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Brief Communication |
Correspondence: 1 Corresponding Author: Fabio Del Piero, DVM, PhD, Dipl. ACVP, Associate Professor of Pathology, University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology and Department of Clinical Studies, New Bolton Center 382, W. Street Road, Kennett Square, PA 19348
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Abstract |
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Key Words: Anticoagulant • chlorophacinone • hemorrhage • ovine • rodenticide
Anticoagulant rodenticide intoxication is frequently diagnosed in dogs, but not in production animals.10 This report describes a case of chlorophacinone toxicosis in lambs, characterized by acute severe hemorrhages. Eleven lambs, approximately 1–2 months of age, suddenly developed epistaxis, respiratory distress, and facial and cervical swelling. They had fresh blood around the nostrils and perineal melena. Affected animals died within 1–2 hours from the onset of clinical signs.
Two lambs were sent to the University of Pennsylvania New Bolton Center for postmortem examination. Both animals were in good nutritional condition with mild autolysis. Lightly pigmented mucosae were very pale. Black, digested, malodorous blood (melena) stained the perineal hair and base of the tail and scant fresh blood stained the hair around the nostrils. The abomasum and the entire intestine contained melena. All of the organs were pale, especially the liver. The lungs were moderately heavier than normal, wet, and slightly brownish. Lamb #1 was affected by a severe acute hemorrhage involving the thymic parenchyma separating the thymic lobules and mild, acute intra-articular right hock joint hemorrhage. Lamb #2 was slightly icteric and was affected by diffuse acute hemorrhage involving the cervical subcutis and muscles infiltrating the interstitium.
For histopathology, tissues from all major organ systems were collected from both lambs, fixed in 10% buffered formalin, dehydrated in alcohol and xylene, and paraffin embedded. Five-micron tissue sections on glass slides were stained with hematoxylin and eosin and coverslipped. There was moderate pulmonary edema characterized by eosinophilic intra-alveolar proteinaceous fluid with a few macrophages. The abomasal wall exhibited acute severe interstitial hemorrhage with separation of the smooth muscle layers. Lamb #2 also had moderate to severe periacinar and pericentrolobular coagulative hepatocellular necrosis. Histological examination of the organs macroscopically affected by acute hemorrhage confirmed the severity of the lesions and the interstitial pattern of hemorrhage.
The signs and lesions were suggestive of several pathological conditions, including anticoagulant rodenticide or moldy sweet clover (Melilotus officinalis and Melilotus alba) intoxication, nitrofurazone intoxication, or an unidentified thrombocytopenic or vasculotropic infectious disease similar to thrombocytopenic pestivirus infection in cattle.
No microorganisms were detected in lung, lymphoid tissue, and intestine samples following virus isolation, immunohistochemistry, and bacterial cultures for ruminant pathogens such as pestivirus, aerobic and anaerobic bacteria, and Salmonella.
Liver, kidney, blood, and ruminal contents were collected for routine toxicologic examination. Metal screens including arsenic, cadmium, chromium, mercury, lead, selenium, thallium, calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, and zinc were performed on liver and kidney tissues by inductively coupled plasma argon emission spectroscopy. All tissue metal results were within normal ranges. Although metal intoxication was not one of the differentials in this case, metal screens are commonly included as part of a routine postmortem examination. Submitted liver and feed samples were analyzed for the presence of brodifacoum, bromodiolone, chlorophacinone, coumafuryl, dicoumarol, difenacoum, difethialone, diphacinone, pindone, valone, and warfarin. The analysis was performed using high-performance liquid chromatography. Briefly, 2 g of tissue and feed were homogenized separately in 6-ml acetonitrile (AN) and centrifuged. Supernatant was collected and applied to an activated alumina/SepPack C-18 cartridge purification setup. The column was prewashed with 4 ml, respectively, of methanol, water, and AN. The effluent was collected from the column, followed by a 6-ml AN wash. The initial effluent and subsequent wash were combined and evaporated under a stream of nitrogen at 55°C. The residue was redissolved in 300 µl of 19.2-M tetrabutylammonium hydroxide (pH = 7), filtered through a 0.45-µm filter, and injected into the high-performance liquid chromatography system with ultraviolet and fluorescent detectors (excitation at 280 nm and emission at 410 nm). The run time was 22 minutes on a reverse-phase C-18 column (26 x 4.6 cm) at room temperature. Gradient elution with methanol and 0.064-M tetrabutylammonium hydroxide (pH = 7) was used to achieve separation.
Chlorophacinone was detected in the submitted livers at 0.58 ppm and 0.50 ppm (wet weight), respectively. None of the other anticoagulants were detected in the liver samples at or above the assay detection limits. The detection of a potent anticoagulant in liver tissue, along with other compatible postmortem findings, confirmed the diagnosis of anticoagulant (chlorophacinone) intoxication. However, the feed did not appear to be the source of exposure because chlorophacinone was not detected in the submitted feed samples.
A subsequent search at the farm led to the identification of a dusty greenish compact material between the wall studs of the building housing the lambs. The interior walls of the pen had been covered with plywood, which prevented the sheep from gaining access to the spaces between the studs. However, the lambs had been noted to be chewing on the plywood and had chewed holes through the plywood in several areas, thus gaining access to the material. This material was identified as chlorophacinone using the same analytical protocol as outlined above. Chlorophacinone was quantitated at 890 ppm. There are 13 products containing chlorophacinone currently approved for use in the United States, and the specific product could not be determined.
Chlorophacinone (2-[(p-chlorophenyl)phenylacetyl]-1,3-indandione) is a second-generation indandione anticoagulant that has been marketed under several trade names (Rozol, Microzul, Ramucide, Ratomet, Raviac, and Topitox). It is formulated as an oil or dust concentrate for incorporation into finished products. Finished products include meal baits, tracking powders, paraffin blocks, pellets, and ground spray.3 It is normally incorporated into finished baits at 50 ppm (50 mg/kg or 0.005%). The much higher concentration of chlorophacinone in the tested sample is most likely because of degradation of the bait carrier over time with resulting concentration of the anticoagulant in the remaining material. Chlorophacinone is designed as a single-feeding rodenticide. It is effective against warfarin-resistant rodents and is used to control pests such as mice, rats, moles, muskrats, voles, and vampire bats in both outdoor and indoor environments.3 Chlorophacinone intoxication is rarely reported.
As with other anticoagulant rodenticides, the anticoagulant action of chlorophacinone is based on inhibition of vitamin K 2,3-epoxide reductase and vitamin K quinine activity.9 Anticoagulant rodenticide toxicity is based on inhibition of vitamin K 2,3-epxoide reductase and vitamin K quinone reductase in the liver, preventing the reduction of inactive vitamin K1 2,3-epoxide to active vitamin K1 (vitamin K quinol). This in turn prevents carboxylation of clotting factors II, VII, IX, and X, which is required for their activity in clot formation. Depletion of activated clotting factors results in hemorrhage. Clinical signs do not become apparent for 2–4 days following a toxic exposure because existing clotting factors need to be used before coagulopathy occurs. To the authors' knowledge, a minimum toxic dose for chlorophacinone has not been determined in sheep. Reported oral lethal doses are 2 mg/kg in the rat, 1 mg/kg in the mouse, 50 mg/kg in the rabbit, and 100 mg/kg in the duck.11 Thus, toxicity varies among species. In general, ruminants are less sensitive than monogastrics to the anticoagulant rodenticides.6 As with other anticoagulant rodenticides, ingestion of smaller doses of chlorophacinone over several days is more toxic than an acute (single) ingestion of an equivalent dose.15
Coumarins, indandiones, and other anticoagulants cause variable clinical signs in most animal species, which are dependent on the location and extent of hemorrhage. Clinical signs can include one or more of the following: hematuria, epistaxis, hematoma formation, melena, weakness, pale mucous membranes, muffled heart or lung sounds, and dyspnea. In humans, nasal and gastrointestinal hemorrhages have occasionally caused death from exsanguination.7 In the current cases, it is hypothesized that the hemorrhage was caused by the clorophacinone vitamin K reductase inhibition as described above; icterus followed posthemorrhagic hemolysis, and lung edema most likely developed following a decrease of osmotic intravascular pressure. Hemorrhage and hemolysis are often complicated by hypoxic hepatocellular necrosis.
Whereas anticoagulant rodenticide intoxication is one of the most frequently encountered toxicoses in dogs, it is relatively uncommon in food animals. This probably is related to a lack of access to sufficient quantities of the rodenticides. Historically, intoxication by dicoumarol found in moldy sweet clover was the most common form of anticoagulant intoxication in livestock. However, this is not commonly reported today because of replacement of sweet clover by other forages. There is one report of chlorophacinone intoxication involving cattle and lambs in 2 separate incidences.2 In addition, there is one case report of brodifacoum intoxication in 2 horses.5
Unless an animal is noted to have ingested an anticoagulant rodenticide, gastrointestinal decontamination procedures are often not helpful, because exposure to the product may have occurred several days before the onset of clinical signs. If the animal has just ingested an anticoagulant, then induction of emesis (in species able to readily vomit) and administration of activated charcoal and/or a saline cathartic may be indicated. If an animal presents with a coagulopathy and is severely anemic, the administration of whole blood plus fresh or fresh-frozen plasma to provide red blood cells and clotting factors can result in rapid improvement. Of course, in the case of food animal intoxication, the limited availability and costs of whole blood and fresh frozen plasma generally precludes such interventions.
Vitamin K1 (phytonadione) is antidotal. A recommended dose for intoxicated dogs is 2.5–5.0 mg/kg given PO as a loading dose followed by 2.5–5.0 mg/kg per day PO, divided BID to TID. Intoxication with second-generation anticoagulant rodenticides such as chlorophacinone will often require 2–4 weeks of daily treatment. Again, in a herd outbreak such as reported here, the lack of availability and prohibitive cost would most likely preclude its use. Alternatively, consideration should be given to incorporating vitamin K3 (menadione) into the ration. However, the efficacy of this approach is highly variable based upon its use in dicoumarol-intoxicated livestock.1,4,5,12,13 If used, a delay in response is expected because vitamin K3 is not physiologically active and needs to be metabolized to active forms of the vitamin. It is best to avoid the intravenous administration of vitamin K (K1 and K3) because of the potential for anaphylactoid reactions or nephrotoxicity.6,8,14
Livestock intoxication from synthetic anticoagulant rodenticides is uncommon; however, their widespread use for rodent control can result in livestock exposure. As illustrated by this case, livestock can gain access to baits believed to be out of their reach. In addition, baits can retain their toxicity long after their placement. Testing of serum or whole blood antemortem and liver postmortem for anticoagulants is critical for confirming exposure. Detection of an anticoagulant along with evidence of a coagulopathy is important for a diagnosis of intoxication.
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