BackgroundThe use of intra-articular (IA) local anaesthetics has proven to be an effective means to treat post-operative pain. The effects of local anaesthetics on equine chondrocytes are mixed with some studies reporting chondrodestruction and others no adverse effects. A liposomal formulation of bupivacaine is used in people and dogs by intra- and peri-articular administration to provide up to 72h of analgesia. The potential uses, side effects including chondrotoxicity, and likelihood of abuse (long-term analgesic effects) has not been evaluated in horses. ObjectivesDescribe bupivacaine concentrations following IA administration and assess biomarkers as indicators of the effects of liposomal bupivacaine on chondrocytes invivo. Study designParallel design. MethodsSixteen exercised horses received a single IA administration of 0.12mg/kg liposomal bupivacaine or 0.9% saline. Blood and urine samples were collected for 96h post-drug administration. Six horses treated with bupivacaine and those receiving saline, underwent daily arthrocentesis. Six additional bupivacaine treated horses underwent arthrocentesis at 96h. Drug concentrations were measured using LC-MS/MS and pharmacokinetic analyses performed. Immunoassays were used to measure markers of collagen degradation (C2C, C12C) and cartilage matrix synthesis (CPII, CS846) in synovial fluid. ResultsThe bupivacaine plasma elimination half-life was 17.85.42 and 11.95.17h for horses from which synovial fluid was collected daily and at 96h respectively. Bupivacaine concentrations in the joint were still detectable at 96h. Significant increases in C12C and C2C were noted at 96h in horses undergoing arthrocentesis at 96h only. CPII was increased at 48h and CS846 at 24 and 48h in horses sampled daily. Main limitationsLimited number of animals and absence of liposome control group. ConclusionsSustained concentrations of IA bupivacaine suggest viability of this medication as an intra-articular analgesic. Effects on equine chondrocytes need further study.

Used in both beef cattle and dairy cows, monensin can provide many health benefits but can, when unintended overexposures occur, result in adverse effects. Information on serum and tissue concentrations following overexposure and/or overt toxicosis which may aid in diagnostics and clinical outcome is lacking. The aim of this study was to determine concentrations of monensin in biological specimens following oral exposure for 10 days to an approved dose (1 mg/kg) and a higher dose (5 mg/kg) of monensin given daily on a body weight basis to 10 dairy cows. No deaths were reported; cows receiving 5 mg/kg showed early signs of toxicosis including depression, decreased feed intake, and diarrhea after 4 days of exposure. Histopathological findings were minimal in most cows. Pharmacokinetic modeling of the detected serum concentrations for the 1 and 5 mg/kg dose groups determined the Cmax , Tmax, and t1/2λ to be 0.87 and 1.68 ng/mL, 2.0 and 1.0 h, and 1.76 and 2.32 days, respectively. Mixed regression models showed that the dose level and days since last dose were significantly associated with monensin concentrations in all four tissues, and with cardiac troponin levels. The high dose resulted in a significant elevation of monensin in tissues at approximately 4.7 times compared to the monensin concentrations in the tissues of animals from the low-dose group. The cTnI concentrations in the high-dose group were 2.1 times that of cTnI in the low-dose group. Thus, the ability to diagnose monensin overexposure and/or toxicosis will improve from knowledge of biological monensin concentrations from this study.

Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1). A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS) detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

BACKGROUND:Dietary supplement use in both human and animals to augment overall health continues to increase and represents a potential health risk due to the lack of safety regulations imposed on the manufacturers. Because there are no requirements for demonstrating safety and efficacy prior to marketing, dietary supplements may contain potentially toxic contaminants such as hepatotoxic microcystins produced by several species of blue-green algae.CASE PRESENTATION:An 11-year-old female spayed 8.95 kg Pug dog was initially presented for poor appetite, lethargy polyuria, polydipsia, and an inability to get comfortable. Markedly increased liver enzyme activities were detected with no corresponding abnormalities evident on abdominal ultrasound. A few days later the liver enzyme activities were persistently increased and the dog was coagulopathic indicating substantial liver dysfunction. The dog was hospitalized for further care consisting of oral S-adenosylmethionine, silybin, vitamin K, and ursodeoxycholic acid, as well as intravenous ampicillin sodium/sulbactam sodium, dolasetron, N-acetylcysteine, metoclopramide, and intravenous fluids. Improvement of the hepatopathy and the dog's clinical status was noted over the next three days. Assessment of the dog's diet revealed the use of a commercially available blue-green algae dietary supplement for three-and-a-half weeks prior to hospitalization. The supplement was submitted for toxicology testing and revealed the presence of hepatotoxic microcystins (MCs), MC-LR and MC-LA. Use of the supplement was discontinued and follow-up evaluation over the next few weeks revealed a complete resolution of the hepatopathy.CONCLUSIONS:To the authors' knowledge, this is the first case report of microcystin intoxication in a dog after using a commercially available blue-green algae dietary supplement. Veterinarians should recognize the potential harm that these supplements may cause and know that with intervention, recovery is possible. In addition, more prudent oversight of dietary supplement use is recommended for our companion animals to prevent adverse events/intoxications.

A 6-year-old Peruvian Paso-Mustang cross mare was presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis during the fall (October) with a 2-day history of lethargy and icterus. The mare was from a herd of 26 mares and 3 foals from which 5 mares had died (leaving 21) during the preceding 7 days. These mares had varying degrees of colic, ataxia, pigmenturia, pale and icteric mucous membranes, lethargy and inappetance; they died within 48 hours of initial signs. Three of the affected mares had been pyrexic with rectal temperatures ranging from 102 to 102.5 °F. Treatment with nonsteroidal anti-inflammatory medications and oral antimicrobials was initiated on the affected mares, with no improvement in clinical signs. The herd had been moved to the current property 6 months previously. It consisted of 40 acres of undulating land comprised of native woodland and a planted Pistacia orchard (containing P. atlantica, P. terebinthus, P. chinensis). Mares with suckling foals were housed in a separate corral and the remaining mares grazed the entire 40 acres and were supplemented with orchard grass hay. All affected horses were part of the latter group. The horses were provided with county irrigation water, which was piped to troughs, and they had access to the irrigation ditch directly. There were no recent changes in herd management or housing, except for felling of the Pistacia orchard shortly before the first horse developed clinical signs; the owner had witnessed the horses eating from trees that had been cut down. The same owner housed 11 stallions on a property 1.5 miles away where they were provided with well water and the same orchard grass hay. None of these horses, or the lactating mares who were housed separately and fed the same hay, exhibited any signs of illness. Physical examination revealed tachycardia (56 beats per minute) and icteric oral, ocular, and vulvar mucous membranes. Pertinent laboratory results are listed in Table 1. Many eccentrocytes were noted on the blood smear. The low hematocrit, presence of nucleated erythrocytes, increased RDW, and indirect hyperbilirubinemia are consistent with hemolytic anemia. The eccentrocytes are indicative of oxidative damage to hemoglobin and erythrocyte membrane proteins. A Coggins test and Babesia caballi and Theileria equi PCR results were negative. Leptospira antibody titers were not indicative of active infection (L. bratislava and L. icterohemorrhagiae titers were positive at 1 : 100, and L. Pomona, canicola, grippothyphosa and hardjo titers were negative). Serum was negative for nitrate, nitrite, monensin and lead, and trace minerals were within acceptable ranges except for an increased iron (3.9 ppm, reference range 0.8–2.5 ppm) consistent with hemolysis, and slightly decreased magnesium (15 ppm, reference range 18–35 ppm) consistent with reduced feed intake. Urinalysis revealed a specific gravity of 1.032, pH = 8, and proteinuria (150 mg/dL, reference range: 0 mg/dL), 6–8 erythrocytes/HPF (reference range 0–2/HPF), and hemoprotein 150 ery/μL (reference range 0 ery/μL) with no other important abnormalities. The urine was negative for the presence of myoglobin, indicating the hemoprotein present was because of hemoglobinuria, also consistent with hemolytic anemia. Polymerase chain reaction test result on urine was negative for Leptospira gene sequences. Urine contained 32 mg/mL of pyrogallol (reference range 0 mg/L). The presence of urinary pyrogallol is indicative of ingestion of gallic acid, present in many trees and plants. The horse was administered ampicillin1 (20 mg/kg IV once) flunixin meglumine2 (0.6 mg/kg, IV once) and isotonic, polyionic fluids3 (25 mL/kg, as a bolus IV). Minocycline4 (4 mg/kg, PO) was administered until Leptospira infection was ruled out 24 hours later. Fluids were administered (isotonic, polyionic, 12.5 mL/kg IV daily) for 3 days. The lethargy, icterus, and pigmenturia gradually resolved over a few days. A 6-year-old Lusitano mare was presented with Case 1. This mare had no obvious clinical signs of illness but the owner was concerned because of the recent deaths of 5 horses. This mare had similar hematologic and biochemical derangements as did Case 1 (see Table 1). Eccentrocytes were evident on cytologic examination. Nitrate and nitrite were not detected in serum and serum Leptospira titer results were not indicative of active infection. A Coomb's test was negative. Urine was negative for Leptospira gene PCR. The urine was positive for pyrogallol at 90 mg/L (reference: 0 mg/L). The mare was treated with intravenous fluids (isotonic, polyionic 44 mL/kg, followed by 2.2 mL/kg/h), ampicillin (20 mg/kg IV), flunixin meglumine (0.55 mg/kg IV), and minocycline (4 mg/kg PO). Antimicrobial treatment was discontinued the next day after receiving negative Leptospira results. Activated charcoal5 (1 mg/kg) was administered to the mare in feed over a 12-hour period in an attempt to adsorb potential toxins from the gastrointestinal tract. The mare remained bright and maintained a good appetite throughout hospitalization. Intravenous fluid therapy was continued for 2 days and oral activated charcoal (0.1 mg/kg q 6 hours) for 3 days. Recheck of creatinine concentration on day 3 was within the reference range (0.9 mg/mL) and there was gradual improvement in PCV up to 21% by day 4 of hospitalization. The mare was discharged after 3 days. The owner was instructed to move the mares away from access to Pistacia trees and to administer activated charcoal to horses observed to ingest Pistacia leaves or seeds. Upon moving the mares to a smaller area of the property with no access to Pistacia trees, there were no additional illnesses or deaths. Necropsies were performed on 3 of the dead mares from the herd. These revealed hemoglobin nephropathy as well as hepatic and splenic hemosiderosis. Pyrogallol was present in the kidneys of 2 of the horses. Other findings included slightly low hepatic selenium concentrations (0.27, 0.25 ppm; ref range: 0.3–1.0 ppm) in 2 of the horses, and high liver iron (1700, 880, 520 ppm; ref range: 100–300 ppm) both on wet-weight basis. A site visit was performed to inspect the property for possible toxin exposure. Sampling of the water, hay, trees, and vegetation was conducted to investigate potential intoxication as the cause of hemolytic anemia. Physical examination and blood sampling of 2 randomly selected herd mates was performed. Physical examination was unremarkable, however both mares were anemic (PCV 22% and 28% respectively, reference 30–46%). Based on the presence of severe anemia, hemoglobinuria, eccentrocytosis, methemoglobinemia, indirect hyperbilirubinemia, presence of urine pyrogallol, negative Coombs test and negative tests for infectious diseases, a diagnosis of hemolytic anemia, likely associated with an oxidant toxin, was made. Examination of the plants retrieved from the property identified 3 varieties of Pistacia tree from the felled orchard, including Pistacia atlantica, Pistacia terebinthus, and Pistacia chinensis. No maple trees, onions, or other plants associated with oxidant damage or hemolysis in horses were found. Two in vitro screening assays using a pyrogallol standard (0.17 mg/mL) as a positive control, confirmed the oxidative properties of P. atlantica, P. terebinthus, and P. chinensis leaf and seed extracts on equine erythrocytes; methemoglobin formation and hemolysis resulted after exposing equine erythrocytes to these plant extracts.1 Two weeks after initial evaluation, repeat CBC and serum biochemistry profiles showed improved HCT of 21% and 27% in cases 1 and 2, respectively (reference 30-46%). There was no evidence of eccentrocytes and all other values were within reference ranges. Serum was also submitted for repeat Leptospira serology, which revealed no evidence of seroconversion. A 10-year-old Quarter Horse gelding was presented to Arizona Equine Medical and Surgical Center for obtundation and pigmenturia. The gelding's pasture mate, a 9-year-old Quarter Horse gelding, had been euthanized earlier the same day for signs consistent with hemolytic anemia and acute renal failure. The horses' pasture consisted of irrigated Bermuda grass and a single tree. The horses were observed to ingest fallen and wilted leaves from this tree. The tree was later identified as Pistacia atlantica. On physical examination, the gelding was obtunded, icteric, slightly tachycardic and had an initial PCV of 20% (reference range 30–46%). The gelding was treated with fluids IV (Lactated Ringers Solution (LRS)6 2 mL/kg/h for day 1, then 1 mL/kg/h for 6 days), activated charcoal (1 mg/kg q 12h for 2 days, then 30 mL paste PO, q 6 hours), and enrofloxacin7 (5 mg/kg, IV q 24 h for 7 days). The PCV decreased to 10% within 24 hours of admission (reference 30–46%). The horse remained hospitalized and showed gradual improvement over a 2-week period. Urine Leptospira PCR, blood piroplasmosis PCR, and a Coggins test were negative. No other toxic plants were identified in the pasture. The leaves and seeds of the Pistacia tree were tested with the same in vitro hemolysis and oxidative assay as for the California cases. On follow-up over 12 months later, a CBC and serum biochemistry panel were unremarkable, and the horse had returned to its previous work level (roping). The severity and acuteness of clinical signs described in these cases provoked detailed investigation to establish the cause of the acute hemolytic anemia. Although the presence of methemoglobinemia and eccentrocytosis on the blood smear were considered highly suggestive of oxidative damage to the red blood cells,3, 2 other potential infectious causes of hemolysis were ruled out. Testing for nitrate, nitrite, and lead, which have been reported to cause oxidative damage, also yielded negative results. Previously reported oxidizing toxins known to cause intravascular hemolysis in horses include wilted red maple, sugar maple, and silver maple leaves as well as onions.4-6 None of these were found on the property. Many of the clinical and clinicopathologic findings of the cases described in this report are similar to those of red maple (Acer rubrum) toxicosis.6 Clinical signs of red maple toxicosis also include weakness, lethargy, icterus, pigmenturia, and even unexpected death.6 The leaves, especially when wilted in the fall, cause severe oxidative damage to equine red blood cells, resulting in methemoglobinemia and hemolytic anemia.7-9 A major component of the Acer leaves which causes methemoglobin formation has been identified as gallic acid. An amount of gallic acid equivalent to that found in A rubrum extract significantly increased methemoglobin concentration, compared to that in extract-free control erythrocytes, but caused less than actual A rubrum extract. A potential co-oxidant, 2,3-dihydro-3,5-dihydroxy-6-methoxy-4H-pyran-4-one, was found in the A rubrum extract, which may have been responsible for increasing methemoglobin formation above the gallic acid alone.4 Pyrogallol has been demonstrated to be a more potent oxidizing agent than either gallic or tannic acid.10 In a previous study, gallic acid was metabolized to pyrogallol in equine ileum contents to a greater extent than in other gastrointestinal tract tissues.10 Incubation of tannic acid and A. rubrum leaves, individually with ileum contents, produced gallic acid and subsequently pyrogallol. Ileum suspensions formed no pyrogallol when passed through a filter which excluded microbes, suggesting a microbial basis to the pathway.10 Bacteria isolated from the ileum were found capable of pyrogallol formation. Therefore, gallotannins and gallic acid present in A. rubrum leaves can be metabolized by K. pneumoniae and E. cloacae, found in the equine ileum, to form pyrogallol.10 The detailed history provided by the owner of the California cases in our report did not suggest exposure to any known or previously reported hemolytic or oxidative toxins. The presence of pyrogallol in the urine of both affected mares, as well as in the kidneys of 2/3 necropsied horses, suggests ingestion of plants containing pyrogallol, gallic acid, or gallotannins. These findings along with the evidence of red blood cell oxidative damage (eccentrocytes, methemoglobin, hemoglobinuria) suggest a similar pathogenesis to red maple toxicosis. The affected horses had been observed to consume Pistacia leaves, which are known to contain gallic acid.11 In addition, in vitro testing performed using extract from Pistacia leaves and seeds, similar to that previously reported to confirm the oxidative and hemolytic properties of A. rubrum leaves, was consistent with oxidative and hemolytic effects on equine erythrocytes.1, 4 The high liver iron concentration in all 3 necropsied horses is an expected finding with intravascular hemolysis. It is interesting to speculate whether the slightly low liver selenium concentrations in 2/3 necropsied horses were associated with an increase predisposition to or a result of increase oxidative damage. P. atlantica, P. terebinthus, and P. chinensis are not native to North America, but are found in California, as well as several other states ranging from the Southwest to the Southeast of the United States. In the Middle East, these trees have been used for nutritional and medicinal purposes.12 Oxidative and hemolytic effects of Pistacia spp. have not been previously reported in horses or other species; the results reported here indicate that these plants are toxic to horses. Interestingly, the California outbreak arose in the fall, the same seasonality as reported with red maple leaf toxicosis, when the leaves are wilted and falling off the trees. The similar incident in Arizona, also associated with access to a Pistacia atlantica tree, also occurred at a time when leaves were falling off the tree. While the horses had access to the trees throughout the remainder of the year, we propose that the pathophysiology of Pistacia spp. toxicosis is similar to that of red maple in that felled and wilted leaves may be of importance. Furthermore, the felled trees in the California outbreak would have allowed for easy access and ingestion of large quantities of wilting leaves and seeds by the horses, thus accentuating these effects. Further research is required to identify the exact pathophysiology of Pistacia tree toxicosis, the toxic principles involved and the quantities required to cause clinical disease in horses. Until that time, it is clear that horses must be isolated from these trees to prevent acute hemolytic anemia and death. Drs. Mark Anderson and Federico Giannitti. This study was supported by the Roberta A. and Carla Henry Endowed Chair in Emergency Medicine and Critical Care, as well as the Center for Equine Health, with funds from the Oak Tree Racing Association, the State of California pari-mutuel wagering fund and contributions from private donors. Conflict of Interest Declaration: Authors disclose no conflict of interest. Off-label Antimicrobial Declaration: Authors declare no off-label use of antimicrobials.

Harmful algal blooms expose humans and animals to microcystins (MCs) through contaminated drinking water. While hepatotoxicity following acute exposure to MCs is well documented, neurotoxicity after sub-lethal exposure is poorly understood. We developed a novel statistical approach using a generalized linear model and the quasibinomial family to analyze neurotoxic effects in adult Caenorhabditis elegans exposed to MC-LR or MC-LF for 24 h. Selective effects of toxin exposure on AWA versus AWC sensory neuron function were determined using a chemotaxis assay. With a non-monotonic response MCs altered AWA but not AWC function, and MC-LF was more potent than MC-LR. To probe a potential role for protein phosphatases (PPs) in MC neurotoxicity, we evaluated the chemotactic response in worms exposed to the PP1 inhibitor tautomycin or the PP2A inhibitor okadaic acid for 24 h. Okadaic acid impaired both AWA and AWC function, while tautomycin had no effect on function of either neuronal cell type at the concentrations tested. These findings suggest that MCs alter the AWA neuron at concentrations that do not cause AWC toxicity via mechanisms other than PP inhibition.

Two horses were referred for methemoglobinemia and hemolytic anemia following 5 acute deaths in their herd from an unidentified toxin source. Horses have a greater risk than other mammalian species of developing methemoglobinemia and hemolytic anemia following ingestion of oxidizing toxins, due to deficiencies in the mechanisms that protect against oxidative damage in erythrocytes. Their susceptibility to oxidative erythrocyte damage is evident in the numerous cases of red maple (Acer rubrum) toxicosis. The suspected toxins causing A. rubrum toxicosis are tannic acid, gallic acid, and a metabolite of gallic acid, pyrogallol. These compounds can be found in a variety of plants, posing a risk to equine health. In order to quickly identify toxin sources, 2 rapid in vitro assays were developed to screen plant extracts for the ability to induce methemoglobin formation or cause hemolysis in healthy equine donor erythrocytes. The plant extract screening focused on 3 species of the genus Pistacia: P. atlantica, P. terebinthus, and P. chinensis, which were located in the horse pasture. Extracts of the seeds and leaves of each species induced methemoglobin formation and resulted in hemolysis, with seed extracts having greater potency. The in vitro assays used in the current study provide a useful diagnostic method for the rapid identification of oxidizing agents from unidentified sources. There is no effective treatment for oxidative erythrocyte damage in horses, making rapid identification and removal of the source essential for the prevention of poisoning.