Renal, gastrointestinal, and hemostatic effects of oral administration of meloxicam to Hispaniolan Amazon parrots (Amazona ventralis)

Bas Dijkstra Department of Companion Animal Medicine, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands.

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David Sanchez-Migallon Guzman Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Kate Gustavsen Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Sean D. Owens Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Carlyle Hass William R. Pritchard Veterinary Medical Teaching Hospital Clinical Laboratory Services, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Philip H. Kass Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Joanne R. Paul-Murphy Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

OBJECTIVE To investigate renal, gastrointestinal, and hemostatic effects associated with oral administration of multiple doses of meloxicam to healthy Hispaniolan Amazon parrots (Amazona ventralis).

ANIMALS 12 Hispaniolan Amazon parrots.

PROCEDURES Birds were assigned to receive meloxicam oral suspension (1.6 mg/kg, PO, q 12 h) and 2.5 mL of tap water inserted into the crop by use of a gavage tube (n = 8) or the equivalent volume of tap water only (control group; 4) for 15 days. Urine and feces were collected 2 hours after treatment administration each day. Feces were evaluated for occult blood. Results of a CBC and serum biochemical analysis and measured N-acetyl-β-d-glucosaminidase (NAG) activity and whole blood clotting time were evaluated before, during, and after completion of treatments. Results of urinalysis and measured urine NAG activity were also evaluated.

RESULTS Birds treated with meloxicam had a significant increase in number of WBCs and decrease in PCV from before to after treatment. The PCV also decreased significantly, compared with results for the control group; however, WBC count and PCV for all birds remained within reference ranges throughout the study. One parrot treated with meloxicam had a single high value for urine NAG activity.

CONCLUSIONS AND CLINICAL RELEVANCE Meloxicam administered orally at the dosage used in this study caused no apparent negative changes in several renal, gastrointestinal, or hemostatic variables in healthy Hispaniolan Amazon parrots. Additional studies to evaluate adverse effects of NSAIDs in birds will be needed.

Abstract

OBJECTIVE To investigate renal, gastrointestinal, and hemostatic effects associated with oral administration of multiple doses of meloxicam to healthy Hispaniolan Amazon parrots (Amazona ventralis).

ANIMALS 12 Hispaniolan Amazon parrots.

PROCEDURES Birds were assigned to receive meloxicam oral suspension (1.6 mg/kg, PO, q 12 h) and 2.5 mL of tap water inserted into the crop by use of a gavage tube (n = 8) or the equivalent volume of tap water only (control group; 4) for 15 days. Urine and feces were collected 2 hours after treatment administration each day. Feces were evaluated for occult blood. Results of a CBC and serum biochemical analysis and measured N-acetyl-β-d-glucosaminidase (NAG) activity and whole blood clotting time were evaluated before, during, and after completion of treatments. Results of urinalysis and measured urine NAG activity were also evaluated.

RESULTS Birds treated with meloxicam had a significant increase in number of WBCs and decrease in PCV from before to after treatment. The PCV also decreased significantly, compared with results for the control group; however, WBC count and PCV for all birds remained within reference ranges throughout the study. One parrot treated with meloxicam had a single high value for urine NAG activity.

CONCLUSIONS AND CLINICAL RELEVANCE Meloxicam administered orally at the dosage used in this study caused no apparent negative changes in several renal, gastrointestinal, or hemostatic variables in healthy Hispaniolan Amazon parrots. Additional studies to evaluate adverse effects of NSAIDs in birds will be needed.

The NSAIDs are the most commonly prescribed veterinary analgesics and exert their therapeutic effect by inhibition of COX enzymes, which block the conversion of arachidonic acid to proinflammatory PG mediators. Meloxicam, an enolic acid derivative and selective COX-2 inhibitor, is an NSAID commonly prescribed for extralabel use1 in companion birds because it is commercially available in both oral and injectable formulations at concentrations amenable for use in avian species. Investigators have evaluated the pharmacokinetics of meloxicam in avian species, including chickens,2 ostriches,2 ducks,2 turkeys,2 pigeons,2 vultures,3 ring-necked parakeets,4 Hispaniolan Amazon parrots (Amazona ventralis),5 African grey parrots (Psittacus erithacus erithacus),6 red-tailed hawks (Buteo jamaicensis),7 and great-horned owls (Bubo virginianus).7 Mean ± SD half-life (15.8 ± 8.6 hours) and peak concentration (3.5 ± 1.2 μg/mL), time to peak concentration (6 hours), and bioavailability (range, 49% to 75%) of meloxicam in Hispaniolan Amazon parrots after oral administration of 1 mg/kg have been determined.5 Authors of a pharmacodynamic study8 concluded that meloxicam at a dosage of 1 mg/kg, IM, every 12 hours significantly improved weight bearing in Hispaniolan Amazon parrots with experimentally induced arthritis, but a dosage of 0.5 mg/kg every 12 hours did not. In addition, several studies have been conducted to evaluate the adverse effects of meloxicam in budgerigars,9 African grey parrots,10 domestic pigeons,11 and Japanese quail.12 However, no significant renal, gastrointestinal, or hemostatic adverse effects were reported at the dosages evaluated. The renal effects of NSAIDs are of particular concern in Asian vultures (Gyps bengalensis, Gyps indicus, and Gyps tenuirostris).13,14 These species are more sensitive to the adverse renal effects of NSAIDs, except for meloxicam.15

Antagonism of COX-1 and COX-2 disrupts conversion of arachidonic acid into physiologic bioactive PGs16 and can cause renal, gastrointestinal, and hemostatic adverse effects.1,17 In the mammalian kidney, PG products of the COX-1 and COX-2 arachidonic acid pathways have vasodilatory effects that increase glomerular filtration rate, sodium excretion, renal blood flow, and renin release.18–20 Because NSAIDs greatly reduce the release of vasodilatory PGs, they increase the risk of irreversible renal damage attributable to ischemia when there is renal hypotension.17 Also, direct toxic effects attributable to reactive oxygen species and interference with uric acid transport have been observed with long-term exposure of wild vultures to diclofenac-contaminated carcasses.21 Variables considered to be useful for assessment of renal damage in avian species include uric acid and BUN concentrations,22,23 urinalysis variables,22–24 urinalysis tubular casts,25–28 and NAG activity.29,30 N-acetyl-β-d-glucosaminidase is an exoglycolytic enzyme found in lysosomes of renal tubular cells. It is released into the urine and plasma of mammals,31 hens,30 and pigeons29 with necrosis of the renal tubular epithelium. In domestic pigeons (Columba livia) given nephrotoxic doses of gentamicin, plasma NAG activity increased 6-fold and urine NAG activity increased 50-fold from baseline values.29

In the gastrointestinal tract, PG products of arachidonic acid metabolism have cytoprotective effects,32–34 including vasodilation,35,36 reduced secretion of gastric acid,32,33,36,37 increased secretion of bicarbonate, and increased secretion of mucus.38,39 Because COX-1 and COX-2 both catalyze the synthesis of these mediators, NSAIDs must antagonize both COX-1 and COX-2 before gastric integrity is compromised and ulcers develop.40,41 Gastrointestinal ulcers with hemorrhage can be evaluated by means of occult blood tests. Given that these tests are based on an oxidative reaction, other oxidative agents can cause false-positive results.42 All NSAIDs undergo hepatic metabolism.43,44 Although exact mechanisms are unknown, drug accumulation, reactive metabolites, mitochondrial damage, and idiosyncratic damage are considered to be causes of NSAID-induced hepatotoxicosis.43,45 In birds, hepatic damage is correlated with increases in enzyme activities and decreases or increases in metabolite concentrations; the hepatic enzymes considered most useful in avian medicine are AST, γ-glutamyl transferase, lactate dehydrogenase, and GDH, and the metabolites considered most specific are albumin and bile acids.22,23,46

In mammalian platelets, inhibition of COX-1 has a prolonged antithrombotic effect because de novo protein synthesis is not possible without a nucleus.47 In contrast, cells of the vascular endothelium recover from COX inhibition within 6 hours.47 Nucleated avian thrombocytes may be more resistant to NSAID effects than are their anuclear mammalian counterparts. Mammalian platelets express only COX-1 and synthesize only thromboxane A2. However, PGE2, PGF2, and PGI2 are found in relevant quantities in avian thrombocytes because the nucleated thrombocytes express both COX-1 and COX-2.48 Because avian species do not express intrinsic clotting factors, whole blood clotting times are used to assess coagulation in birds.49,50

Despite meloxicam's large therapeutic range and relative safety, compared with those of other NSAIDs,44,51–53 potential species-specific adverse effects need to be evaluated. The purpose of the study reported here was to evaluate possible adverse effects of meloxicam administered at a dosage (1.6 mg/kg, PO, q 12 h for 15 days) that has been found to be therapeutic on the basis of pharmacodynamic and pharmacokinetic data available in Hispaniolan Amazon parrots. The hypothesis was that meloxicam administered at this dosage would not result in renal, gastrointestinal, or hemostatic adverse effects in this species.

Materials and Methods

Animals

Twelve adult Hispaniolan Amazon parrots of unknown sex, with body weights ranging from 280 to 300 g, were included in the study. All birds were considered healthy on the basis of clinical history and results of a physical examination, CBC, and serum biochemical analysis. Animals were treated and evaluated at the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis. Birds were housed individually in wire cages (61 × 58 × 66 cm) with wooden perches and hanging toys; a cycle of 12 hours of light to 12 hours of darkness was provided. Birds had ad libitum access to water and a commercial pelleted diet.a All procedures were approved by the University of California-Davis Institutional Animal Care and Use Committee.

Study design

During the 14 days preceding the study, all birds received 2.5 mL of water inserted into the crop by use of a gavage tube each morning. Ad libitum water intake was restricted for three hours. This resulted in urination within 3 hours after gavage for most birds. By use of a random integer list generator,b birds were assigned to receive meloxicam oral suspensionc (1.6 mg/kg, PO, q 12 h) and 2.5 mL of tap water inserted into the crop by use of a gavage tube (n = 8) or the equivalent volume of tap water only (control group; 4) for 15 days. First day of treatment was designated as day 0.

Blood collection

Blood samples were collected on days 17, 14, and 12 before treatment; 6 hours after the first treatment; every 48 hours during the treatment period; and 32 days after the conclusion of treatment. Blood samples were collected by jugular or medial metatarsal venipuncture with a 1- or 3-mL syringe and 27-gauge needle. On days when multiple analyses were performed, 0.1 mL of blood was collected in a microhematocrit tube for determination of whole blood clotting time, which was followed by collection of 0.5 mL of blood in a K2EDTA tubed and 0.6 mL of blood in a serum separator tube.e Samples in K2EDTA tubes were used for CBCs; they were maintained on ice and submitted within 2 hours after collection to the University of California-Davis Veterinary Medical Teaching Hospital Clinical Pathology Service. Samples for serum analysis were allowed to clot for at least 1 hour at room temperature (20°C) and then centrifuged at 1,843 × g for 6 minutes. Serum was harvested, placed in cryopreservation tubes,f and stored at −80°C.

Whole blood clotting time

Whole blood clotting time was measured in accordance with a modification of a method reported previously.49,50 Measurements were obtained on days 20, 17, and 12 before treatment and treatment days 7 and 15. Blood was collected with a 1- or 3-mL syringe and 27-gauge needle and immediately injected slowly into 14 microhematocrit tubes.g Beginning 90 seconds after completion of venipuncture, 1 tube was manually broken every 30 seconds. Tubes were broken at a distance of approximately 4 cm from a vertically mounted opaque plastic sheet. A sample was considered clotted if a clot was visible between the halves of the broken tube or if there was no spatter of liquid blood on the plastic sheet. Clotting time was recorded as the first of 3 consecutively clotted tubes. In some cases, difficulty with venipuncture or a prolonged blood collection resulted in clot formation in the syringe prior to filling of the microhematocrit tubes; these results were excluded from further analysis.

Hematologic evaluation and serum biochemical analysis

Hematologic evaluation was performed on all birds 14 days before treatment, serum biochemical analysis was performed on all birds 12 days before treatment, and both examinations were performed on all birds on day 15 of treatment and 32 days after the conclusion of treatment. Hematologic evaluation and serum biochemical analysis were performed by the University of California-Davis Veterinary Medical Teaching Hospital Clinical Pathology Service in accordance with standard operating procedures.54 Hematologic variables measured were RBC count, hemoglobin concentration, PCV (centrifuged), mean cellular volume (calculated), mean cell hemoglobin content, mean cell hemoglobin concentration, WBC count (manual), WBC differential count, and plasma protein concentration with fibrinogen concentration. Blood smears were prepared by laboratory hematology technicians and stained with Wright-Giemsa stain. For calculation of the WBC count, both sides of a standard hemacytometer were charged with a 1:100 dilution of whole blood. At 500× magnification, 9 squares were counted on each side of the hemacytometer, and the mean value was calculated as the WBC count. If values for the sides differed by > 10%, counts were repeated with another sample of blood. A 200-cell WBC differential count was performed by the same technician who performed the WBC count. Absolute counts were calculated from differential percentages and the total WBC count. To verify the manual count, an estimate of the WBC count was made by screening a monolayer blood smear, determining the mean number of WBCs at 400× magnification in 10 fields, and multiplying this value by 2,500. Thrombocyte counts were estimated as low, adequate, or high.

Blood biochemical analysis was performed with a clinical chemistry analyzer.h Total protein, albumin, cholesterol, and uric acid concentrations as well as GDH, AST, and ALP activities were evaluated. All assays were performed in accordance with the manufacturer's instructions.

Serum and urine NAG activity were determined with an analyzeri by use of a commercially available assay.j In that assay, NAG hydrolyzed 2-methoxy-4-(2′nitrovinyl)-phenyl2-acetamido-2-deoxy-β-d-glucopyranoside to 2-methoxy-4-(2′-nitrovinyl)-phenol. The product was detected by development of color at 505 nm by the addition of an alkaline (pH, 10) sodium bicarbonate buffer solution. The assay was validated for use on urine and serum samples from Hispaniolan Amazon parrots on the basis of a recovery evaluation and repeated measurements of identical samples obtained during the pretreatment period. Recovery of purified NAG added to pretreatment samples was highly efficient (r = 0.999). During validation, the duration of incubation of the sample and substrate was adjusted on the basis of NAG activity in the test samples. Precision and repeatability of the assay were assessed by evaluating interassay and intra-assay variability for urine samples with low, medium, and high NAG activity. The assay was performed in accordance with the manufacturer's instructions.

Serum samples for measurement of NAG activity were obtained on days 17, 14, and 12 before treatment; at 6 hours after the first treatment; and on days 3, 7, and 15. All serum samples were frozen at −80°C within 4 hours after collection and analyzed as a batch on day 16. Serum samples were obtained 32 days after the conclusion of treatment, frozen at −80°C within 4 hours, and analyzed the next day. Samples were thawed only once (at the time of analysis).

Urine collection and analysis

Each morning between 7 and 9 am, before the room lights were turned on for the day, water bottles and food were removed from each cage. Meloxicam or control doses were administered, and transparent plastic sheets were placed over the cage bedding to collect urine and feces. Three hours after treatment, urine was collected in disposable plastic pipettes. Urine samples were centrifuged at 485 × g for 10 minutes, cooled at 4°C for 30 minutes to facilitate precipitation of mucus, and centrifuged again at 485 × g for 2 minutes. Urine specific gravity was determined with a refractometer. Urine was analyzed (pH and protein, glucose, ketones, and hemoglobin concentrations) with a human urinalysis dipstick testk every 48 hours beginning on treatment day 2. The remainder of the supernatant was stored at −80°C until analysis for NAG activity. Recovery of purified NAG added to pretreatment samples was highly efficient (r = 0.999). Urine samples for analysis of NAG activity were collected on days 17, 14, and 12 before treatment and on days 3, 7, and 15. All urine samples were stored at −80°C and simultaneously analyzed as described for serum.

Urine sediment was examined for the presence of casts, somatic or inflammatory cells, and crystals in samples obtained 12 and 13 days before treatment and on days 3 through 6. Sediment was resuspended in approximately 200 μL of supernatant or, when insufficient supernatant remained, deionized water. A drop of resuspended sediment was mixed with a drop of new methylene blue stain on a glass slide, covered with a coverslip, and examined immediately with light microscopy.

Fecal collection and occult blood analysis

Fresh fecal samples were collected from the plastic sheets at the same time as collection of urine samples. Fecal samples were applied directly onto occult blood test slides.l All positive results were confirmed by a second observer at the time of the test.

Statistical analysis

Data were evaluated for normality by means of a Shapiro-Wilk test. Because several variables were not normally distributed and the sample size was 12 birds, descriptive statistics were reported as median, IQR (25th to 75th percentile), and range.55 Data were analyzed via multilevel mixed-effects linear regression to evaluate effects of treatment, time, or the treatment-by-time interaction. If no significant interaction was found, the analysis was repeated without the interaction term to reduce the degrees of freedom and increase sensitivity for detecting treatment and time effects. Scatterplots of data were visually examined, and noticeable outliers were screened by means of the Tukey method for outlier detection.56 Values of P ≤ 0.05 were considered significant. All data were analyzed with commercially available software.m

Results

Birds

Pretreatment body weight (median, 293.4 g; IQR, 278.1 to 301.2 g) and posttreatment body weight (median, 295.7 g; IQR, 279.2 to 304.7 g) for all 12 birds did not differ significantly.

Whole blood clotting time

Whole blood clotting time decreased significantly (P < 0.001) over time for both groups during the pretreatment period (days 20, 17, and 12 before treatment). There were no significant effects of treatment (P = 0.35), time (P = 0.53), or the treatment-by-time interaction (P = 0.51) during the treatment period. Mean ± SE difference between treatments was 0.58 ± 0.61 seconds (power to detect this difference between the 2 groups, 0.13). One parrot from the meloxicam group had a significant (as determined with the Tukey method) increase in clotting time during the treatment period.

Hematologic evaluation

The meloxicam group had a significant (P = 0.026) increase in median WBC count, from 4,094 × 106 WBCs/L (IQR, 3,527 × 106 WBCs/L to 6,200 × 106 WBCs/L) before treatment to 6,056 × 106 WBCs/L (IQR, 5,122 × 106 WBCs/L to 6,725 × 106 WBCs/L) after treatment, and a significant (P = 0.001) decrease in median PCV, from 56.0% before treatment (IQR, 54.0% to 56.3%) to 50.5% (IQR, 49.9% to 57.6%) after treatment (Table 1). There were no other significant effects of treatment or time and no significant interactions among hematologic variables.

Table 1—

Statistical evaluation of results for hematologic evaluation and serum biochemical analysis of samples obtained from Hispaniolan Amazon parrots (Amazona ventralis) before, during, and after receiving meloxicam (1.6 mg/kg, PO, q 12 h) and 2.5 mL of tap water by use of a gavage tube (n = 8) or an equal volume of tap water (control group; 4) for 15 days.

VariableGroupEffectP value
Hematologic evaluation
 WBC countTreatmentTime effect: increase over time0.026
 HctTreatmentInteraction: decrease over time, compared with values for the control group0.001
 TreatmentTime effect: decrease over time< 0.001
Serum biochemical analysis
 Total protein concentrationNS
 Albumin concentrationNS
 Cholesterol concentrationNS
 GDH activityControlInteraction: increase over time, compared with values for the treatment group0.001
 AST activityNS
 ALP activityNS
 Uric acid concentrationControl and treatmentTime: decrease over time< 0.001

NS = Not significant (P > 0.05). — = Not applicable.

Serum biochemical analysis

A significant (P = 0.014) treatment-by-time interaction was detected for GDH activity, which was caused by a significant (P = 0.001) increase from a median of 0.50 U/L (IQR, 0 to 1.25 U/L) before treatment to 2.50 U/L (IQR, 1.00 to 4.25 U/L) after treatment in the control group only. There was a significant (P < 0.001) decrease in median uric acid concentration over time in both groups, from 8.20 mmol/L (IQR, 6.50 to 8.23 mmol/L) before treatment to 6.25 mmol/L (IQR, 5.98 to 7.50 mmol/L) after treatment for the meloxicam group and from 7.55 mmol/L (IQR, 5.85 to 9.13 mmol/L) before treatment to 5.35 mmol/L (IQR, 5.05 to 5.88 mmol/L) after treatment for the control group; however, there was no significant effect of treatment. There were no other significant effects of treatment or time and no significant interactions among the remaining biochemical variables.

Median serum NAG activity increased significantly (P = 0.007) for both groups, from 33.30 U/L (IQR, 29.45 to 41.53 U/L; range, 25.70 to 52.30 U/L) before treatment to 41.00 U/L (IQR, 37.30 to 56.70 U/L); however, there was no significant treatment effect. This increase was not correlated with urine specific gravity (Figures 1 and 2).

Figure 1—
Figure 1—

Serum NAG activity in samples obtained from Hispaniolan Amazon parrots (Amazona ventralis) before and after receiving meloxicam (1.6 mg/kg, PO, q 12 h) plus 2.5 mL of tap water by use of a gavage tube (n = 8 [black circles]) or an equal volume of tap water (control group; 4 [white squares]) for 15 days. The solid and dashed lines represent median NAG activity for the meloxicam and control groups, respectively. First day of treatment was designated as day 0.

Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.308

Figure 2—
Figure 2—

Serum NAG activity versus urine specific gravity in samples obtained from the 12 Hispaniolan Amazon parrots of Figure 1. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.308

Urinalysis

A significant (P = 0.035) treatment-by-time interaction was detected for urine specific gravity during the pretreatment period, which was caused by a significant (P < 0.001) decrease in urine specific gravity in the control group only. There were no significant effects of treatment (P = 0.66), time (P = 0.43), or the treatment-by-time interaction (P = 0.81) for any urinalysis variables during the treatment period. Median urine NAG activity for both groups before treatment was 2.55 U/L (IQR, 1.75 to 3.48 U/L; range, 1.10 to 9.20 U/L). There were no significant effects of treatment (P = 0.96), time (P = 0.83), or the treatment-by-time interaction (P = 0.75) for urine NAG activity. One parrot in the meloxicam group had a significant (as determined with the Tukey method) increase in urine NAG activity at 1 point during the treatment period.

Fecal occult blood testing

Positive fecal occult blood test results were detected for both the meloxicam and control groups. One parrot in the control group had a positive result on the last day of the pretreatment period. Three parrots in the meloxicam group had positive results on the first day of treatment (sample obtained 3 hours after the first administration of meloxicam). One parrot in the treatment group had positive results on days 13 and 14 but negative results on day 15. No patterns over time were evident. Daily testing yielded positive test results for individual birds on the last day of pretreatment and days 1, 13, and 14 for both the control and treatment groups. No bird had repeated positive test results over a prolonged period.

Discussion

In the present study, no clinically relevant adverse effects, including whole blood clotting time or results of hematologic evaluation, serum biochemical analysis, urinalysis, and fecal occult blood tests, were detected during and after a period during which Hispaniolan Amazon parrots received meloxicam (1.6 mg/kg, PO, q 12 h for 15 days). Although some significant differences were found for results of the hematologic evaluation and serum biochemical analysis, values were within the respective reference ranges and were considered not to be clinically important.

A decrease in clotting time was observed for both groups. We propose that this may have been caused by local irritation of the jugular vein and surrounding area attributable to repeated venipuncture. One parrot had prolonged clotting times before and during the study and was statistically assessed as an outlier, which could have been attributed to individual sensitivity to meloxicam-induced blood clotting or to individual sensitivity for repeated venipuncture and blood sample collection. Prolonged clotting time after the use of NSAIDs in mammals is attributed to irreversible inhibition of COX-1 in platelets. Mammalian platelets lack a nucleus, so no de novo synthesis of COX-1 can be initiated, which results in long-lasting inhibition of thromboxane.48 However, avian and reptilian thrombocytes have a nucleus49 and therefore are theoretically able to synthesize new COX enzymes, which could decrease the effect of NSAIDs on avian and reptilian clotting times. De novo synthesis might explain the relatively short duration of thromboxane inhibition after flunixin administration to mallard ducks (12 hours),57 compared with results after flunixin administration to horses (48 hours).58 Testing methods available to evaluate avian clotting times are limited. Because of the subsidiary role of the intrinsic clotting pathway,49 activated partial thromboplastin time does not provide an accurate estimation of avian hemostasis. Prothrombin time has been evaluated in Amazon parrots and umbrella cockatoos but has not been standardized.59 Only whole blood clotting times have been used clinically.49 Unfortunately, determination of whole blood clotting time yields only crude results. For the study reported here, a standard method49,50 was modified so that a smaller blood volume could be used. The standard method relies on observation of a clot between broken segments of the microhematocrit tubes, but because clots were rarely seen during preliminary experiments, blood spatter when breaking the tube was included in the assessment. In the present study, a prolonged duration for blood collection or venipuncture through a hematoma resulted in accelerated blood clotting. This was thought to be caused by contamination with tissue thromboplastin (factor III), which can substantially decrease whole blood clotting time.60 Overall, values were within the reference range49 (< 5 minutes) for this test, which, combined with the absence of time, treatment, and individual effects, indicated that the dosage of meloxicam used in this study did not cause serious delays in coagulation.

There was a significant decrease in PCV and increase in WBC count for the meloxicam group before and after treatment. Although these were significant changes, the median PCV and WBC count were within established reference ranges for Hispaniolan Amazon parrots,61 which makes the importance of these findings unclear. Both groups had the same volume of blood collected throughout the study, and there was no evidence of renal or gastrointestinal blood loss. Immune-mediated hemolysis has been attributed to the use of NSAIDs in humans,62,63 but in the present study, no erythrogram abnormalities were found that would support a finding of hemolysis or direct NSAID toxic effects on RBCs. The concurrent increase in WBC count makes bone marrow suppression less likely as a cause of the decreased PCV. An increase in WBC count combined with a decrease in PCV may represent a systemic response to low-grade inflammation, as seen in mammals. Considering that all variables remained within their reference ranges, clinical importance was considered to be minimal.

Activity of GDH increased significantly for the control group, which was considered most likely to be a spurious and clinically irrelevant event. No increase was detected for the treatment group. Activity of AST remained constant, although 1 parrot had increased AST activity both before and after treatment. This phenomenon was attributed to individual variation within the birds. Activity of ALP did not change significantly. No changes in cholesterol, total protein, and albumin concentrations were detected. The enzyme GDH, which is present in hepatic mitochondria, is regarded as the most specific indicator of hepatocellular necrosis in avian species.22,64 Given that hepatic necrosis is required to increase plasma GDH activity, its sensitivity is low.22,65 In addition, GDH is highly active in renal tissue; however, when renal damage occurs, GDH is excreted into the urine without increasing activity in the plasma.66 Activity of AST is a sensitive but relatively nonspecific marker for hepatocellular and muscular damage.23,65,67,68 Liver diseases, exposure to chemical toxins, and some drugs (eg, doxycycline and antifungal drugs) will cause an increase of plasma AST activity.69,70 In a study71 in which investigators compared hepatotoxic effects of ketoprofen (3 mg/kg, IM) with those of diclofenac (2.5 mg/kg, IM) in broiler chickens, AST activity increased in chickens treated with diclofenac because of hepatic toxicosis but not in chickens treated with ketoprofen, which did not develop clinical signs. Activity of AST was not significantly increased after treatment with meloxicam in the birds of the study reported here. Other possible variables (eg, cholesterol and albumin concentrations) that could be suggestive of liver disease also did not change significantly.

Concentrations of uric acid decreased over time for both groups. Considering that this effect was evident in both groups, it was most likely caused by improved hydration as a result of oral administration of water. Birds in the treatment group received meloxicam and 2.5 mL of water twice daily, whereas birds in the control group received the same volume of water twice daily. We estimated water maintenance to be 50 mL/kg/d. Free water intake was restricted during the first 3 hours after lights were turned on each morning. A maximum weight of 330 g was used to establish a maintenance water dosage of 2 mL every 3 hours for each bird. An additional 0.5 mL of water was added to ensure reliable urine production. During preliminary experiments, this amount of water was able to sustain sufficient urine production for collection and testing. In avian species, uric acid is the most important nitrogen-containing waste product.72 Uric acid concentrations can be influenced by species, diet, and age.69 Only 10% is excreted by glomerular filtration, whereas 90% is excreted by active secretion in the proximal convoluted tubules.73 Therefore, uric acid concentrations in noncarnivorous birds are affected when dehydration is so severe that condensed uric acid cannot flow into the convoluted tubules.73 Another reason for an elevated uric acid concentration is severe renal damage, in which > 70% of renal function has been lost.74 Also, severe tissue necrosis or starvation can increase nitrogenous catabolism, which leads to increased uric acid concentrations.69,75,76 Although commonly used for evaluation of avian renal function, uric acid concentrations are not consistently reliable. Elevated uric acid concentrations are consistent with extensive damage of the proximal tubules or severe dehydration, but not all birds with extensive renal damage will have increased uric acid concentrations.74

Serum NAG activity increased significantly over time for both the control and treatment groups, but no interaction or treatment effect was detected. Stress attributable to handling and diminished water intake just prior to sample collection were thought to cause this result in both groups. N-acetyl-β-d-glucosaminidase is a lysosomal enzyme found in high concentrations in avian renal tubular cells,29 and it is released into blood and urine when there is cell necrosis. It has proven to be a good indicator of acute renal damage in mammalian77–80 and avian29,30 species, which creates an alternative for evaluation of renal damage. The diagnostic value of NAG activity has been examined in hens30 and pigeons.29 In that study29 of pigeons, investigators detected a 6-fold increase in serum NAG activity and a 50-fold increase in urine NAG activity with complete renal failure after administration of high doses of gentamicin. However, other investigators did not detect a significant increase in urine NAG activity after 10 days of 3-fold overdosing of cholecalciferol to hens.30 Given that the increase in NAG activity is more pronounced in urine after renal damage, urine NAG activity might be a more reliable variable than serum NAG activity for assessing renal damage, although serum is easier to obtain.29

No change in urinalysis variables or urine specific gravity was detected during the study. Urine specific gravity significantly decreased before treatment in the control group. This was most likely an incidental finding because we expected no differences between treatment groups prior to the study. Hemoglobin concentrations were consistently high, which was considered to be a false-positive result because no blood cells were observed in the sediment and positive results for fecal occult blood tests were not consistently observed. Urine sediment was screened for casts, but none were observed, which suggested that there was no substantial renal tubular damage.25–28,81 There was no increase in urine NAG activity for both groups.

There were sporadic and inconsistent positive results for the fecal occult blood tests of both groups. Urine consistently had elevated hemoglobin concentrations for both the treatment and control groups, which was not consistent with the fecal occult blood test results. A previous pharmacokinetic study8 of Hispaniolan Amazon parrots with a comparable dose of meloxicam (1 mg/kg, IM, q 12 h for 4 doses) provided negative results of fecal occult blood tests for all Amazon parrots treated with meloxicam, although the birds were not treated for prolonged periods. Considering that gastrointestinal ulcerations would cause blood loss for prolonged periods, we attributed the sporadic positive results of the fecal occult blood tests to transient mucosal damage caused by gavage. Esophageal damage is possible when oral gavage is used, although this is more common in passerine species and less frequently observed in psittacine species.82 Another possibility was the presence of fruit acids in the fecal samples, which can cause false-positive results.42,83 Although the birds were fed a pelleted feed, these pellets contained ingredients extracted from fruits, which might have contained fruit acids.83 In addition to sporadic positive results for fecal occult blood tests, there was a consistent strong positive result for hemoglobin on the urine dipstick test. The urine test for hemoglobin relies on the peroxidase principle, in which hemoglobin catalyzes the oxidation of the indicator by incorporating organic peroxide.84 Although reducing substances inhibit this reaction,85 other oxidative substances can enhance peroxidase activity.86 Bacterial peroxides can also increase the total peroxidase activity of urine.87 Given that urine is mixed with feces before excretion in birds, fecal contamination of the urine with bacterial peroxidase or chlorophyll, a phytol-esterified magnesium porphyrin that is a structural analogue of the heme group in hemoglobin,88 could increase the peroxidase activity of urine.

During this study, no evidence of renal, gastrointestinal, or hemostatic adverse effects were found, as determined on the basis of whole blood clotting time and results of hematologic evaluation and serum biochemical analysis, including NAG activity, urinalysis results, and results of fecal occult blood tests. Changes that were detected, such as a decrease in PCV, increased NAG activity for individual birds, and sporadic positive results for fecal occult blood tests, were considered not to be clinically relevant. It is important to remember that this study was performed with healthy parrots without clinical signs of organ dysfunction. Clinicians are advised to assess function to ascertain suitability of meloxicam use for individual patients. Results of this study suggested that the use of meloxicam oral suspension at a dosage of 1.6 mg/kg twice daily in healthy Hispaniolan Amazon parrots was safe. Additional studies are needed to fully evaluate the effects of meloxicam and other NSAIDs in birds.

Acknowledgments

Supported in part by an Association of Avian Veterinarians research grant and the Merial Veterinary Scholar Program.

Presented in abstract form at the 34th Annual Association of Avian Veterinarians Conference, Jacksonville, Fla, August 2013.

ABBREVIATIONS

ALP

Alkaline phosphatase

AST

Aspartate aminotransferase

COX

Cyclooxygenase

GDH

Glutamate dehydrogenase

IQR

Interquartile range

NAG

N-acetyl-β-d-glucosaminidase

PG

Prostaglandin

Footnotes

a.

ZuPreem FruitBlend, Premium Nutritional Products, Shawnee, Kan.

b.

Random.org. True random number service. Available at: www.random.org. Accessed Apr 9, 2014.

c.

Metacam, Boehringer Ingelheim Vetmedica, St Joseph, Mo.

d.

Microtainer EDTA tube, BD, Franklin Lakes, NJ.

e.

Microtainer SST, BD, Franklin Lakes, NJ.

f.

Cryogenic sterilized tubes, Bio Plas Inc, San Rafael, Calif.

g.

Statspin HT9U-10, Iris Sample Processing Inc, Westwood, Mass.

h.

Cobas 6000 C501, Roche Diagnostics GmbH, Mannheim, Germany.

i.

Cobas C311, Roche Diagnostics GmbH, Mannheim, Germany.

j.

Diazyme Laboratories, Poway, Calif.

k.

Roche Diagnostics Ltd, Burgess Hill, West Sussex, England.

l.

Hemoccult, Clinical Diagnostics Division, Beckman Coulter Inc, Brea, Calif.

m.

Stata, version 12.1, StataCorp, College Station, Tex.

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