HEPATOCELLULAR TOXICITY OF THE METABOLITE EMODIN PRODUCED BY THE COMMON BUCKTHORN (RHAMNUS CATHARTICA) IN GREEN FROG (LITHOBATES CLAMITANS) TADPOLES.

The secondary metabolite emodin, produced by the widely distributed invasive shrub known as the common buckthorn (Rhamnus cathartica), has been shown to produce deformities and mortality in invertebrates, fish, and amphibian larvae. Here, we describe the effects on the liver of green frog (Lithobates clamitans) tadpoles after 21 d of exposure to high concentrations of emodin in a controlled environment. Histopathologic analysis showed fibrosis, bile duct proliferation, hepatocellular swelling, and accumulations of flocculent material consistent with emodin within the gall bladder and bile ducts of exposed individuals. The extensive fibrosis produced probably impeded the blood flow within the portal triads, limiting the detoxification function of the liver and resulting in hepatocellular necrosis and premature death for the individuals exposed. Exposure to emodin in the environment could represent a significant threat to developing amphibian larvae and contribute to local declines of populations.

Intrahistiocytic Storage of Clofazimine Crystals in a Cat.

A 13-year-old castrated male Maine coon cat with a 5-year history of atypical mycobacteriosis was euthanized and submitted for necropsy. The cat had been kept in clinical remission since diagnosis using a combination of the antimycobacterial drug clofazimine and additional multimodal antimicrobial therapy. Grossly, tissues were diffusely discolored red-brown to yellow. Histologically, the myocardial interstitum was expanded by numerous, often multinucleated cells, which were distended by uniformly shaped acicular cytoplasmic spaces. These cells were immunopositive for CD18 and immunonegative for desmin, suggesting a histiocytic rather than muscular origin. Macrophages in other tissues contained similar acicular spaces. Ultrastructurally, the spaces were surrounded by 2 lipid membranes, resembling an autophagosome. Based on the clinical history and histologic, immunohistochemical, and ultrastructural data, we diagnosed clofazimine crystal storage. To our knowledge, this is the first report of clofazimine storage in a cat or within myocardial interstitial macrophages.

Rare Osteochondroma Associated with Natural Mortality of a Wild White-Tailed Deer ( Odocoileus virginianus).

Within a 6-mo period, a radio-collared, white-tailed deer ( Odocoileus virginianus) doe from Delaware, US, developed a fatal head mass consistent with osteochondroma. We suspected a retroviral etiology, but test results were negative. Population implications were not suspected, but this case is concerning as these tumors are not thought to cause mortality.

Impact of prasugrel pretreatment and timing of coronary artery bypass grafting on clinical outcomes of patients with non-ST-segment elevation myocardial infarction: From the A Comparison of Prasugrel at PCI or Time of Diagnosis of Non-ST-Elevation Myocardial Infarction (ACCOAST) study.

OBJECTIVES: We evaluated impact of timing of coronary artery bypass grafting (CABG) and prasugrel pretreatment in patients with non-ST-segment elevation myocardial infarction undergoing CABG in the ACCOAST study. METHODS: Of 4033 enrolled patients, 314 (7.8%) underwent isolated CABG through 30 days. Primary efficacy end point for this analysis was any cardiovascular death, myocardial infarction, stroke, urgent revascularization, or glycoprotein IIb/IIIa inhibitor bailout through 30 days. RESULTS: More CABG versus percutaneous coronary intervention or medically managed patients were men, diabetic, or had peripheral arterial disease. Per randomization, 157 of 314 patients received a 30-mg prasugrel loading dose before CABG, and 157 of 314 received placebo. Patients were stratified by tertile of time from randomization to CABG: <2.98 days (n = 104), ≥2.98 and <6.95 days (n = 106), and ≥6.95 days (n = 104). Primary end point occurred in 12.5%, 4.7%, and 4.8%, respectively (<2.98 days vs other tertiles, hazard ratio [HR] = 2.80; P = .011). Similarly, the rate of all TIMI major bleeding was highest in the lowest tertile (26.0% vs 10.4% and 4.8%; P < .001), but no difference in all-cause death was observed through 30 days (3.9% vs 1.9% and 1.9%; P = .30). Time from randomization to CABG (HR = 0.84 for each day delay), left main disease (HR = 1.76), region of enrollment (Non-Eastern Europe vs Eastern Europe; HR = 3.83), but not prasugrel pretreatment and baseline troponin ≥3× upper limit of normal, were independent predictors of combined 30-day end point of all-cause death/myocardial infarction/stroke/TIMI major bleeding. CONCLUSIONS: In ACCOAST, early (<2.98 days) surgical revascularization carried increased risk of bleeding and ischemic complications without affecting all-cause mortality through 30 days. Baseline troponin and prasugrel pretreatment did not impact ischemic clinical outcomes.

Predictors of bleeding in patients with acute coronary syndromes treated with prasugrel.

BACKGROUND: When considering antiplatelet therapy for acute coronary syndrome (ACS), it is essential to balance benefits (less thrombotic/ischaemic events) versus bleeding risks related to intense platelet inhibition via antagonism of P2Y12 receptors. This analysis aimed to identify predictors of bleeding events among A Comparison of Prasugrel at the Time of PCI or as Pretreatment at the Time of Diagnosis in Patients with NSTEACS (ACCOAST) study population. METHODS AND RESULTS: The ACCOAST study randomised 4033 patients with non-ST-segment elevation myocardial infarction (NSTEMI) to (A) a 30 mg prasugrel loading dose (LD) followed by coronary angiography with an additional 30 mg prasugrel at the time of percutaneous coronary intervention (PCI) or (B) a placebo LD followed by a 60 mg prasugrel at the time of PCI. Patients received standard of care, including use of aspirin. Independent predictors of Thrombolysis in Myocardial Infarction (TIMI) major bleeding not related to coronary artery bypass grafting (CABG) within 7 days were assessed using stepwise Cox proportional model for time to first occurrence of the event. Non-CABG-related TIMI major or minor bleeding was similarly analysed. Non-CABG-related TIMI major bleeding occurred in 36 (0.9%) patients, and TIMI major or minor bleeding occurred in 81 (2.0%) patients. Independent predictors for TIMI major bleeding alone were pretreatment with prasugrel LD (HR 3.02; 95% CI 1.42 to 6.43), femoral access (HR 2.45; 95% CI 1.11 to 5.38), female sex (HR 2.57; 95% CI 1.32 to 5.00), placement of >1 stent (HR 2.50; 95% CI 1.26 to 4.95) and age (HR 1.05; 95% CI 1.02 to 1.09). Pretreatment with prasugrel LD (HR 3.05; 95% CI 1.84 to 5.07), femoral access (HR 3.06; 95% CI 1.74 to 5.38), female sex (HR 2.62; 95% CI 1.67 to 4.12), performed PCI (HR 2.21; 95% CI 1.23 to 3.99), therapy with glycoprotein IIb/IIIa inhibitors (HR 1.88; 95% CI 1.06 to 3.33) and age (increased bleed per year of age HR 1.04; 95% CI 1.02 to 1.06) were independent predictors of TIMI major or minor bleeding through 7 days. CONCLUSIONS: Pretreatment, age, gender and procedural variables predicted bleeding risk in patients with NSTEMI. CLINICAL REGISTRATION NO: NCT01015287.

Parasitology, virology, and serology of free-ranging coyotes (Canis latrans) from central Georgia, USA.

We examined 31 free-ranging coyotes (Canis latrans) from central Georgia, USA, for select parasites and viral agents. Sixteen coyotes had adult heartworms (Dirofilaria immitis). Serum samples from 27 animals revealed antibodies against canine parvovirus (100%), canine distemper virus (48%), canine adenovirus (37%), and Trypanosoma cruzi (7%); none were detected against Leishmania spp. Twenty-two of 24 (92%) coyotes were positive for Toxoplasma gondii. Real-time PCR of feces revealed 32% of coyotes were shedding canine parvovirus, and sequencing revealed type 2b and 2c. Because coyotes could be a spillover host of domestic dog (Canis lupus familiaris) pathogens, studies of the transmission of pathogens between coyotes and domestic dogs are warranted.

Pretreatment with prasugrel in non-ST-segment elevation acute coronary syndromes.

BACKGROUND: Although P2Y12 antagonists are effective in patients with non-ST-segment elevation (NSTE) acute coronary syndromes, the effect of the timing of administration--before or after coronary angiography--is not known. We evaluated the effect of administering the P2Y12 antagonist prasugrel at the time of diagnosis versus administering it after the coronary angiography if percutaneous coronary intervention (PCI) was indicated. METHODS: We enrolled 4033 patients with NSTE acute coronary syndromes and a positive troponin level who were scheduled to undergo coronary angiography within 2 to 48 hours after randomization. Patients were randomly assigned to receive prasugrel (a 30-mg loading dose) before the angiography (pretreatment group) or placebo (control group). When PCI was indicated, an additional 30 mg of prasugrel was given in the pretreatment group at the time of PCI and 60 mg of prasugrel was given in the control group. RESULTS: The rate of the primary efficacy end point, a composite of death from cardiovascular causes, myocardial infarction, stroke, urgent revascularization, or glycoprotein IIb/IIIa inhibitor rescue therapy (glycoprotein IIb/IIIa bailout) through day 7, did not differ significantly between the two groups (hazard ratio with pretreatment, 1.02; 95% confidence interval [CI], 0.84 to 1.25; P=0.81). The rate of the key safety end point of all Thrombolysis in Myocardial Infarction (TIMI) major bleeding episodes, whether related or not related to coronary-artery bypass grafting (CABG), through day 7 was increased with pretreatment (hazard ratio, 1.90; 95% CI, 1.19 to 3.02; P=0.006). The rates of TIMI major bleeding and life-threatening bleeding not related to CABG were increased by a factor of 3 and 6, respectively. Pretreatment did not reduce the rate of the primary outcome among patients undergoing PCI (69% of the patients) but increased the rate of TIMI major bleeding at 7 days. All the results were confirmed at 30 days and in prespecified subgroups. CONCLUSIONS: Among patients with NSTE acute coronary syndromes who were scheduled to undergo catheterization, pretreatment with prasugrel did not reduce the rate of major ischemic events up to 30 days but increased the rate of major bleeding complications. (Funded by Daiichi Sankyo and Eli Lilly; ACCOAST ClinicalTrials.gov number, NCT01015287.).

Effects of theobromine and caffeine on mood and vigilance.

Like caffeine, theobromine crosses the blood-brain barrier and binds to adenosine receptors, suggesting it might share caffeine's beneficial effects on mood and vigilance. Therefore, the purpose of this study was to assess the effect of theobromine doses commonly found in foods on mood and vigilance parameters sensitive to caffeine. Caffeine was tested as a positive control. Twenty-four men (age, 23 [3] years) completed 6 double-blind trials during which they consumed experimental beverages, assessed their mood using standardized self-report questionnaires, and completed a 2-hour visual vigilance task. Three experimental doses (100, 200, and 400 mg theobromine) were delivered in a cocoa-based beverage; 3 matched control treatments (0 mg theobromine, 400 mg theobromine, and 100 mg caffeine) were delivered in a non-cocoa beverage. Mean salivary concentrations of theobromine exhibited significant dose-dependent differences (400 mg trials > 200 mg trial > 100 mg trial > 0 mg trials; P < 0.005). At every dose tested, theobromine failed to consistently affect mood state or vigilance (P > 0.05), but 100-mg caffeine significantly decreased lethargy/fatigue and increased vigor (P = 0.006 and 0.011, respectively). These findings indicate theobromine does not influence mood and vigilance when administered in nutritionally relevant doses, despite sharing many of caffeine's structural characteristics.

Histologic findings in free-ranging Sarasota Bay bottlenose dolphin (Tursiops truncatus) skin: mercury, selenium, and seasonal factors.

Full-thickness epidermal biopsy samples were collected from free-ranging common bottlenose dolphins (Tursiops truncatus) in Sarasota Bay, Florida, USA. Season (summer or winter) of collection, mercury (Hg) concentration, and selenium (Se) concentration were compared to histologic parameters. Epidermal Hg concentration was positively related to age (P<0.001) and negatively related to height of the stratum spinosum (P<0.05). The mitotic index and heights of the stratum externum and intermedium were lower in summer than in winter (P<0.01). Transmission electron microscopic examination revealed variation in the diameters (60-138 nm) and arrangements of collagen fibers, regardless of age or concentrations of Hg and Se. The significance of the variation in height of the stratum spinosum and the perivascular collagen degeneration to dolphin health need further investigation.

Intrinsic platelet reactivity before P2Y12 blockade contributes to residual platelet reactivity despite high-level P2Y12 blockade by prasugrel or high-dose clopidogrel. Results from PRINCIPLE-TIMI 44.

It was the objective of this study to determine whether the intrinsic platelet response to adenosine diphosphate (ADP) before thienopyridine exposure contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade by prasugrel (60 mg loading dose [LD]), 10 mg daily maintenance dose [MD]) or high-dose clopidogrel (600 mg LD, 150 mg daily MD). High residual platelet function during clopidogrel therapy is associated with poor clinical outcomes. It remains unknown whether the relationship between platelet reactivity prior to treatment with clopidogrel (300 mg LD, 75 mg daily MD) and residual on-treatment platelet reactivity is maintained after more potent P2Y12 inhibition. PRINCIPLE-TIMI 44 was a randomised, double-blind, two-phase crossover study of prasugrel compared with high-dose clopidogrel in 201 patients undergoing cardiac catheterisation for planned percutaneous coronary intervention. ADP-stimulated platelet-monocyte aggregates, platelet surface P-selectin and platelet aggregation were measured pre-treatment, during LD (6 h and 18-24 h) and MD (15 d). Correlations of pre-treatment to on-treatment values were determined by Spearman rank order. Prasugrel resulted in greater platelet inhibition than high-dose clopidogrel for each measure. However, for both drugs, pre-treatment reactivity to ADP predicted 6 h, 18-24 h and 15 day reactivity to ADP (correlations 0.24-0.62 for platelet-monocyte aggregates and P-selectin). In conclusion, a patient's intrinsic platelet response to ADP before exposure to thienopyridines contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade with high-dose clopidogrel or even higher level P2Y12 blockade with prasugrel. Patients who are hyper-responsive to ADP pre-treatment are more likely to be hyper-responsive to ADP on-treatment, which may be relevant to therapeutic strategies.

A comparison of prasugrel at the time of percutaneous coronary intervention or as pretreatment at the time of diagnosis in patients with non-ST-segment elevation myocardial infarction: design and rationale for the ACCOAST study.

BACKGROUND: The precise risk/benefit of thienopyridine pretreatment and the optimal dosage and timing of a thienopyridine loading dose (LD) for patients presenting with non-ST-segment elevation (NSTE) acute coronary syndromes are still being debated. Prasugrel, a novel thienopyridine, is an appropriate drug to address this issue as it provides predictably high and rapid inhibition of platelet aggregation. STUDY DESIGN: ACCOAST is a phase 3, multicenter, parallel-group, double-blind, and event-driven study designed to compare 2 prasugrel LD schedules in patients with NSTE myocardial infarction who are scheduled for coronary angiography/percutaneous coronary intervention (PCI). Approximately 4,100 patients will be randomly assigned to an initial LD of 30 mg of prasugrel after the diagnosis followed by coronary angiography with an additional dose of 30 mg of prasugrel given at the time of PCI (pretreatment) or an LD of 60 mg of prasugrel given to patients undergoing PCI at the time of the procedure (non-pretreatment). All patients undergoing PCI will receive 5 or 10 mg of prasugrel daily. The primary objective is to test the hypothesis that prasugrel pretreatment is superior to prasugrel non-pretreatment as measured by a reduction in the composite end point of cardiovascular death, myocardial infarction, stroke, urgent revascularization, or glycoprotein IIb/IIIa inhibitor bailout through 7 days from randomization. Key safety end points include TIMI (Thrombolysis In Myocardial Infarction) major and minor bleeding risks. CONCLUSIONS: The ACCOAST study will provide important evidence with regard to the benefits and risks of prasugrel pretreatment compared with administration of prasugrel at the time of PCI in patients with NSTE myocardial infarction.

Cacao seeds are a "Super Fruit": A comparative analysis of various fruit powders and products.

BACKGROUND: Numerous popular media sources have developed lists of "Super Foods" and, more recently, "Super Fruits". Such distinctions often are based on the antioxidant capacity and content of naturally occurring compounds such as polyphenols within those whole fruits or juices of the fruit which may be linked to potential health benefits. Cocoa powder and chocolate are made from an extract of the seeds of the fruit of the Theobroma cacao tree. In this study, we compared cocoa powder and cocoa products to powders and juices derived from fruits commonly considered "Super Fruits". RESULTS: Various fruit powders and retail fruit products were obtained and analyzed for antioxidant capacity (ORAC (µM TE/g)), total polyphenol content (TP (mg/g)), and total flavanol content (TF (mg/g)). Among the various powders that were tested, cocoa powder was the most concentrated source of ORAC and TF. Similarly, dark chocolate was a significantly more concentrated source of ORAC and TF than the fruit juices. CONCLUSIONS: Cocoa powder and dark chocolate had equivalent or significantly greater ORAC, TP, and TF values compared to the other fruit powders and juices tested, respectively. Cacao seeds thus provide nutritive value beyond that derived from their macronutrient composition and appear to meet the popular media's definition of a "Super Fruit".

The onset of inhibition of platelet aggregation with prasugrel compared with clopidogrel loading doses using gatekeeping analysis of integrated clinical pharmacology data.

The purpose of this analysis was to determine the time by which a prasugrel 60-mg loading dose (LD) achieved significantly greater inhibition of platelet aggregation (IPA) than the peak IPA after a clopidogrel 300-mg LD or 600-mg LD. Data were pooled from nine studies representing 587 individuals: 274 healthy subjects and 313 patients with stable coronary artery disease. The primary pharmacodynamic measure was IPA using 20 [mu]M adenosine-5'-diphosphate as the agonist. Gatekeeping analysis compared the peak IPA at 4, 6, and 24 hours after a clopidogrel 300-mg or 600-mg LD with IPA at various prior time points backwards after a prasugrel LD until a statistically nonsignificant difference was reached. Prasugrel 60-mg LD produced greater IPA by 30 minutes than the peak IPA after a clopidogrel 300-mg LD (P < 0.0001). Significantly greater IPA was achieved at 1 hour after prasugrel 60-mg LD compared with the peak IPA after 600-mg clopidogrel LD (P < 0.0001), regardless of sex, body weight, or age and as early as 30 minutes in the diabetic subgroup. A prasugrel 60-mg LD produces significantly faster onset and greater IPA compared with a clopidogrel 300-mg LD or 600-mg LD.

Epizootic ulcerative syndrome caused by Aphanomyces invadans in captive bullseye snakehead Channa marulius collected from south Florida, USA.

Epizootic ulcerative syndrome (EUS) caused by the oomycete Aphanomyces invadans is an invasive, opportunistic disease of both freshwater and estuarine fishes. Originally documented as the cause of mycotic granulomatosis of ornamental fishes in Japan and as the cause of EUS of fishes in southeast Asia and Australia, this pathogen is also present in estuaries and freshwater bodies of the Atlantic and gulf coasts of the USA. We describe a mass mortality event of 343 captive juvenile bullseye snakehead Channa marulius collected from freshwater canals in Miami-Dade County, Florida. Clinical signs appeared within the first 2 d of captivity and included petechiae, ulceration, erratic swimming, and inappetence. Histological examination revealed hyphae invading from the skin lesions deep into the musculature and internal organs. Species identification was confirmed using a species-specific PCR assay. Despite therapeutic attempts, 100% mortality occurred. This represents the first documented case of EUS in bullseye snakehead fish collected from waters in the USA. Future investigation of the distribution and prevalence of A. invadans within the bullseye snakehead range in south Florida may give insight into this pathogen-host system.

Gingival osteogenic melanoma in two dogs.

Osteogenic melanoma is a rare variant of metaplastic malignant melanoma in human medicine and appears to be a similarly rare variant in dogs. Two dogs with oral malignant melanoma with neoplastic bone formation are reported in this study. Both tumors were characterized by malignant melanocytes that transitioned into neoplastic bone at the deep margins of the neoplasm. Immunohistochemical analysis revealed S100- and Melan-A-positive neoplastic cells adjacent to, and occasionally embedded within, an osteoid and chondroblastic matrix. Scattered clusters of neoplastic cells were also positive for osteocalcin. The findings indicate that in dogs, as in humans, neoplastic melanocytes have metaplastic potential and can be osteogenic.

Population health of Fallow deer (Dama dama) on Little St. Simons Island, Georgia, USA.

Fallow deer (Dama dama) were introduced to Little St. Simons Island, Georgia, USA in the 1920s and thrive at high population densities, to the exclusion of white-tailed deer (Odocoileus virginina). The presence of introduced pathogens and parasites as a result of their introduction is currently unknown, as is the impact of native disease on the exotic fallow deer. Hunter-killed fallow deer from 2003-2005 were necropsied and surveyed for evidence of infectious disease, parasitic agents, and toxicologic parameters. Fallow deer were positive for antibodies to bovine virus diarrhea virus I and II, bluetongue virus, and bovine adenovirus. Twenty species of bacteria were isolated from the internal organs, and 14 species of parasites were recovered including one abomasal nematode, Spiculopteragia asymmetrica, which is not known to occur in native North American ungulates. Concentrations of liver and copper were low, while lead, zinc, and iron were considered within normal levels. No clinical signs of disease were noted, and the overall health of the insular fallow deer was considered good.

Amphibian ocular malformation associated with frog virus 3.

During an on-going amphibian ecology study, a free-ranging American bullfrog (Rana catesbeiana) metamorph was captured in a pitfall trap adjacent to a constructed farm pond at the Plateau Research and Education Center (PREC) on the Cumberland Plateau near Crossville, Tennessee, USA. Grossly, the right eye was approximately 50% the size of the left. Stereo and light microscopic examination revealed two granulomas within the orbit. Electron microscopic examination revealed virus particles scattered throughout one structure but mostly aggregated toward the center. Subsequent PCR and sequencing (GenBank accession Number EF175670) confirmed frog virus 3 (FV3). This represents the first report of a malformation in an anuran associated with FV3.

Occurrence of Piscirickettsiosis-like syndrome in tilapia in the continental United States.

From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 +/- 0.1 x 0.8 +/- 0.2 microm, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.

B-cell conjunctival lymphoma in a cat.

A bilateral conjunctival neoplasm was surgically removed from a 13-year-old Domestic Short-haired female cat and examined by histopathology. The neoplasm was infiltrative, nonencapsulated and composed of large sheets of densely packed, round to polygonal cells. Neoplastic cells were variably sized with scant to modest amounts of cytoplasm and oval to round nuclei. On immunohistochemistry the neoplastic cells stained positively for BLA.36 and negative for CD-3 antibodies. On the basis of histopathology and immunohistochemical findings, a diagnosis of conjunctival B-cell lymphoma was made. This is the first case with immunohistochemical characterization of conjunctival lymphoma in a cat.

West Nile virus in farmed alligators.

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia. Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in 2002. Reverse transcription-polymerase chain reaction (RT-PCR) results on all submitted case samples were positive for West Nile virus for one of four cases associated with the 2001 epizootic and three of three cases associated with the 2002 epizootic. RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak.