt, 1988, pp 44-51 4. Puschner B, Galey FD: Diagnosis and approach to poisoning in the horse. Vet Clin North Am (Equine Pract) 17:399-409, 2001 5. Johnson BJ: Handling forensic necropsy cases. Vet Clin North Am (Equine Pract) 17:411-418, 2001 6. Osweiler GD: Diagnostic toxicology, in Osweiler GD (ed): Toxicology. Philadelphia, PA, Williams & Wilkins, 1996, pp 37-44 7. Blodgett DJ: The investigation of outbreaks of toxicologic disease. Vet Clin North Am (Food Anim Pract) 4:145-158, 1988 8. Smith RD: Epidemiology for the equine practitioner. Vet Clin North Am (Equine Pract) 17:419-432, 2001 9. Hancock DD, Blodgett D, Gay CC: The collection and submission of samples for laboratory testing. Vet Clin North Am (Food Anim Pract) 4:33-60, 1988 10. Oehme FW, Pickrell JA: An analysis of chronic oral toxicity of polyether ionophore antibiotics in animals. Vet Human Toxicol 41:251-257, 1999 11. Novilla MN: The veterinary importance of the toxic syndrome induced by ionophores. Vet Human Toxicol 34:66-70, 1992
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